DLL3 single domain antibody and therapeutic composition of the same

Abstract

To provide a binding polypeptide that specifically binds to DLL3.SOLUTION: Provided is fusion protein that binds to DLL3 and contains a polyvalent and / or multi-specific construct and a chimeric antigen receptor. Further, a polypeptide thereof, a nucleic acid molecule encoding the polypeptide, and a pharmaceutical composition containing a vector and cell thereof, a method for using a provided DLL3 binding polypeptide for treating diseases and conditions such as cancer, and use thereof are also provided.SELECTED DRAWING: Figure 3A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to the following U.S. provisional applications, the contents of which are incorporated by reference in their entireties for all purposes: Serial No. 62 / 744,638, filed October 11, 2018, entitled "DLL3 SINGLE DOMAIN ANTIBODIES AND THERAPEUTIC COMPOSITIONS THEREOF"; Serial No. 62 / 832,265, filed April 10, 2019, entitled "DLL3 SINGLE DOMAIN ANTIBODIES AND THERAPEUTIC COMPOSITIONS THEREOF"; and Serial No. 62 / 877,815, filed July 23, 2019, entitled "DLL3 SINGLE DOMAIN ANTIBODIES AND THERAPEUTIC COMPOSITIONS THEREOF."

[0002] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING This application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided in a file named 744952000640SeqList.TXT, created on October 8, 2019, which is 431 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present disclosure generally provides binding polypeptides that specifically bind to DLL3. More specifically, the present disclosure relates to fusion proteins, such as multivalent and / or multispecific constructs and chimeric antigen receptors, that at least bind to DLL3. The present disclosure also provides nucleic acid molecules encoding the polypeptides, as well as vectors and cells thereof, and methods and uses of the provided DLL3-binding polypeptides for treating diseases and conditions such as cancer. [Background technology]

[0004] background Delta-like ligand 3 (DLL3) is an inhibitory Notch pathway ligand that is highly upregulated and aberrantly expressed on the cell surface of tumor cells and cancer cells, for example, in cells of small cell lung cancer (SCLC) and high-grade neuroendocrine tumors.Due to the expression of DLL3 in various cancers in humans, for example, solid tumors, DLL3 is a desirable therapeutic target.There is a need for improved therapeutic molecules and drugs that target DLL3.Embodiments that meet this need are provided herein. Summary of the Invention

[0005] overview Provided herein are DLL3-binding polypeptide constructs comprising at least one heavy chain-only variable domain (DLL3 VHH domain) that specifically binds to DLL3. In some embodiments, the DLL3-binding construct comprises one or more additional binding domains that bind to targets other than DLL3.

[0006] At least one DLL3 VHH domain comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, and 456; a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 384, 410, and 411; and Provided herein is a DLL3-binding polypeptide construct that comprises a complementarity-determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of NOs: 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 395, and 412-415, and that binds to DLL3.

[0007] Complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, and 456; Complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 384, 410, and 411; and At least one heavy chain-only variable domain (DLL3 VHH domain) that specifically binds to DLL3 and comprises a complementarity-determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of NOs: 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 395, and 412 to 415, and binds to DLL3. Provided herein is a DLL3-binding polypeptide construct comprising:

[0008] In some of any of the provided embodiments, at least one DLL3 VHH domain comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, and 456; a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, and 353; and a complementarity determining region 3 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, and 353. It comprises a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of NOs: 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, and 367, and binds to DLL3.

[0009] In some of any of the provided embodiments, the DLL3 is human DLL3. In some embodiments, the DLL3 has the sequence set forth in SEQ ID NO:86 or its mature form lacking the signal sequence. In some embodiments, the DLL3 has the sequence set forth in SEQ ID NO:87 or its mature form lacking the signal sequence.

[0010] In some embodiments, at least one DLL3 VHH domain is humanized. In some embodiments, the DLL3 VHH is a camelid VHH. In some embodiments, the DLL3 VHH is a humanized version of a camelid VHH.

[0011] In some of the provided embodiments, the one or more additional binding domains bind to an activating receptor on an immune cell. In some embodiments, the immune cell is a T cell. In some of the provided embodiments, the activating receptor is CD3 (CD3ε). In some examples, the DLL3-binding polypeptide construct of the embodiment is bispecific for DLL3 and CD3. In some embodiments, the immune cell is a natural killer (NK) cell.

[0012] In some of any of the provided embodiments, the activating receptor is CD16 (CD16a). In some instances, the DLL3-binding polypeptide construct is bispecific for DLL3 and CD16a.

[0013] In some of any of the embodiments provided, the one or more additional binding domains bind to a cytokine receptor.

[0014] In some of any of the provided embodiments, the one or more additional binding domains comprise an antibody or antigen-binding fragment thereof. In some embodiments, the one or more additional binding domains are monovalent. In some embodiments, the antibody or antigen-binding fragment thereof is an Fv, disulfide-stabilized Fv (dsFv), scFv, Fab, single-domain antibody (sdAb), VNAR, or VHH. In some embodiments, the single-domain antibody (sdAb) is a camelid VHH. In some embodiments, the single-domain antibody (sdAb) is a humanized version of a camelid VHH.

[0015] In some of any of the provided embodiments, the one or more additional binding domains are cytokines or truncated fragments or variants thereof capable of binding to cytokine receptors. In some embodiments, the cytokine is an interferon or a truncated fragment or variant of an interferon. In some embodiments, the interferon is a type I interferon or a type II interferon, a truncated fragment or variant of a type I interferon, or a truncated fragment or variant of a type II interferon. In some embodiments, the type I interferon is IFNα or IFNβ, or a truncated fragment or variant thereof; or the type II interferon is IFNγ, or a truncated fragment or variant thereof.

[0016] In some of the provided embodiments, the polypeptide comprises an immunoglobulin Fc region. In some embodiments, the polypeptide comprises an immunoglobulin Fc region linking at least one VHH domain and one or more additional binding domains. In some embodiments, the DLL3-binding polypeptide construct is a dimer. In some embodiments, the Fc region is a homodimeric Fc region.

[0017] In some of any of the provided embodiments, the Fc region comprises a sequence of amino acids set forth in any of SEQ ID NOs:8, 10, 11, 12, or 13, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:8, 10, 11, 12, or 13. In some embodiments, the Fc region is a human IgG1.

[0018] In some of the embodiments provided, the DLL3-binding polypeptide construct is a dimer. In some embodiments, the Fc region is a homodimeric Fc region.

[0019] In some of any of the embodiments provided, the Fc region is human IgG1.

[0020] In some of any of the provided embodiments, the Fc region comprises a sequence of amino acids set forth in SEQ ID NO:8, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:8.

[0021] In some embodiments, the Fc region is a heterodimeric Fc region. In some embodiments, the Fc region exhibits effector function. In some embodiments, the Fc region comprises a polypeptide comprising one or more amino acid modifications that reduce effector function and / or reduce binding to an effector molecule selected from an Fcγ receptor or C1q. In some embodiments, the one or more amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235.

[0022] In some of any of the provided embodiments, the Fc region comprises a sequence of amino acids set forth in SEQ ID NO:9, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:9.

[0023] In some of any of the provided embodiments, at least one DLL3 VHH domain comprises a VHH domain sequence set forth in any of SEQ ID NOs:244-318 and 455, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:244-318 and 455, and binds DLL3.

[0024] In some of any of the provided embodiments, at least one DLL3 VHH domain comprises a VHH domain sequence set forth in any of SEQ ID NOs: 102, 244-318, 401-409, 416, 455, 476-480-488, and 507-518, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 102, 244-318, 401-409, 416, 455, 476-480-488, and 507-518, and binds DLL3.

[0025] In some of any of the provided embodiments, at least one DLL3 VHH domain is a VHH domain sequence set forth in any of SEQ ID NOs: 102, 244-275, 277-300, 302-305, 314, 401, 416, 455, 476-480-488, and 507-518, or a VHH domain sequence set forth in any of SEQ ID NOs: 102, 244-275, 277-300, 302-305, 314, 401, 416, 455, 476-480-488, and 507-518. It comprises an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of NOs: 102, 244-275, 277-300, 302-305, 314, 401, 416, 455, 476-480-488, and 507-518, and binds to DLL3.

[0026] In some of any of the provided embodiments, at least one DLL3 VHH domain comprises (i) the sequence set forth in SEQ ID NO:244, (ii) a humanized variant of SEQ ID NO:244, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:244, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 319, 320, 321, 322, 323, 324, 325, and 326; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 336, 337, and 338; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 354, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 319, 336, and 354, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 319, 337, and 354, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:319, 338, and 354, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:320, 338, and 354, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:321, 338, and 354, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:322, 338, and 354, respectively.In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:323, 338, and 354, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:324, 338, and 354, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:325, 338, and 354, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:326, 338, and 354, respectively. In some of any of the foregoing embodiments, at least one DLL3 VHH domain binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:245-257, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:245-257, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:245-257, and binds to DLL3.

[0027] In some of any of the provided embodiments, at least one DLL3 VHH domain comprises (i) the sequence set forth in SEQ ID NO:258, (ii) a humanized variant of SEQ ID NO:258, or (iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:258, and binds DLL3. In some embodiments, at least one DLL3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:327; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:339; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:355, and binds DLL3. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:327, 339, and 355, respectively, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises the sequence of amino acids set forth in any one of SEQ ID NOs:259-263, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:259-263, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises the sequence of amino acids set forth in any one of SEQ ID NOs:259-263, and binds to DLL3.

[0028] In some of any of the provided embodiments, at least one DLL3 VHH domain comprises (i) the sequence set forth in SEQ ID NO:264, (ii) a humanized variant of SEQ ID NO:264, or (iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:264, and binds DLL3. In some embodiments, at least one DLL3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:328, 329, or 456; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:340; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:356, and binds DLL3. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 328, 340, and 356, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 329, 340, and 356, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 456, 340, and 356, respectively. In some of any of the foregoing embodiments, at least one DLL3 VHH domain binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:265-274, 416, or 455, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:265-274 or 416 or 455, and binds to DLL3.In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs: 265-274, or 416, or 455, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs: 265-274, 416, 455, or 476-478, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 265-274, 416, 455, or 476-478, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs: 265-274, 416, 455, or 476-478, and binds to DLL3.

[0029] In some of any of the provided embodiments, at least one DLL3 VHH domain comprises (i) the sequence set forth in SEQ ID NO:275, (ii) a humanized variant of SEQ ID NO:275, or (iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:275, and binds DLL3. In some embodiments, at least one DLL3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:320; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:341; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:357, and binds DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:276-279, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:276-279, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:276-279, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:277-279 and 479, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs:277-279 and 479, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:277-279 and 479, and binds to DLL3.

[0030] In some of any of the provided embodiments, at least one DLL3 VHH domain comprises (i) the sequence set forth in SEQ ID NO:280, (ii) a humanized variant of SEQ ID NO:280, or (iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:280, and binds DLL3. In some embodiments, at least one DLL3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:330; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:342; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:358, and binds DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:281-286, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:281-286, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:281-286, and binds to DLL3.

[0031] In some of any of the provided embodiments, at least one DLL3 VHH domain comprises (i) the sequence set forth in SEQ ID NO:287, (ii) a humanized variant of SEQ ID NO:287, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:287, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:320; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:345, 346, and 347; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:359, 360, and 361, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:320, 345, and 359, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:320, 346, and 359, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:320, 347, and 359, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:320, 345, and 360, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:320, 345, and 361, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 320, 347, and 360, respectively. In some embodiments, any of the provided DLL3 VHH domains binds to DLL3.In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:288-298 or 102, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:288-298 or 102, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:288-298 or 102, and binds to DLL3.

[0032] In some of any of the provided embodiments, at least one DLL3 VHH domain comprises (i) the sequence set forth in SEQ ID NO:299, (ii) a humanized variant of SEQ ID NO:299, or (iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:299, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:331; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:348, 349, and 350; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:356, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 331, 348, and 356, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 331, 349, and 356, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 331, 350, and 356, respectively. In some embodiments, any of the foregoing provided DLL3 VHH domains binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:300-305, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:300-305, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:300-305, and binds to DLL3.In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs: 300, 302-305, and 480, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 300, 302-305, and 480, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs: 300, 302-305, and 480, and binds to DLL3.

[0033] In some of any of the provided embodiments, at least one DLL3 VHH domain comprises (i) the sequence set forth in SEQ ID NO:507, (ii) a humanized variant of SEQ ID NO:507, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:507, and binds to DLL3. At least one DLL3 VHH domain comprises (i) the sequence set forth in SEQ ID NO:306, (ii) a humanized variant of SEQ ID NO:306, or (iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:306, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:332; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:348, 349, and 350; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:362, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 332, 348, and 362, respectively. In some embodiments, at least one DLL3 VHH domain comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 332, 349, and 362, respectively. In some embodiments, at least one DLL3 VHH domain comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 332, 350, and 362, respectively.In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:300-305, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:307-313, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:307-313, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:508-514, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:508-514, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:508-514, and binds to DLL3.

[0034] In some of any of the provided embodiments, at least one DLL3 VHH domain comprises (i) the sequence set forth in SEQ ID NO:401, (ii) a humanized variant of SEQ ID NO:401, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:401, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:320; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:384, 410, and 411; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:395, 412, 413, 414, and 415, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:320, 384, and 395, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:320, 410, and 395, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:320, 411, and 395, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:320, 384, and 412, respectively. In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:320, 384, and 413, respectively. In some embodiments, at least one DLL3 VHH domain comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 320, 384, and 414, respectively.In some embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:320, 384, and 415, respectively, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises the sequence of amino acids set forth in any one of SEQ ID NOs:402-409, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:402-409, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises the sequence of amino acids set forth in any one of SEQ ID NOs:402-409, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:481-488, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:481-488, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any one of SEQ ID NOs:481-488, and binds to DLL3.

[0035] In some of the provided embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 333, 351, and 363, respectively. In some of the provided embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in 334, 352, and 364, respectively. In some of the provided embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in 320, 353, and 365, respectively. In some of the provided embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in 334, 339, and 366, respectively. In some of the provided embodiments, at least one DLL3 VHH domain comprises the CDR1, CDR2, and CDR3 set forth in 335, 348, and 367, respectively. In some of any of the provided embodiments, the DLL3 VHH domain binds to DLL3. In some embodiments, at least one DLL3 VHH domain comprises (i) the sequence set forth in SEQ ID NO:314, 315, 316, 317, or 318, (ii) a humanized variant of SEQ ID NO:314, 315, 316, 317, or 318, or (iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:314, 315, 316, 317, or 318, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain is set forth in SEQ ID NO:314, 315, 316, 317, or 318 and binds to DLL3.In some embodiments, at least one DLL3 VHH domain comprises (i) a sequence set forth in SEQ ID NO:314, 518, 515, 516, or 517, (ii) a humanized variant of SEQ ID NO:314, 518, 515, 516, or 517, or (iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:314, 518, 515, 516, or 517, and binds to DLL3. In some embodiments, at least one DLL3 VHH domain is set forth in SEQ ID NO:314, 518, 515, 516, or 517, and binds to DLL3.

[0036] In some embodiments, the DLL3 VHH domain comprises the VHH domain sequence set forth in SEQ ID NO:244. In some embodiments, the DLL3 VHH domain comprises the VHH domain sequence set forth in SEQ ID NO:251. In some embodiments, the DLL3 VHH domain comprises the VHH domain sequence set forth in SEQ ID NO:264. In some embodiments, the DLL3 VHH domain comprises the VHH domain sequence set forth in SEQ ID NO:268. In some embodiments, the DLL3 VHH domain comprises the VHH domain sequence set forth in SEQ ID NO:275. In some embodiments, the DLL3 VHH domain comprises the VHH domain sequence set forth in SEQ ID NO:287. In some embodiments, the DLL3 VHH domain comprises the VHH domain sequence set forth in SEQ ID NO:299. In some embodiments, the DLL3 VHH domain comprises the VHH domain sequence set forth in SEQ ID NO:507. In some embodiments, the DLL3 VHH domain comprises the VHH domain sequence set forth in SEQ ID NO:314. In some embodiments, the DLL3 VHH domain comprises the VHH domain sequence set forth in SEQ ID NO:518. In some embodiments, the DLL3 VHH domain comprises the VHH domain sequence set forth in SEQ ID NO:515. In some embodiments, the DLL3 VHH domain comprises the VHH domain sequence set forth in SEQ ID NO:516. In some embodiments, the DLL3 VHH domain comprises the VHH domain sequence set forth in SEQ ID NO:517. In some embodiments, the DLL3 VHH domain comprises the VHH domain sequence set forth in SEQ ID NO:455.

[0037] In some of the provided embodiments, the DLL3 VHH domain may include additional amino acids at the N-terminus and / or C-terminus for linkage to other amino acid sequences, such as other polypeptides. In some of the provided embodiments, the DLL3 VHH domain may include a flexible linker, such as a linker composed primarily of the amino acids glycine and serine, referred to herein as a glycine linker or GS linker. Such linkers of the present disclosure may be of various lengths, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length. In some embodiments, the linker is TIFF2025060787000002.tif23162. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of: (GGGGS)n (n is 1 to 5) (SEQ ID NO:123); (GGGGGS)n (n is 1 to 4) (SEQ ID NO:124); TIFF2025060787000003.tif16143. In some embodiments, the linker is a GG linker. In some embodiments, the DLL3-binding polypeptide comprises a combination of a GS linker and a glycine linker. In some embodiments, the DLL3 VHH domain can comprise an additional linker at the C-terminus, for example, for linking to other amino acid sequences, such as other polypeptides. In some of any of the provided embodiments, the DLL3 VHH domain can comprise a linker at the N-terminus, for linking to other amino acid sequences, such as other polypeptides.

[0038] In some of any of the provided embodiments, at least one DLL3 VHH domain comprises (i) the sequence set forth in SEQ ID NO:244, (ii) a humanized variant of SEQ ID NO:244, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:244, and binds to DLL3.

[0039] Provided herein is a multispecific polypeptide construct comprising: (a) a first component comprising a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide; and (b) a second component comprising an anti-CD3 antibody or antigen-binding fragment comprising a variable heavy chain region (VH) and a variable light chain region (VL), wherein the VH and VL constituting the anti-CD3 antibody or antigen-binding fragment are linked to opposing polypeptides of the heterodimeric Fc; the first and second components are coupled by a linker, and the heterodimeric Fc region is positioned N-terminal to the anti-CD3 antibody; and one or both of the first and second components comprises at least one antigen-binding domain comprising a VHH domain that specifically binds to DLL3 (DLL3 VHH domain). In a specific embodiment, the DLL3 VHH domain can comprise any of the provided DLL3 VHH domain sequences, for example, any of those described above or elsewhere herein.

[0040] In some embodiments, the multispecific polypeptide construct comprises at least (i) a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and a VH or VL domain of an anti-CD3 antibody or antigen-binding fragment; and (ii) a second polypeptide comprising a second Fc polypeptide of a heterodimeric Fc region, a linker, optionally the same linker as present in the first polypeptide, and the other of the VH or VL domain of the anti-CD3 antibody or antigen-binding fragment, wherein one or both of the first and second polypeptides comprises at least one DLL3 VHH domain.

[0041] In some embodiments, one or both of the first and second Fc polypeptides of the heterodimeric Fc region comprise at least one modification that induces heterodimerization compared to the polypeptide of the homodimeric Fc region, and optionally compared to the Fc polypeptide set forth in SEQ ID NO:8, or an immunologically active fragment thereof. In some embodiments, each of the first and second Fc polypeptides of the heterodimeric Fc region independently comprises at least one amino acid modification. In some embodiments, each of the first and second Fc polypeptides of the heterodimeric Fc region comprises a knob-into-hole modification or a charge mutation that increases the electrostatic complementarity of the polypeptides.

[0042] In some embodiments, the amino acid modification is a knob-into-hole modification. In some embodiments, the first Fc polypeptide of the heterodimeric Fc region comprises a modification selected from among Thr366Ser, Leu368Ala, Tyr407Val, and a combination thereof, and the second Fc polypeptide of the heterodimeric Fc region comprises the modification Thr366Trp. In such embodiments, the first and second Fc polypeptides may further comprise a non-cysteine ​​residue to cysteine ​​residue modification, wherein the modification of the first Fc polypeptide is at one of positions Ser354 and Tyr349, and the modification of the second Fc polypeptide is at the other of positions Ser354 and Tyr349.

[0043] In some embodiments, the amino acid modification is a charge mutation that increases the electrostatic complementarity of the polypeptides, hi some embodiments, the first and / or second Fc polypeptide, or each of the first and second Fc polypeptides, comprises a modification at a complementary position, where the modification is a substitution with an amino acid having an opposite charge to the complementary amino acid of the other polypeptide.

[0044] In some of any of the provided embodiments, one of the first or second Fc polypeptides of the heterodimeric Fc further comprises a modification at residue Ile253. In some embodiments, the modification is Ile253Arg. In some embodiments, one of the first or second Fc polypeptides of the heterodimeric Fc further comprises a modification at residue His435. In some embodiments, the modification is His435Arg.

[0045] In some embodiments, the Fc region of any of the provided polypeptides or constructs comprises a polypeptide lacking Lys447.

[0046] In some embodiments, the Fc region of any of the provided polypeptides or constructs comprises at least one modification to enhance FcRn binding. In some embodiments, the modification is at a position selected from the group consisting of Met252, Ser254, Thr256, Met428, Asn434, and combinations thereof. In some embodiments, the modification is selected from the group consisting of Met252Y, Ser254T, Thr256E, Met428L, Met428V, Asn434S, and combinations thereof. In some embodiments, the modification is at positions Met252 and Met428. In some embodiments, the modification is Met252Y and Met428L. In some embodiments, the modification is Met252Y and Met428V.

[0047] In some of any of the provided embodiments, the first Fc polypeptide of the heterodimeric Fc comprises a sequence of amino acids set forth in any of SEQ ID NOs: 103, 107, 115, or 117, and the second Fc polypeptide of the heterodimeric Fc comprises a sequence of amino acids set forth in any of SEQ ID NOs: 104, 108, 111, 113, 119, or 121.

[0048] In some of any of the provided embodiments, the Fc region of the provided polypeptide or construct comprises a polypeptide comprising at least one amino acid modification that reduces effector function and / or reduces binding to an effector molecule selected from an Fcγ receptor or C1q. In some embodiments, the one or more amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235.

[0049] In some of any of the provided embodiments, the first Fc polypeptide of the heterodimeric Fc comprises a sequence of amino acids set forth in any of SEQ ID NOs: 105, 109, 116, or 118, and the second Fc polypeptide of the heterodimeric Fc comprises a sequence of amino acids set forth in any of SEQ ID NOs: 106, 110, 112, 114, 120, or 122.

[0050] In some of any of the provided embodiments, the anti-CD3 antibody or antigen-binding fragment is monovalent. In some embodiments, the anti-CD3 antibody or antigen-binding fragment is an Fv antibody fragment. In some embodiments, the Fv antibody fragment comprises a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv).

[0051] In some embodiments, the anti-CD3 antibody or antigen-binding fragment is not a single chain antibody, and optionally is not a single chain variable fragment (scFv).

[0052] In some embodiments, the anti-CD3 antibody or antigen-binding fragment comprises a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO:29); Amino acid sequence VH CDR2 containing TIFF2025060787000004.tif4128; Amino acid sequence VH CDR3 containing TIFF2025060787000005.tif4128; Amino acid sequence VL CDR1 containing TIFF2025060787000006.tif4128; VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO:33); and and a VL having the amino acid sequence of any of SEQ ID NOs:66-84 and 368, or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:66-84 and 368. In some embodiments, the anti-CD3 antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO:47 and the amino acid sequence of SEQ ID NO:75. In some embodiments, the anti-CD3 antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO:47 and the amino acid sequence of SEQ ID NO:368.

[0053] In some embodiments, the VL of the anti-CD3 antibody or antigen-binding fragment is linked to a first Fc polypeptide of the heterodimeric Fc region and the VH of the anti-CD3 antibody or antigen-binding fragment is linked to a second Fc polypeptide of the heterodimeric Fc region.

[0054] In some embodiments, at least one DLL3 VHH domain is positioned amino terminal to the Fc region of the multispecific construct and / or carboxy terminal to the CD3 binding region of said multispecific construct.

[0055] In some embodiments, the multispecific polypeptide construct comprises a first DLL3 VHH domain that specifically binds to DLL3 and a second DLL3 VHH domain that specifically binds to DLL3. In specific embodiments, the first DLL3 VHH domain or the second DLL3 VHH domain can independently comprise any of the provided DLL3 VHH domains, for example, any of those described above or elsewhere herein. In some embodiments, the first and second DLL3 VHH domains are identical. In some embodiments, the first and second DLL3 VHH domains are different. In some embodiments, the first and second DLL3 VHH domains bind to distinct or non-overlapping epitopes of DLL3 and / or do not compete for binding to DLL3.

[0056] In some embodiments, the first or second DLL3 VHH domain is positioned amino terminal to the Fc region of the multispecific construct, and the other of the first or second DLL3 VHH domain is positioned carboxy terminal to the CD3 binding region of the multispecific construct.

[0057] In some embodiments of any of the provided multispecific polypeptide constructs, the first component comprises, in order from N-terminus to C-terminus, a first DLL3 VHH domain that binds DLL3, a first Fc polypeptide of the heterodimeric Fc region, a linker, a VH or VL domain of an anti-CD3 antibody or antigen-binding fragment, and a second DLL3 VHH domain that binds DLL3; and the second polypeptide comprises, in order from N-terminus to C-terminus, a second Fc polypeptide of the heterodimeric Fc region, a linker, optionally the same linker as present in the first component, and another VH or VL domain of the anti-CD3 antibody or antigen-binding fragment.

[0058] In some embodiments, one or both of the first and second components comprises at least one costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor. In some embodiments, the at least one costimulatory receptor binding region (CRBR) is positioned amino-terminal to the Fc region of the multispecific polypeptide construct and / or carboxy-terminal to the CD3 binding region of the multispecific polypeptide construct. In some embodiments, the multispecific polypeptide construct comprises only one costimulatory receptor binding region (CRBR).

[0059] In some embodiments of any of the provided multispecific polypeptide constructs, the first component comprises, in order from N-terminus to C-terminus, a first DLL3 VHH domain that binds DLL3, a first Fc polypeptide of the heterodimeric Fc region, a linker, a VH or VL domain of an anti-CD3 antibody or antigen-binding fragment, and a second DLL3 VHH domain that binds DLL3; the second component comprises a CRBR and, in order from N-terminus to C-terminus, a second Fc polypeptide of the heterodimeric Fc region, a linker, optionally the same linker as present in the first component, the other of the VH or VL domain of the anti-CD3 antibody or antigen-binding fragment, where the CRBR is positioned amino-terminal to the Fc region of the second component or carboxy-terminal to the anti-CD3 antibody or antigen-binding fragment.

[0060] In some embodiments, at least one costimulatory receptor binding region (CRBR) is or comprises the extracellular domain of a natural cognate binding partner of a costimulatory receptor or a binding fragment thereof, or a variant thereof that exhibits binding activity with a costimulatory receptor. In some embodiments, at least one costimulatory receptor binding region (CRBR) is an antibody or an antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a Fv fragment, an scFv, a scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. In some embodiments, the antibody or antigen-binding fragment thereof is an Fv, scFv, Fab, a single-domain antibody (VHH domain), a VNAR, or a VHH. In some embodiments, the antibody or antigen-binding fragment is a VHH domain. In some embodiments, the VHH domain is a human VHH domain or a humanized VHH domain.

[0061] In some of any of the provided embodiments, at least one costimulatory receptor binding region (CRBR) binds to a costimulatory receptor selected from among 41BB (CD137), OX40 (CD134), CD27, glucocorticoid-inducible TNFR-related protein (GITR), CD28, ICOS, CD40, B-cell activating factor receptor (BAFF-R), B-cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI), and NKG2D. In some embodiments, at least one costimulatory receptor binding region (CRBR) binds to a costimulatory receptor selected from among 41BB (CD137), OX40 (CD134), and glucocorticoid-inducible TNFR-related protein (GITR).

[0062] In some embodiments, at least one costimulatory receptor binding region (CRBR) comprises a sequence of amino acids set forth in SEQ ID NO:210, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:210, and binds to 4-1BB.

[0063] In some embodiments, at least one costimulatory receptor binding region (CRBR) comprises the sequence of amino acids set forth in SEQ ID NO:210 and binds to 4-1BB.

[0064] In some embodiments, at least one costimulatory receptor binding region (CRBR) comprises a sequence of amino acids set forth in SEQ ID NO:470 and binds to 4-1BB.

[0065] In some embodiments, one or both of the first and second components comprises at least one inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor. In some embodiments, the at least one inhibitory receptor binding region (IRBR) is positioned amino-terminal to the Fc region of the multispecific polypeptide construct and / or carboxy-terminal to the CD3 binding region of the multispecific polypeptide construct. In some embodiments, the multispecific polypeptide construct comprises only one inhibitory receptor binding region (IRBR).

[0066] In some embodiments of any of the provided multispecific polypeptide constructs, the first component comprises, in order from N-terminus to C-terminus, a first DLL3 VHH domain that binds DLL3, a first Fc polypeptide of the heterodimeric Fc region, a linker, a VH or VL domain of an anti-CD3 antibody or antigen-binding fragment, and a second DLL3 VHH domain that binds DLL3; the second component comprises an IRBR and, in order from N-terminus to C-terminus, a second Fc polypeptide of the heterodimeric Fc region, a linker, optionally the same linker as present in the first component, and the other of the VH or VL domain of the anti-CD3 antibody or antigen-binding fragment, wherein the IRBR is positioned amino-terminal to the Fc region of the second component or carboxy-terminal to the anti-CD3 antibody or antigen-binding fragment.

[0067] In some embodiments, at least one IRBR is or comprises the extracellular domain of a natural cognate binding partner of an inhibitory receptor or a binding fragment thereof, or a variant thereof that exhibits binding activity with the inhibitory receptor. In some embodiments, at least one IRBR is an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a Fv fragment, an scFv, a scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. In some embodiments, the antibody or antigen-binding fragment thereof is an Fv, an scFv, a Fab, a single-domain antibody (VHH domain), a VNAR, or a VHH. In some embodiments, the antibody or antigen-binding fragment is a VHH domain. In some embodiments, the VHH domain is a human VHH domain or a humanized VHH domain. In some embodiments, at least one IRBR binds to an inhibitory receptor selected from PD-1, CTLA-4, TIGIT, VISTA, or TIM3. In some embodiments, at least one IRBR binds to PD-1.

[0068] In some of any of the provided embodiments of the multispecific polypeptide construct, the first component comprises, in order from N-terminus to C-terminus, a first DLL3 VHH domain that binds DLL3, a first Fc polypeptide of the heterodimeric Fc region, a linker, a VH or VL domain of an anti-CD3 antibody or antigen-binding fragment, and a second DLL3 VHH domain that binds DLL3; and the second component comprises, in order from N-terminus to C-terminus, one of IRBR or CRBR, a second Fc polypeptide of the heterodimeric Fc region, a linker, optionally the same linker as present in the first component, the other of the VH or VL domain of the anti-CD3 antibody or antigen-binding fragment, and the other of CRBR or IRBR.

[0069] In some of the embodiments provided, the linker is a peptide or polypeptide linker. In some embodiments, the linker is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0070] In some embodiments, the linker is TIFF2025060787000007.tif4128, and combinations thereof. In some embodiments, the linker is a non-cleavable linker such as a linker comprising the sequence Is or contains TIFF2025060787000008.tif4128.

[0071] In some embodiments, the linker is a cleavable linker, such as a polypeptide that functions as a substrate for a protease. In some embodiments, the protease is produced by an immune effector cell, by a tumor, or by a cell present in the tumor microenvironment. In some embodiments, the protease is produced by an immune effector cell, and the immune effector cell is an activated T cell, a natural killer (NK) cell, or an NK T cell. In some embodiments, the protease is matriptase, a matrix metalloproteinase (MMP), granzyme B, or a combination thereof. In some embodiments, the cleavable linker has the amino acid sequence Includes TIFF2025060787000009.tif4128.

[0072] Provided herein are isolated single domain antibodies that bind to DLL3 and contain any of the DLL3 VHH domain sequences provided herein, such as any of those described above or elsewhere herein.

[0073] Complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, and 456; Complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 384, 410, and 411; and Provided herein is an isolated single domain antibody that binds to DLL3, comprising a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of NOs:354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, and 367, 395, and 412-415.

[0074] Provided herein are polynucleotides encoding any of the DLL3-binding polypeptide constructs provided herein.

[0075] Also provided herein are polynucleotides encoding any of the multispecific polypeptide constructs provided herein, or a first or second polypeptide thereof. In some embodiments, the polynucleotide comprises a first nucleic acid encoding the first polypeptide and a second nucleic acid encoding the second polypeptide of the provided multispecific constructs, wherein the first and second nucleic acid sequences are separated by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, such as T2A, P2A, E2A, or F2A.

[0076] Polynucleotides encoding any of the provided single domain antibodies are also provided.

[0077] Also provided are vectors, such as expression vectors, encoding any of the provided polynucleotides.

[0078] Provided herein are cells comprising any one or more of the provided polynucleotides or any one or more of the provided vectors.

[0079] Provided herein are methods for producing a polypeptide, comprising introducing any of the provided one or more polynucleotides or vectors into a cell and culturing the cell under conditions to produce a multispecific polypeptide construct. Provided herein are polypeptides produced by any of the methods provided herein.

[0080] Provided herein are engineered immune cells comprising a chimeric antigen receptor comprising an extracellular domain comprising any of the provided DLL3 VHH domain sequence single domain antibodies; a transmembrane domain; and an intracellular signaling domain.

[0081] Provided herein are pharmaceutical compositions comprising any of the provided DLL3-binding polypeptides, multispecific polypeptide constructs, single domain antibodies, or engineered immune cells.

[0082] Provided herein are methods of stimulating or inducing an immune response in a subject, comprising administering to a subject in need thereof any of the provided DLL3-binding polypeptides, multispecific polypeptide constructs, single-domain antibodies, or engineered immune cells, or pharmaceutical compositions.

[0083] Also provided herein are methods of treating a disease or condition in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of any of the DLL3-binding polypeptides described herein, any of the multispecific polypeptide constructs described herein, any of the single domain antibodies described herein, any of the engineered immune cells described herein, or any of the pharmaceutical compositions described herein. [Brief explanation of the drawings]

[0084] [Figure 1] Figure 1 shows a graph illustrating the ability of various anti-DLL3 single domain antibodies (sdAbs) to bind to cell surface DLL3. Binding was assessed by flow cytometry in the DLL3-positive cell line SHP-77, where the DLL3 sdAbs 10D9, 10E5, 8E7, 5A7, 5A8, 5H8, 3G3, 6C5, 6F1, 3B4, 3B12, or 6B4 were operably linked to human Fc. [Figure 2-1]Figures 2A-L show a series of graphs illustrating the ability of DLL3-targeting sdAbs and their humanized variants to bind to cell surface DLL3. Binding was assessed by flow cytometry in the DLL3-positive cell line SHP-77 or HEK-293 freestyle cells transiently transfected with a vector encoding DLL3. Figure 2A shows the binding of 3G3 and its humanized variants in SHP-77. Figure 2B shows the binding of 5A7 and its humanized variants in SHP-77. Figure 2C shows the binding of 3C5 and its humanized variants in SHP-77. Figure 2D shows the binding of 6C5 and its humanized variants in SHP-77. Figures 2E-G show the binding of 5A8 and its humanized variants in SHP-77 (Figures 2E and 2F) and DLL3-transfected 293FS(FL) (Figure 2G). Figure 2H shows the binding of 10D9 and its humanized variants in SHP-77. Figure 2I shows the binding of 10E5 and its humanized variants in SHP-77. Figures 2J and 2K show the binding of 8E7 and its humanized variants in 293FS cells transfected with SHP-77 and DLL3, respectively. Figure 2L shows the binding of 6F1 and its humanized variants in SHP-77. Here, the DLL3 sdAb was functionally linked to human Fc. [Figure 2-2] See the description of Figure 2-1. [Figure 2-3] See the description of Figure 2-1. [Figure 2-4] See the description of Figure 2-1. [Figure 2-5] See the description of Figure 2-1. [Figure 2-6] See the description of Figure 2-1. [Figure 3A]3A-3E illustrate a series of schematic diagrams depicting various DLL3-targeted restricted CD3-engaging constructs. The basic components of the disclosed DLL3-targeted restricted CD3-engaging constructs with restricted CD3 binding are shown. The antigen-binding domain is located at the amino-terminus and / or carboxy-terminus. The Fc region, such as a heterodimeric Fc region, is located N-terminal to the CD3-binding region. This positioning of the Fc in close proximity to the CD3-binding region prevents CD3 binding. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 4] Figures 4A-4B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx4720 to DLL3-positive cells SHP-77 (Figure 4A) and lack of binding to primary human T cells (Figure 4B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 5] Figures 5A-5B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx3715 to DLL3-positive cells SHP-77 (Figure 5A) and lack of binding to primary human T cells (Figure 5B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 6]Figures 6A-6B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx4422 to DLL3-positive cells SHP-77 (Figure 6A) and lack of binding to primary human T cells (Figure 6B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 7] Figures 7A-7B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx3708 to DLL3-positive cells SHP-77 (Figure 7A) and lack of binding to primary human T cells (Figure 7B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 8] Figures 8A-8B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx4052 to DLL3-positive cells SHP-77 (Figure 8A) and lack of binding to primary human T cells (Figure 8B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 9] Figures 9A-9B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx3985 to DLL3-positive cells SHP-77 (Figure 9A) and lack of binding to primary human T cells (Figure 9B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 10]Figures 10A-10B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx4059 to DLL3-positive cells SHP-77 (Figure 10A) and lack of binding to primary human T cells (Figure 10B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 11] Figures 11A-11B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx4087 to DLL3-positive cells SHP-77 (Figure 11A) and lack of binding to primary human T cells (Figure 11B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 12] Figures 12A-12B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx4088 to DLL3-positive cells SHP-77 (Figure 12A) and lack of binding to primary human T cells (Figure 12B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 13] Figures 13A-13B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx4895 to DLL3-positive cells SHP-77 (Figure 13A) and lack of binding to primary human T cells (Figure 13B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 14]Figures 14A-14B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx3991 to DLL3-positive cells SHP-77 (Figure 14A) and lack of binding to primary human T cells (Figure 14B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 15] Figures 15A-15B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx3711 to DLL3-positive cells SHP-77 (Figure 15A) and lack of binding to primary human T cells (Figure 15B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 16] Figures 16A-16B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx4887 to DLL3-positive cells SHP-77 (Figure 16A) and lack of binding to primary human T cells (Figure 16B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 17] Figures 17A-17B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx4896 to DLL3-positive cells SHP-77 (Figure 17A) and lack of binding to primary human T cells (Figure 17B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 18]Figures 18A-18B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx4899 to DLL3-positive cells SHP-77 (Figure 18A) and lack of binding to primary human T cells (Figure 18B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 19] Figures 19A-19B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx4406 to DLL3-positive cells SHP-77 (Figure 19A) and lack of binding to primary human T cells (Figure 19B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 20] Figures 20A-20B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx3707 to DLL3-positive cells SHP-77 (Figure 20A) and lack of binding to primary human T cells (Figure 20B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 21] Figures 21A-21B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx3710 to DLL3-positive cells SHP-77 (Figure 21A) and lack of binding to primary human T cells (Figure 21B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 22]Figures 22A-22B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx4888 to DLL3-positive cells SHP-77 (Figure 22A) and lack of binding to primary human T cells (Figure 22B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 23] Figures 23A-23B demonstrate the binding of the DLL3-targeted restricted CD3-engaging construct cx4890 to DLL3-positive cells SHP-77 (Figure 23A) and lack of binding to primary human T cells (Figure 23B). Binding was assessed by flow cytometry using an anti-human IgG APC secondary antibody. Histograms show normalized cell number versus fluorescence at 200 nM of each construct unless otherwise noted. [Figure 24] Figures 24A-24B depict graphs demonstrating the ability of DLL3-targeted restricted CD3-engaging constructs to induce DLL3-dependent T cell activation. The Jurkat CD3 NFAT-GFP reporter cell line was used to monitor T cell activation. SHP-77 cells (Figure 24A) and HEK-293 freestyle cells (Figure 24B) were used as antigen-positive and -negative cell lines, respectively. [Figure 25-1] Figures 25A-25D depict graphs demonstrating the ability of biepitope DLL3-targeted restricted CD3-engaging constructs to induce DLL3-dependent T cell activation. The Jurkat CD3 NFAT-GFP reporter cell line was used to monitor T cell activation. SHP-77 cells (Figures 22A and 22C) and HEK-293 freestyle cells (Figures 22B and 22D) were used as antigen-positive and -negative cell lines, respectively. [Figure 25-2] See the description of Figure 25-1. [Figure 26]Figures 26A-26B depict graphs demonstrating the ability of DLL3-targeted restricted CD3-engaging constructs to mediate antigen-specific T cell cytotoxicity in DLL3-positive SHP-77 cells (Figure 26A) and DLL3-negative HEK-293 Freestyle cells (Figure 26B). [Figure 27-1] Figures 27A-27B depict graphs demonstrating the ability of DLL3-targeted restricted CD3-engaging constructs to mediate antigen-specific T cell activation in DLL3-positive SHP-77 cells (Figures 27A and 27C) and DLL3-negative HEK-293 Freestyle cells (Figures 27B and 27D). [Figure 27-2] See the description of Figure 27-1. [Figure 28] Figure 28 demonstrates the ability of DLL3-targeted restricted CD3-engaging constructs to induce cytokine production from T cells in an antigen-dependent manner. Cytokine production was monitored using ELISA. [Figure 29A] Figure 29A is a schematic diagram of three constrained CD3 constructs targeting DLL3, composed of two polypeptides, chain 1 and chain 2. Chain 1 contains the heterodimeric Fc "hole" linked via a non-cleavable linker to a G100C-modified anti-CD3 VL domain linked to a costimulatory receptor-targeting sdAb. Chain 2 contains either a DLL3-targeting sdAb linked to a complementary heterodimeric Fc "knob" linked via the linker to an anti-CD3 VH domain modified at G44C, which is linked to a second DLL3-targeting sdAb (top); a heterodimeric Fc "knob" linked via the linker to an anti-CD3 VH domain modified at G44C, which is linked to a DLL3-targeting sdAb (middle); or a DLL3-targeting sdAb linked to a complementary heterodimeric Fc "knob" linked via the linker to an anti-CD3 VH domain modified by G44C (bottom). The resulting constructs engage DLL3 either bivalently (top) or monovalently (middle and bottom). All constructs herein contain costimulatory receptor-targeting sdAbs. [Figure 29B] Figure 29B is a schematic diagram of the DLL3-targeting constrained CD3 construct cx5499, composed of two polypeptides, chain 1 and chain 2. cx5499 is identical to cx5352 shown in Figure 29A (top), except that it lacks the costimulatory receptor-targeting sdAb at the C-terminus of chain 1. When coexpressed, the CD3-binding domains assemble properly through VL:VH association at the hole and knob, respectively. The VH:VL interaction is stabilized by an engineered disulfide bond between modified residues G44C in the VH domain and G100C in the VL domain. [Figure 30] Figures 30A-30B demonstrate that representative monovalent (cx5800 and cx5801) and bivalent (cx5352) restricted CD3 engaging constructs targeting DLL3 bind to the DLL3-expressing cell line SHP-77 (Figure 30A) but not to isolated T cells (Figure 30B). Binding was assessed by flow cytometry. Figure 30C illustrates the ability of representative restricted CD3 engaging constructs targeting DLL3 to agonize CD3 signaling in the presence of DLL3-positive SHP-77 cells. Engagement of DLL3-positive cells with a bivalent, biepitopic construct for DLL3 (cx5352) induced more potent T cell activation than constructs monovalent for DLL3 (cx5800 and cx5801). A Jurkat CD3 NFAT luciferase reporter cell line was used to assess CD3 agonism. [Figure 31-1]Figures 31A-31E demonstrate the ability of a representative restricted CD3 engaging construct targeting DLL3, cx5499, to induce T cell-mediated cytotoxicity and T cell activation in the presence of DLL3-positive SHP-77 cells. Figure 31A demonstrates the ability of a representative restricted CD3 engaging construct targeting DLL3 to induce T cell-mediated cytotoxicity in the presence of DLL3-positive SHP-77 cells. Figures 31B-31E demonstrate the ability of a representative restricted CD3 engaging construct targeting DLL3 to induce T cell activation in the presence of DLL3-positive SHP-77 cells, as assessed by CD25 expression on CD4+ T cells (Figure 31B), CD69 expression on CD4+ T cells (Figure 31C), CD25 expression on CD8+ T cells (Figure 31D), and CD69 expression on CD8+ T cells (Figure 31E). [Figure 31-2] See the description of Figure 31-1. [Figure 32] Figure 32 shows that the DLL3-targeted restricted CD3-engaging construct cx5352, which contains the 41BB-binding domain, can mediate 41BB signaling, whereas the identical construct cx5499, which lacks the 41BB-binding domain, cannot. DLL3-dependent 41BB signaling was monitored using Jurkat 41BB NFkB luciferase reporter cells cocultured with either parental HEK-293 cells or HEK-293 cells transiently expressing a truncated version of DLL3. Activity is shown as relative luciferase units (RLU). [Figure 33] Figures 33A-B illustrate the efficacy of T cell-mediated cytotoxicity driven by an exemplary DLL3-targeted restricted CD3-engaging construct, cx5352, containing the 41BB-binding domain against the DLL3-positive cell line, SHP-77 (Figure 33A) or the DLL3-negative cell line, HEK-293FS (Figure 33B). Three different T cell donors were used as sources of effector cells in this assay. [Figure 34]Figures 34A-C show the differential efficacy of T cell-mediated cytotoxicity driven by exemplary DLL3-targeted restricted CD3-engaging constructs, cx5352 containing the 41BB-binding domain and cx5499, a construct lacking the 41BB-binding domain. Various effector (T cells) to target cell (SHP-77) ratios (effector:target cell ratios) of 10:1 (Figure 34A), 5:1 (Figure 34B), or 1.25:1 (Figure 34C) were compared. Human PBMCs were used as the source of T cells. [Figure 35] Figure 35 shows a comparison of IFNγ production by T cells treated with titrations of representative DLL3-targeted restricted CD3-engaging constructs, cx5352 containing the 41BB binding domain and cx5499 not containing the 41BB binding domain, in the presence of the DLL3-positive cell line SHP-77. [Figure 36] Figures 36A-B illustrate an exemplary TAA-targeted restricted CD3 engager comprising a costimulatory receptor binding region (CRBR). The construct has an antigen-targeting sdAb positioned N- and C-terminal to the Fc knob on one chain of the heterodimer, and a costimulatory receptor binding region (CRBR) positioned C-terminal to the Fc hole on the other chain of the heterodimer, but with the VH and VL of the CD3-binding Fv positioned opposite each other. [Figure 37-1] Figures 37A-D illustrate the results of T cell reporter assays for the exemplary constructs described in Figures 36A-B. Figures 37A and 37B illustrate the mean fluorescence intensity (MFI) of the GFP reporter when the TAA-positive cell line A375 or the TAA-negative cell line CCRF-CEM, respectively, were co-cultured with Jurkat CD3 NFAT-GFP reporter cells. Figures 37C and 37D illustrate the relative light units (RLU) of the luciferase reporter when the TAA-positive cell line A375 or the TAA-negative cell line CCRF-CEM, respectively, were co-cultured with Jurkat CD3 NFAT luciferase reporter cells. [Figure 37-2] See the description of Figure 37-1. [Figure 38]Figures 38A-B illustrate the results of a T cell reporter assay using a Jurkat reporter cell line engineered to stably express CD16a along with an NFAT-driven luciferase reporter gene. Figure 38A illustrates the relative light units (RLU) of the luciferase reporter when DLL3-expressing CHO cells were co-cultured with the reporter cells and treated with a titration of a DLL3-targeted construct containing IgG1 Fc. Figure 38B illustrates the RLU of the luciferase reporter when treated with a titration of a DLL3-targeted construct containing IgG1 Fc in the absence of DLL3-expressing cells. DETAILED DESCRIPTION OF THE INVENTION

[0085] Detailed Description Polypeptides that specifically bind to DLL3, hereinafter also referred to as DLL3-binding polypeptides, are provided herein. In some embodiments, the provided binding polypeptides comprise at least one VHH domain that binds to DLL3. In some embodiments, the DLL3-binding polypeptides provided herein comprise 1, 2, 3, 4, 5, 6, 7, or 8 VHH domains, each of which individually binds to DLL3. In some embodiments, the DLL3-binding polypeptides provided herein comprise 1, 2, 3, or 4 VHH domains that bind to DLL3. In some embodiments, the DLL3-binding polypeptides are monospecific. In some embodiments, the DLL3-binding polypeptides are multispecific. For example, the provided DLL3-binding polypeptides include polypeptides that may comprise at least one VHH domain that binds to DLL3 and one or more additional binding domains, such as one or more additional VHH domains that bind to one or more target proteins other than DLL3.

[0086] In some embodiments, the DLL3-binding polypeptide comprises at least one VHH domain that binds to DLL3 and an Fc domain. In some embodiments, the DLL3-binding polypeptide provided herein comprises one, two, three, or four VHH domains that bind to DLL3 and an Fc domain. In some embodiments, the Fc domain mediates dimerization of the DLL3-binding polypeptide under physiological conditions, thus forming a dimer and doubling the number of DLL3-binding sites. For example, a DLL3-binding polypeptide comprising three VHH domains that bind to DLL3 and an Fc region is trivalent as a monomer, but under physiological conditions, the Fc region mediates dimerization, and therefore, under such conditions, the DLL3-binding polypeptide exists as a hexavalent dimer.

[0087] DLL3 is a member of the delta protein ligand family. The delta protein ligand family functions as a Notch ligand, characterized by a DSL domain, EGF repeats, and a transmembrane domain. DLL3 is highly expressed in fetal brain, but not in normal adult tissues. In contrast, DLL3 is expressed on the surface of various tumor cells and tumor vasculature, including, but not limited to, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), and ovarian cancer. In contrast to Delta protein family members that activate Notch during normal development, DLL3 inhibits both cis- and trans-acting Notch pathway activation by interacting with Notch and DLL1, redirecting or retaining them in endosomal / lysosomal compartments or the Golgi, respectively, thereby preventing their translocation to the cell surface (Chapman et al., 2011, Hum Mol Genet. 20(5):905-16; Serth et al., 2015, PLoS One. 10(4):e0123776). Notably, in neuroendocrine tumors, Notch activation suppresses tumor growth (Kunnimalaiyaan and Chen, 2007, Oncologist. 12(5):535-42). These observations suggest that DLL3 may be associated with a neuroendocrine phenotype by downregulating Notch signaling and thus contribute to neuroendocrine tumorigenesis. DLL3 expression is highly restricted in normal adult tissues but widespread in malignant SCLC, LCNEC, melanoma, glioblastoma, and extrapulmonary neuroendocrine carcinoma (NEC) (Saunders et al., 2015, Sci Transl Med. 2015, 7(302):302ra136; Peng et al., 2016, J. Clin. Oncol., 34, no. 15_suppl 11611-11611).Furthermore, high levels of DLL3 expression in tumor tissue correlated with advanced tumor stage and / or poor survival in glioblastoma, medullary thyroid carcinoma, and neuroendocrine pancreatic cancer (Peng et al., 2016, J. Clin. Oncol., 34, no. 15_suppl 11611-11611), SCLC and LCNEC (Saunders et al., 2015, Sci Transl Med. 2015, 7(302):302ra136), and some ovarian cancers (Hu et al., 2014, Cancer Res., 74(12):3282-3293).

[0088] An exemplary sequence of authentic human DLL3 is shown below: TIFF2025060787000010.tif41155

[0089] An exemplary sequence of a non-canonical human DLL3 is shown below: TIFF2025060787000011.tif41155

[0090] In some cases, the provided DLL3-binding polypeptides directly block or inhibit the activity of DLL3, which in some aspects can be used as therapeutic agents to inhibit or reduce the growth or survival of tumor cells.

[0091] Various DLL3 polypeptide binding formats are provided. In some examples, the DLL3 binding polypeptide comprises a DLL3 VHH-Fc polypeptide. In some embodiments, the Fc exhibits one or more immune effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC).

[0092] In some embodiments, the provided DLL3-binding polypeptides can be used to stimulate an immune response in a subject, which in some aspects treats a disease or disorder, such as cancer, in the subject. In some aspects, the DLL3-binding polypeptides provided herein, such as DLL3-Fc, can bind to DLL3-expressing tumor cells and induce an active immune response against the DLL3-expressing tumor cells. In some cases, the active immune response can cause the death of cancerous cells (e.g., antibody binding to cancer cells induces apoptotic cell death) or suppress the growth of cancerous cells (e.g., block cell cycle progression). In other cases, the DLL3-binding polypeptides provided herein, such as DLL3 VHH-Fc, can bind to cancerous cells and antibody-dependent cellular cytotoxicity (ADCC) can eliminate cancerous cells to which the DLL3-binding polypeptide binds. In some cases, the provided DLL3 VHH-binding polypeptides can also activate both cellular and humoral immune responses, recruiting more natural killer cells or increased production of cytokines (e.g., IL-2, IFN-γ, IL-12, TNF-α, TNF-β, etc.) that further activate an individual's immune system to destroy cancerous cells. In yet another embodiment, a DLL3-binding polypeptide, such as a DLL3 VHH-Fc, can bind to cancerous cells, allowing macrophages or other phagocytic cells to opsonize the cancerous cells, for example, via the CDC or ADCP process.

[0093] In other aspects, VHH-binding polypeptides that exhibit multispecific binding are also provided herein. In some cases, the binding polypeptides include polypeptides that exhibit dual affinity for DLL3 and T cell antigens such as CD3. In some aspects, such dual affinity molecules can engage or activate T cells at tumor sites upon binding to DLL3 expressed on tumors. In particular, among such molecules provided herein are molecules that exhibit restricted CD3 binding. Also provided herein are engineered cells, such as engineered T cells, that express chimeric antigen receptors containing DLL3-binding polypeptides.

[0094] All publications, including patent documents, scientific articles, and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. To the extent that the definitions set forth herein conflict or are otherwise inconsistent with the definitions set forth in the patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over the definitions incorporated herein by reference.

[0095] The techniques and procedures described or referenced herein are generally well understood and can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M. Ausubel, et al. eds., (2003)); METHODS IN ENZYMOLOGY (Academic Press, Inc.) series: PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984);Methods in Molecular Biology, Humana Press;Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press;Animal Cell Culture (RI Freshney), ed., 1987);Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press;Cell and Tissue Culture Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DMWeir and CC Blackwell, eds.);Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987);PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (JE Coligan et al., eds., 1991);Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (CA Janeway and P. Travers, 1997);Antibodies (P. Finch, 1997);Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989);Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993); and the latest editions thereof, using conventional methodologies commonly used by those skilled in the art.

[0096] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0097] I. Definition Unless otherwise defined, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context or expressly indicated, singular terms shall include pluralities and plural terms shall include the singular. In the event of any discrepancy in definitions among various sources or references, the definitions provided herein shall control.

[0098] It is understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments. As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise indicated. The use of the term "or" herein is not intended to imply that alternatives are mutually exclusive.

[0099] In this application, unless expressly stated or understood by a person skilled in the art, the use of "or" means "and / or." In the context of multiple dependent claims, the use of "or" refers back to more than one such independent or dependent claim.

[0100] The term "about" as used herein refers to a normal error range for each value, which is readily apparent to those skilled in the art. Reference to a value or parameter with "about" herein includes (and describes) aspects directed to the value or parameter itself. For example, a description that refers to "about X" includes the description of "X".

[0101] The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" may be used interchangeably and refer to a polymer of nucleotides. Such a polymer of nucleotides may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0102] As used herein, the term "isolated polynucleotide" is intended to mean a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, that, by reason of its origin, (1) is not associated with all or a portion of polynucleotides found in nature, (2) is operably linked to polynucleotides to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

[0103] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of this disclosure, "polypeptide" refers to proteins containing modifications, such as deletions, additions, and substitutions (generally conservative in nature), relative to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, such as through site-directed mutagenesis, or accidental, such as through mutations of the host producing the protein or errors due to PCR amplification.

[0104] The term "isolated protein" as referred to herein means that the subject protein (1) is free from at least some other proteins with which it is typically found in nature, (2) is essentially free from other proteins from the same source, e.g., from the same species, (3) is expressed by cells from a different species, (4) is separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated, (5) the "isolated protein" is not associated (by covalent or non-covalent interactions) with portions of proteins with which it is naturally associated, (6) is functionally associated (by covalent or non-covalent interactions) with polypeptides with which it is not naturally associated, or (7) is not naturally occurring. Such isolated proteins can be encoded by genomic DNA, cDNA, mRNA, or other RNA, can be of synthetic origin, or any combination thereof. In certain embodiments, an isolated protein is substantially pure or substantially free from proteins, polypeptides, or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or other).

[0105] As used herein, "substantially pure" means that the species of interest is the predominant species present (i.e., on a molar basis, it is more abundant than any other individual species in the composition), and a substantially purified fraction is a composition in which the species of interest constitutes at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will constitute greater than about 80% of all macromolecular species present in the composition, e.g., in some embodiments, greater than about 85%, 90%, 95%, and 99%. In some embodiments, the species of interest is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods), such that the composition consists essentially of a single macromolecular species.

[0106] As used herein, the term "operably linked" refers to the positioning of the components so described, in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0107] The term "specifically binds" to an antigen or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with higher affinity than with alternative cells or substances. A single domain antibody (sdAb) or VHH-containing polypeptide "specifically binds" or "preferentially binds" to a target if it binds with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. For example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to a DLL3 epitope is an sdAb or VHH-containing polypeptide that binds to this epitope with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other DLL3 epitopes or non-DLL3 epitopes. For example, it is understood by reading this definition that an sdAb or VHH-containing polypeptide that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding refers to preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0108] As used herein, the term "epitope" refers to a site on a target molecule (e.g., an antigen, such as a protein, nucleic acid, carbohydrate, or lipid) to which an antigen-binding molecule (e.g., an sdAb or VHH-containing polypeptide) binds. Epitopes often comprise chemically active surface arrangements of molecules, such as amino acids, polypeptides, or sugar side chains, and possess specific three-dimensional structural and charge characteristics. Epitopes can be formed from both contiguous and / or juxtaposed noncontiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of a target molecule. Epitopes formed from contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes can include, but are not limited to, at least 3, at least 5, or 8-10 residues (e.g., amino acids or nucleotides). In some embodiments, an epitope is less than 20 residues (e.g., amino acids or nucleotides) in length, less than 15 residues, or less than 12 residues. Two antibodies may bind to the same epitope within an antigen if they exhibit competitive binding to the antigen. In some embodiments, an epitope can be identified by a certain minimum distance to the CDR residues on the antigen-binding molecule. In some embodiments, an epitope can be identified by the above distance and further limited to those residues involved in binding (e.g., hydrogen bonding) between the antigen-binding molecule residues and the antigen residues. Epitopes can also be identified by various scans; for example, an alanine scan or an arginine scan can reveal one or more residues with which the antigen-binding molecule can interact. Unless explicitly stated, a set of residues as an epitope does not exclude other residues from being part of the epitope for a particular antigen-binding molecule. Rather, the existence of such a set indicates a minimum series (or species set) of epitopes. Thus, in some embodiments, the set of residues identified as an epitope represents the minimal epitope associated with the antigen, rather than an exclusive list of residues for the epitope on the antigen.

[0109] A "nonlinear epitope" or "conformational epitope" comprises non-contiguous polypeptides, amino acids, and / or sugars within an antigen protein to which an epitope-specific antigen-binding molecule binds. In some embodiments, at least one of the residues will be non-contiguous with other referenced residues of the epitope; however, one or more of the residues may also be contiguous with other residues.

[0110] A "linear" epitope comprises adjacent polypeptides, amino acids, and / or sugars in an antigen protein to which an epitope-specific antigen-binding molecule binds. It is noted that in some embodiments, not all of the residues in a linear epitope need to directly bind (or participate in binding) to an antigen-binding molecule. In some embodiments, a linear epitope can be derived from immunization with a peptide that effectively consists of the sequence of the linear epitope, or from a structural segment of the protein that has exactly that sequence segment and is relatively isolated from the rest of the protein (so that the antigen-binding molecule can at least primarily interact).

[0111] The terms "antibody" and "antigen-binding molecule" are used interchangeably in the broadest sense and encompass a variety of polypeptides containing antibody-like antigen-binding domains, including, but not limited to, conventional antibodies (typically comprising at least one heavy chain and at least one light chain), single-domain antibodies (sdAbs, typically comprising only one chain similar to a heavy chain), VHH-containing polypeptides (polypeptides comprising at least one heavy-chain-only antibody variable domain, i.e., VHH), and fragments of any of the foregoing, so long as they exhibit the desired antigen-binding activity. In some embodiments, an antibody comprises a dimerization domain. Such dimerization domains include, but are not limited to, a heavy-chain constant domain (comprising a CH1, hinge, CH2, and CH3; CH1 typically pairs with a light-chain constant domain, CL, while the hinge mediates dimerization), and an Fc domain (comprising a hinge, CH2, and CH3; the hinge mediates dimerization).

[0112] The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as camelids (including llamas), sharks, mice, humans, cynomolgus monkeys, etc.

[0113] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains of a native antibody (V H and V L ) generally have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single V H or V L A single domain may be sufficient to confer antigen-binding specificity to a single domain antibody, such as a VHH. Furthermore, antibodies that bind to a particular antigen can be identified by combining VHHs derived from antibodies that bind to the antigen. H or V Ldomains to form complementary V L or V H Libraries of domains may be screened and isolated (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0114] "Antibody fragment" or "antigen-binding fragment" refers to a molecule other than a conventional or intact antibody that contains a portion of a conventional or intact antibody that contains at least the variable region that binds to an antigen. Examples of antibody fragments include Fv, single-chain Fv (sdFv), Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; V H This includes, but is not limited to, single domain antibodies (VHH) which contain only the region.

[0115] As used herein, "monovalent" with respect to a binding molecule refers to a binding molecule that has a single antigen recognition site that is specific to a target antigen. Examples of monovalent binding molecules include, for example, monovalent antibody fragments, proteinaceous binding molecules with antibody-like binding properties, or MHC molecules. Examples of monovalent antibody fragments include, but are not limited to, Fab fragments, Fv fragments, and single-chain Fv fragments (scFv).

[0116] The terms "single domain antibody," "sdAb," and "VHH" are used interchangeably herein to refer to an antibody having an antigen-binding / recognition domain that is a single monomer domain. Such antibodies include camelid antibodies or shark antibodies. In some embodiments, a VHH comprises three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, a VHH may be truncated at the N- or C-terminus to comprise only partial FR1 and / or FR4, or to lack one or both of these framework regions, so long as the VHH substantially maintains antigen binding and specificity.

[0117] The term "VHH-containing polypeptide" refers to a polypeptide comprising at least one VHH domain. In some embodiments, a VHH polypeptide comprises two, three, four, or more VHH domains, and each VHH domain may be the same or different. In some embodiments, a VHH-containing polypeptide comprises an Fc domain. In some such embodiments, a VHH polypeptide may form a dimer. Non-limiting structures of a VHH-containing polypeptide include VHH1-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc, where VHH1, VHH2, and VHH3 may be the same or different. In some embodiments of such structures, one VHH may be connected to another VHH by a linker, or one VHH may be connected to an Fc by a linker. In some such embodiments, the linker comprises 1-20 amino acids, preferably 1-20 amino acids composed primarily of glycine, and optionally serine. In some embodiments, when a VHH-containing polypeptide comprises an Fc, it forms a dimer. Thus, the structure VHH1-VHH2-Fc is considered tetravalent when it forms a dimer (i.e., the dimer has four VHH domains). Similarly, the structure VHH1-VHH2-VHH3-Fc is considered hexavalent when it forms a dimer (i.e., the dimer has six VHH domains).

[0118] As used herein, a DLL3-binding polypeptide is a polypeptide or protein that specifically binds to DLL3. Typically, the DLL3-binding polypeptide herein is a VHH-containing polypeptide that contains at least one VHH domain that binds to DLL3. The DLL3-binding polypeptide includes a conjugate, including a fusion protein. The DLL3-binding polypeptide includes a fusion protein, including a fusion protein that contains an Fc domain. In some embodiments, the DLL3-binding polypeptide contains two, three, four, or more VHH domains, each of which specifically binds to DLL3, and each VHH domain may be the same or different. In some embodiments, the DLL3-binding polypeptide is multivalent. In some embodiments, the DLL3-binding polypeptide is multispecific. In some cases, the DLL3-binding polypeptide may contain one or more additional domains that bind to one or more additional or additional antigens other than DLL3.

[0119] The term "monoclonal antibody" refers to an antibody (including an sdAb or VHH-containing polypeptide) from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Thus, a sample of monoclonal antibodies may bind to the same epitope on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods, e.g., as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example.

[0120] The term "CDR" refers to complementarity determining region as defined in at least one particular manner by those skilled in the art. The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described in: Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 Jan;27(1):55-77 ("IMGT" numbering scheme); Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8;309(3):657-70 ("Aho" numbering scheme); and Martin et al., "Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23):9268-9272 ("AbM" numbering scheme).

[0121] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Both the Kabat and Chothia numbering schemes are based on the length of the most common antibody region sequences, and insertions and deletions, coordinated by an insertion character such as "30a," occur in some antibodies. The two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme. The AbM scheme is a compromise between the Kabat and Chothia definitions, based on that used by Oxford Molecular's AbM antibody modeling software.

[0122] In some embodiments, CDRs can be defined according to any of the Chothia numbering scheme, Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, and / or the Contact definition. VHHs contain three CDRs designated CDR1, CDR2, and CDR3. Table 1 below lists exemplary boundary positions for CDR-H1, CDR-H2, and CDR-H3 as identified by the Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat numbering scheme and the Chothia numbering scheme. FRs are located between the CDRs; for example, FR-H1 precedes CDR-H1, FR-H2 is located between CDR-H1 and CDR-H2, and FR-H3 is located between CDR-H2 and CDR-H3. It is noted that the Kabat numbering scheme shown places the insertion at H35A and H35B, so the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention shown varies between H32 and H34 depending on the length of the loop.

[0123] Table 1. CDR boundaries according to various numbering schemes TIFF2025060787000012.tif311481 - Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 2 - Al-Lazikani et al., (1997) JMB 273,927-948

[0124] Thus, unless otherwise specified, the "CDR" or "complementarity determining region" of a given antibody or region thereof, e.g., its variable region, or individual designated CDRs (e.g., CDR-H1, CDR-H2, CDR-H3) should be understood to encompass a (or specific) complementarity determining region as defined by any of the aforementioned schemes. For example, when a particular CDR (e.g., CDR-H3) is stated to contain the amino acid sequence of a corresponding CDR in a given VHH amino acid sequence, it is understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) within the VHH as defined by any of the aforementioned schemes. In some embodiments, specific CDR sequences are specified. Exemplary CDR sequences of the provided antibodies are described using various numbering schemes (see, e.g., Table 1), however, it is understood that the provided antibodies may include CDRs as described according to any of the other aforementioned numbering schemes or other numbering schemes known to those of skill in the art.

[0125] As used herein, "conjugate," "conjugation," or grammatical variations thereof refer to the joining or linking of two or more compounds to each other, resulting in the formation of another compound, by any joining or linking method known in the art.It can also refer to a compound produced by joining or linking two or more compounds to each other.For example, a VHH domain directly or indirectly linked to one or more chemical moieties or polypeptides is an exemplary conjugate.Such conjugates include fusion proteins, those produced by chemical conjugation, and those produced by any other method.

[0126] Immunoglobulin Fc fusions (Fc fusions), such as VHH-Fc, are molecules comprising one or more VHH domains operably linked to the Fc region of an immunoglobulin. The immunoglobulin Fc region may be indirectly or directly linked to one or more VHH domains. Various linkers are known in the art and can optionally be used to link the Fc to a fusion partner to create an Fc fusion. In some such embodiments, the linker comprises 1-20 amino acids, preferably 1-20 amino acids consisting primarily of glycine and optionally serine. Fc fusions of the same species can be dimerized to form Fc fusion homodimers, or with non-identical species to form Fc fusion heterodimers. In some embodiments, the Fc is a mammalian Fc, such as a human Fc.

[0127] As used herein, the term "heavy chain constant region" refers to a region of at least three heavy chain constant domains, C H 1. Hinge, C H 2, and C H 3. Of course, deletions and modifications that do not alter function within the domain are encompassed within the scope of the term "heavy chain constant region" unless otherwise specified. Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting exemplary heavy chain constant regions also include epsilon and mu. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody containing a gamma constant region is an IgG antibody, an antibody containing a delta constant region is an IgD antibody, and an antibody containing an alpha constant region is an IgA antibody. Furthermore, an antibody containing a mu constant region is an IgM antibody, and an antibody containing an epsilon constant region is an IgE antibody. A particular isotype can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing a gamma 1 constant region), IgG2 (containing a gamma 2 constant region), IgG3 (containing a gamma 3 constant region), and IgG4 (containing a gamma 4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (containing an alpha 1 constant region) and IgA2 (containing an alpha 2 constant region) antibodies; IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0128] As used herein, "Fc region" refers to a portion of a heavy chain constant region comprising CH2 and CH3. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. In various embodiments, when the Fc region comprises a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region may be of any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0129] A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include Fc receptor binding; C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of assays.

[0130] A "native-sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native-sequence human Fc regions include native-sequence human IgG1 Fc regions (non-A and A allotypes); native-sequence human IgG2 Fc regions; native-sequence human IgG3 Fc regions; and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0131] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification. In some embodiments, a "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification, yet retains at least one effector function of the native-sequence Fc region. In some embodiments, the variant Fc region has at least one amino acid substitution, e.g., about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions, in the native-sequence Fc region or in the Fc region of the parent polypeptide, compared to the native-sequence Fc region or the Fc region of the parent polypeptide. In some embodiments, the variant Fc region herein will retain at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the native-sequence Fc region and / or the Fc region of the parent polypeptide.

[0132] Generally, the numbering of residues in an immunoglobulin heavy chain or portion thereof, such as the Fc region, is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0133] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcγR is a native human FcR. In some embodiments, the FcR is one that binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. For example, the term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the control of maternal IgG transfer to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and immunoglobulin homeostasis.Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.)).

[0134] As used herein, an "acceptor human framework" refers to a heavy chain variable domain (V) derived from a human immunoglobulin framework or a human consensus framework, as discussed herein. H ) framework. The acceptor human framework derived from a human immunoglobulin framework or a human consensus framework can contain the same amino acid sequence or can contain amino acid sequence changes. In some embodiments, the number of amino acid changes is less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3 across all of the human frameworks in a single antigen-binding domain such as a VHH.

[0135] As used herein, "chimeric antigen receptor" or "CAR" refers to an engineered receptor that introduces antigen specificity via an antigen-binding domain onto a cell into which it is engineered (e.g., a T cell, such as a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof), thus combining the antigen-binding properties of the antigen-binding domain with the T cell activity of the T cell (e.g., lytic ability and self-renewal). CARs typically comprise an extracellular antigen-binding domain (ectodomain), a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain generally contains at least one ITAM signaling domain, e.g., derived from CD3ζ, and optionally at least one costimulatory signaling domain, e.g., derived from CD28 or 4-1BB. In the CARs provided herein, the VHH domain forms the antigen-binding domain and is located on the extracellular side when expressed in a cell.

[0136] "Affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or VHH-containing polypeptide) and its binding partner (e.g., an antigen). The affinity or apparent affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) or K D-見かけ Affinity can be measured by common methods known in the art, including those described herein (e.g., ELISA K D , KinExA, flow cytometry, and / or surface plasmon resonance instrumentation, etc. Such methods include, but are not limited to, BIAcore®, Octet®, or methods involving flow cytometry.

[0137] As used herein, "K D The term "K" refers to the equilibrium dissociation constant of an antigen-binding molecule / antigen interaction. D As used herein, the term " D and K.D-見かけ Includes:

[0138] In some embodiments, the K D is measured by flow cytometry using an antigen-expressing cell line and fitting the mean fluorescence measured at each antibody concentration to a nonlinear one-site binding equation (Prism Software graphpad). D is K D-見かけ is.

[0139] The term "biological activity" refers to any one or more biological properties of a molecule (whether naturally occurring as found in vivo or provided or enabled by recombinant means). Biological properties include, but are not limited to, binding of a ligand, induction or increase in cell proliferation (e.g., T cell proliferation), and induction or increase in cytokine expression.

[0140] An "affinity matured" VHH-containing polypeptide refers to a VHH-containing polypeptide that has one or more alterations in one or more CDRs compared to a parent VHH-containing polypeptide that does not possess such alterations, which alterations result in improved affinity of the VHH-containing polypeptide for its antigen.

[0141] As used herein, "humanized VHH" refers to a VHH in which one or more framework regions have been substantially replaced with human framework regions. In some instances, certain framework region (FR) residues of a human immunoglobulin are replaced with corresponding non-human residues. Furthermore, a humanized VHH may contain residues that are not found in either the original VHH or human framework sequence, but are included to further improve and optimize the performance of the VHH or VHH-containing polypeptide. In some embodiments, a humanized VHH-containing polypeptide comprises a human Fc region. As will be appreciated, a humanized sequence can be identified by its primary sequence and does not necessarily represent the process by which the antibody was created.

[0142] As used herein, the term "substantially similar" or "substantially the same" refers to a sufficiently high degree of similarity between two or more numerical values ​​that those skilled in the art would consider the difference between the two or more values ​​to be small or to be without any biological and / or statistical significance within the context of the biological characteristic measured by the values. In some embodiments, two or more substantially similar values ​​differ by only about 5%, 10%, 15%, 20%, 25%, or 50%.

[0143] A polypeptide "variant" refers to a biologically active polypeptide having at least about 80% amino acid sequence identity to a native sequence polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, not counting any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues have been added or deleted at the N- or C-terminus of the polypeptide. In some embodiments, a variant has at least about 80% amino acid sequence identity. In some embodiments, a variant has at least about 90% amino acid sequence identity. In some embodiments, a variant has at least about 95% amino acid sequence identity to a native sequence polypeptide.

[0144] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared.

[0145] Amino acid substitutions can include, but are not limited to, replacing one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 2. Amino acid substitutions can be introduced into an antibody of interest, and the products can be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0146] (Table 2) TIFF2025060787000013.tif90128

[0147] Amino acids may be grouped according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0148] Non-conservative substitutions entail exchanging a member of one of these classes for another class.

[0149] The term "vector" is used to describe a polynucleotide that can be manipulated to contain one or more cloned polynucleotides that can be propagated in a host cell. A vector can include one or more of the following elements: an origin of replication, one or more control sequences (such as a promoter and / or enhancer) that control the expression of a polypeptide of interest, and / or one or more selectable marker genes (such as antibiotic resistance genes and genes that can be used in colorimetric assays, such as β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0150] "Host cell" refers to a cell that can be or has been the recipient of a vector or isolated polynucleotide. Host cells may be prokaryotic or eukaryotic. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells, as well as their derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. Host cells include the progeny of a single host cell; progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected in vivo with the polynucleotides provided herein.

[0151] As used herein, the term "isolated" refers to a molecule that is separated from at least some of the components with which it is typically found or produced in nature. For example, a polypeptide is said to be "isolated" when it is separated from at least some of the components of the cell in which it is produced. If a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell in which it was produced is considered to "isolate" the polypeptide. Similarly, a polynucleotide is said to be "isolated" when it is not part of a larger polynucleotide with which it is typically found in nature (e.g., in the case of a DNA polynucleotide, genomic DNA or mitochondrial DNA, etc.) or, for example, in the case of an RNA polynucleotide, when it is separated from at least some of the components of the cell in which it was produced. Thus, a DNA polynucleotide contained in a vector within a host cell can be said to be "isolated."

[0152] The terms "individual" and "subject" are used interchangeably herein to refer to animals, e.g., mammals. The term patient includes humans and veterinary subjects. In some embodiments, methods are provided for treating mammals, including but not limited to humans, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian livestock, mammalian sport animals, and mammalian pets. The subject can be male or female and can be of any suitable age, including infants, juveniles, adolescents, adults, and geriatric subjects. In some examples, "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject receiving treatment can be a patient, which refers to the fact that the subject has been identified as having or being at sufficient risk of suffering from a disorder related to the treatment. In certain embodiments, the subject is a human, such as a human patient.

[0153] As used herein, a "disease" or "disorder" refers to a condition for which treatment is needed and / or desired.

[0154] The terms "tumor cell," "cancer cell," "cancer," "tumor," and / or "neoplasm," unless otherwise specified, are used interchangeably herein to refer to a cell (or cells) that exhibit uncontrolled growth and / or abnormally increased cell survival and / or inhibited apoptosis that interferes with the normal functioning of the body's organs and systems. Benign and malignant cancers, polyps, hyperplasias, and dormant tumors or micrometastases are included in this definition.

[0155] The terms "cancer" and "tumor" encompass solid cancers and hematologic / lymphatic cancers, and also encompass malignant, premalignant, and benign growths, such as dysplasia. Also included in this definition are cells (e.g., virally infected cells) with abnormal growth that is not prevented by the immune system (e.g., immune evasion and immune escape mechanisms). Exemplary cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatocellular carcinoma; intraepithelial neoplasia; kidney or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and pulmonary carcinoma). squamous cell carcinoma); melanoma; myeloma; neuroblastoma; oral cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma, and B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL)); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with nevus syndrome, edema (e.g., associated with brain tumors), and Meigs syndrome.

[0156] The term "non-tumor cell" as used herein refers to normal cells or tissues. Exemplary non-tumor cells include, but are not limited to: T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, monocytes, macrophages, epithelial cells, fibroblasts, hepatocytes, renal stromal cells, fibroblast-like synoviocytes, osteoblasts, and cells located in breast, skeletal muscle, pancreas, stomach, ovary, small intestine, placenta, uterus, testis, kidney, lung, heart, brain, liver, prostate, colon, lymphoid organs, bone, and bone-derived mesenchymal stem cells. The term "peripherally located cells or tissues" as used herein refers to non-tumor cells that are not located near tumor cells and / or within the tumor microenvironment.

[0157] As used herein, the term "cells or tissues in the tumor microenvironment" refers to the cells, molecules, extracellular matrix, and / or blood vessels that surround and / or feed tumor cells. Exemplary cells or tissues in the tumor microenvironment include, but are not limited to, tumor vasculature; tumor-infiltrating lymphocytes; fibroblastic reticular cells; endothelial progenitor cells (EPCs); cancer-associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen-presenting cells; T cells; regulatory T cells (Treg cells); macrophages; neutrophils; myeloid-derived suppressor cells (MDSCs) and other immune cells located in the vicinity of tumors. Methods for identifying tumor cells and / or cells / tissues located in the tumor microenvironment are well known in the art, as described herein below.

[0158] In some embodiments, "increase" or "decrease" refers to a statistically significant increase or decrease, respectively.As will be clear to those skilled in the art, "modulation" can also include: causing a change (can be either increase or decrease) in the affinity, avidity, specificity, and / or selectivity of a target or antigen to one or more of its ligands, binding partners, homo- or hetero-multimeric association partners, or substrates, compared with the same conditions but without the presence of a test substance; causing a change (can be either increase or decrease) in the sensitivity of a target or antigen to one or more conditions (e.g., pH, ionic strength, the presence of cofactors, etc.) in the medium or surroundings in which the target or antigen is present; and / or cell proliferation or cytokine production.This can be determined in any suitable manner and / or by any suitable assay known per se or described herein, depending on the target involved.

[0159] As used herein, "immune response" is intended to encompass a cellular and / or humoral immune response that is sufficient to suppress, prevent the onset of, or ameliorate the symptoms of a disease (e.g., cancer or cancer metastasis). An "immune response" can encompass aspects of both the innate and adaptive immune systems.

[0160] As used herein, the terms "treating," "treatment," and "therapy" of a disease, disorder, or condition are approaches to obtain beneficial or desired clinical results. As used herein, "treatment" covers any administration or application of a therapeutic agent to a disease in a mammal, including a human. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, reduction in the extent of the disease, prevention or delay of disease spread (e.g., metastasis, e.g., to the lungs or lymph nodes), prevention or delay of disease recurrence, delay or slowing of disease progression, amelioration of the disease state, inhibition of the disease or disease progression, inhibition or slowing of the disease or its progression, cessation of its occurrence, and remission (either partial or complete). By "treatment," reduction of the pathological consequences of a proliferative disease is also encompassed. The methods provided herein contemplate any one or more of these aspects of treatment. Consistent with the above, the term treatment does not require 100 percent elimination of all aspects of the disorder.

[0161] As used herein in the context of cancer, the terms "treat" or "inhibit," "inhibiting," or "inhibition" of cancer refer to at least one of the following: a statistically significant decrease in the rate of tumor growth, a cessation of tumor growth, or a reduction in tumor size, mass, metabolic activity, or volume, as measured by standard criteria such as, but not limited to, Response Evaluation Criteria for Solid Tumors (RECIST), or a statistically significant increase in progression-free survival (PFS) or overall survival (OS).

[0162] "Ameliorating" refers to a decrease or improvement in one or more symptoms compared to not administering a therapeutic agent. "Ameliorating" also includes a shortening or reduction in the duration of a symptom.

[0163] "Preventing," "prophylaxis," or "prevention" of a disease or disorder refers to the administration of a pharmaceutical composition, either alone or in combination with another compound, to prevent the appearance or development of a disease or disorder or some or all of the symptoms of a disease or disorder, or to reduce the likelihood of developing a disease or disorder.

[0164] The term "inhibition" or "inhibiting" refers to a reduction or cessation of any phenotypic trait, or a reduction or cessation of the incidence, degree, or likelihood of that trait. "Reducing" or "inhibiting" is to decrease, reduce, or stop an activity, function, and / or amount compared to a reference. In some embodiments, "reducing" or "inhibiting" refers to the ability to cause an overall decrease of 10% or more. In some embodiments, "reducing" or "inhibiting" refers to the ability to cause an overall decrease of 50% or more. In some embodiments, "reducing" or "inhibiting" refers to the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or more. In some embodiments, the amount is inhibited or reduced over a period of time compared to a control over the same period of time.

[0165] As used herein, "delaying the onset of disease" means to postpone, prevent, slow down, retard, stabilize, suppress, and / or postpone the onset of a disease (e.g., cancer). This delay can be of varying lengths of time, depending on the disease history and / or the individual being treated. As will be apparent to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, the onset of late-stage cancer, such as metastasis, can be delayed.

[0166] As used herein, "preventing" includes providing protection against the appearance or recurrence of a disease in a subject who may have a predisposition to the disease but has not yet been diagnosed with the disease. Unless otherwise specified, the terms "reducing," "suppressing," or "preventing" do not represent or require complete prevention all the time, but only over the period of time being measured.

[0167] The term " anticancer agent " is used herein in the broadest sense to refer to the agent used in the treatment of one or more cancers.The exemplary types of such agents include but are not limited to chemotherapeutic agents, anticancer biologics (for example, cytokines, receptor extracellular domain-Fc fusions and antibodies), radiation therapy, CAR-T therapy, therapeutic oligonucleotides (for example, antisense oligonucleotides and siRNA) and oncolytic viruses.

[0168] The term "biological sample" refers to any number of substances derived from living or formerly living organisms, including, but not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0169] The term "control" or "reference" refers to a composition known to not contain the analyte (a "negative control") or a composition known to contain the analyte (a "positive control"). A positive control can contain a known concentration of the analyte.

[0170] The term "effective amount" or "therapeutically effective amount" refers to the amount and / or concentration of a composition containing an active ingredient (e.g., an sdAb or VHH-containing polypeptide) that, when administered to a patient either alone (i.e., as monotherapy) or in combination with an additional therapeutic agent, results in a statistically significant reduction in disease progression, e.g., by ameliorating or eliminating the symptoms and / or causes of the disease. An effective amount can be an amount that relieves, reduces, or alleviates at least one symptom or biological response or effect associated with a disease or disorder, prevents the progression of a disease or disorder, or improves the patient's physical function. A therapeutically effective amount of a composition containing an active agent can vary according to factors such as the individual's disease state, age, sex, and weight, and the ability of the active agent to elicit a desired response in an individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the active agent are outweighed by the therapeutically beneficial effects. A therapeutically effective amount can be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective to achieve the desired therapeutic and / or prophylactic result, at the dosages and for the period of time necessary.

[0171] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells, which may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0172] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and does not contain any additional components that are unacceptably toxic to the subject to which the formulation is administered. Thus, it is a composition that is suitable for pharmaceutical use in mammalian subjects, often humans. A pharmaceutical composition typically comprises an effective amount of an active agent (e.g., an sdAb or VHH-containing polypeptide) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient, or diluent, respectively. Such formulations may be sterile.

[0173] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventional in the art for use with therapeutic agents that together constitute a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations used and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation being used.

[0174] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0175] The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents where at least some of the administration overlaps in time, or where the administration of one therapeutic agent is within a short period of time relative to the administration of the other therapeutic agent, or where the therapeutic effects of both agents overlap for at least some period of time.

[0176] The term "sequentially" is used herein to refer to the administration of two or more therapeutic agents that do not overlap in time or where the therapeutic effects of the agents do not overlap.

[0177] As used herein, "in conjunction with" refers to the administration of one treatment modality in addition to another treatment modality. As such, "in conjunction with" refers to the administration of one treatment modality to an individual before, during, or after the administration of another treatment modality.

[0178] The term "package insert" is used to refer to instructions customarily included in commercial packaging of therapeutic products that contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications, and / or warnings pertaining to the use of such therapeutic product.

[0179] An "article of manufacture" is any article of manufacture (e.g., package or container) or kit that includes at least one reagent, e.g., a drug for treating a disease or disorder (e.g., cancer), or a probe for specifically detecting a biomarker described herein. In some embodiments, the article of manufacture or kit is promoted, distributed, or sold as a unit for performing a method described herein.

[0180] The terms "label" and "detectable label" refer to a moiety added to an antibody or antigen to, for example, make the reaction (e.g., binding) between members of a specific binding pair detectable. A labeled member of a specific binding pair is said to be "detectably labeled." Thus, the term "labeled binding protein" refers to a protein having an incorporated label that provides identification of the binding protein. In some embodiments, the label is a detectable marker capable of generating a signal that is detectable by visual or instrumental means, for example, by incorporation of a radiolabeled amino acid or addition of a biotinyl moiety to the polypeptide that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C. 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153Sm); chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic substances, e.g., gadolinium chelates. Representative examples of labels commonly used in immunoassays include light-producing moieties, e.g., acridinium compounds, and fluorescence-producing moieties, e.g., fluorescein. In this regard, the moiety itself need not be detectably labeled, but may become detectable upon reaction with yet another moiety.

[0181] II. VHH domains that bind to DLL3 Provided herein are DLL3-binding polypeptides that are VHH-containing polypeptides containing at least one VHH domain that specifically binds to DLL3. In some embodiments, the VHH domain binds to human DLL3. In some of the provided embodiments, the VHH domain binds to DLL3 having the sequence set forth in SEQ ID NO:86 or its mature form lacking a signal sequence. In some of the provided embodiments, the VHH domain binds to DLL3 having the sequence set forth in SEQ ID NO:87 or its mature form lacking a signal sequence.

[0182] In some embodiments, a VHH-containing polypeptide incorporates multiple copies of a VHH domain provided herein. In such embodiments, a VHH-containing polypeptide may incorporate multiple copies of the same VHH domain. In some embodiments, a VHH-containing polypeptide may incorporate multiple copies of different VHH domains that recognize the same epitope on DLL3. VHH-containing polypeptides may be formatted in a variety of formats, including any of those described in Section III below.

[0183] VHH domains are antibody fragments that contain a single monomeric variable antibody domain capable of selectively binding to a specific antigen. VHH domains (also called single-domain antibodies) have a molecular weight of only 12-15 kDa, much smaller than a typical antibody (150-160 kDa), which is composed of two heavy and two light protein chains, and even smaller than a Fab fragment (approximately 50 kDa, one light chain and half a heavy chain) and a single-chain variable fragment (approximately 25 kDa, two variable domains, one from the light chain and one from the heavy chain).

[0184] A single-domain antibody is an antibody whose complementarity-determining region is part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, antibodies naturally lacking light chains, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. Single-domain antibodies can be derived from any species, including, but not limited to, mouse, human, camel, llama, alpaca, vicuna, guanaco, shark, goat, rabbit, and / or cow. In some embodiments, the single-domain antibody used herein is a naturally occurring single-domain antibody known as a heavy-chain antibody lacking light chains. For clarity, this variable domain derived from a heavy-chain antibody naturally lacking light chains is referred to herein as VHH to distinguish it from the conventional VH of four-chain immunoglobulins. Such VHH molecules can be derived from antibodies raised in Camelidae species, such as camel, llama, dromedary, alpaca, vicuna, and guanaco. Besides Camelidae, other species may produce heavy chain antibodies that are naturally devoid of light chains; such VHHs are within the scope of the present disclosure.

[0185] Methods for screening VHH domains, including VHH-binding polypeptides, that possess the desired specificity for DLL3 include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs), enzyme assays, flow cytometry, and other immunologically mediated techniques known in the art.

[0186] Among the VHH domains provided herein are DLL3 VHH (llama-derived) and humanized sequences, such as any of the following:

[0187] In some embodiments, the VHH domain that binds to DLL3 may be humanized. Humanized antibodies (e.g., VHH-containing polypeptides) are useful as therapeutic molecules because they reduce or eliminate immune responses to antibody therapeutics and human immune responses to non-human antibodies, which can result in reduced efficacy of the therapeutic. Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. Optionally, the humanized antibody also comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0188] Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633, and are also described, for example, in Riechmann et al., (1988) Nature 332:323-329; Queen et al., (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033; U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., (2005) Methods 36:25-34; Padlan, (1991) Mol. Immunol. 28:489-498 (describing "resurfacing"); Dall'Acqua et al., (2005) Methods 36:43-60 (describing "FR shuffling"); and Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (describing a "guided selection" approach to FR shuffling).

[0189] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. (1993) J. Immunol. 151:2296); framework regions derived from consensus sequences of human antibodies of particular subgroups of heavy chain variable regions (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol, 151:2623); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633); and framework regions derived from screening of FR libraries (see, e.g., Baca et al., (1997) J. Biol. Chem. 272: (See Rosok et al., (1996) J. Biol. Chem. 271:22611-22618). Typically, the FR region of a VHH is replaced with a human FR region to create a humanized VHH. In some embodiments, certain FR residues of the human FR are replaced to improve one or more properties of the humanized VHH. A VHH domain having such replaced residues is still referred to herein as "humanized."

[0190] A VHH amino acid sequence selected from any of SEQ ID NOs: 102, 244 to 318, 401 to 409, 416, 455, and 476 to 480 to 488, 507 to 518, or a VHH amino acid sequence selected from any of SEQ ID NOs: 102, 244 to 318, 401 to 409, 416, 455, and 476 to 480 to 488, 507 to 518 HProvided herein is a VHH domain that binds to DLL3, comprising CDR1, CDR2, and CDR3 contained in an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the H region amino acids. In some embodiments, the DLL3 VHH domain provided herein contains a CDR1 set forth in any of SEQ ID NOs: 319-335 or 456, a CDR2 set forth in any of SEQ ID NOs: 336-353, 384, 410, and 411, and a CDR3 set forth in any of SEQ ID NOs: 354-367, 395, and 412-415. In some embodiments, the DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs: 102, 244-318, 401-409, 416, 455, and 476-480-488, 507-518, or a VHH domain selected from any of SEQ ID NOs: 102, 244-318, 401-409, 416, 455, and 476-480-488, 507-518. H In some embodiments, the DLL3 VHH domain has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the H-region amino acids. In some embodiments, the DLL3 VHH domain has a sequence of amino acids set forth in any one of SEQ ID NOs: 102, 244-318, 401-409, 416, 455, and 476-480-488, 507-518.

[0191] A VHH amino acid sequence selected from any of SEQ ID NOs: 244 to 318 and 455, or a VHH amino acid sequence selected from any of SEQ ID NOs: 244 to 318 and 455 HProvided herein are VHH domains that bind to DLL3, comprising CDR1, CDR2, and CDR3 contained in an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the H region amino acids. In some embodiments, the DLL3 VHH domains provided herein contain a CDR1 set forth in any of SEQ ID NOs:319-335, a CDR2 set forth in any of SEQ ID NOs:336-353, and a CDR3 set forth in any of SEQ ID NOs:354-367. The provided DLL3 VHH domains comprise a VHH domain selected from the amino acid sequence set forth in any of SEQ ID NOs:1-114, or any of SEQ ID NOs:244-318. H In some embodiments, the DLL3 VHH domain has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the H region amino acids. In some embodiments, the DLL3 VHH domain has a sequence of amino acids set forth in any one of SEQ ID NOs:244-318 and 455.

[0192] In some embodiments, the DLL3 VHH domain provided herein contains CDR1, CDR2, and CDR3 contained in the VHH domain set forth in SEQ ID NO: 244, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH region amino acids set forth in SEQ ID NO: 244. In some embodiments, the DLL3 VHH domain has the amino acid sequence set forth in SEQ ID NO: 244, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acids set forth in SEQ ID NO: 244. In some embodiments, the DLL3 VHH domain is a humanized variant of the amino acid sequence set forth in SEQ ID NO: 244.

[0193] In some embodiments, the DLL3 VHH domain provided herein contains a CDR1 set forth in any of SEQ ID NOs: 319, 320, 321, 322, 323, 324, 325, 326, a CDR2 set forth in any of SEQ ID NOs: 336, 337, 338, and a CDR3 set forth in SEQ ID NO: 354.

[0194] In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 319, 336, and 354, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 319, 337, and 354, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 319, 338, and 354, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 320, 338, and 354, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 321, 338, and 354, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 322, 338, and 354, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 323, 338, and 354, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 324, 338, and 354, respectively. In some embodiments, the DLL3 VHH domain provided herein contains the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 325, 338, and 354, respectively.In some embodiments, the DLL3 VHH domain provided herein contains the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 326, 338, and 354, respectively.

[0195] In some aspects, the VHH domain that binds to DLL3 comprises a VHH amino acid sequence selected from any of SEQ ID NOs: 245-257, or a VHH amino acid sequence selected from any of SEQ ID NOs: 245-257. H It comprises CDR1, CDR2, and CDR3 contained in an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the H region amino acids.

[0196] In some cases, the provided DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs: 245-257, or a VHH domain selected from any of SEQ ID NOs: 245-257. H In some embodiments, the DLL3 humanized VHH domain has a sequence of amino acids set forth in any one of SEQ ID NOs:245-257.

[0197] In some embodiments, the DLL3 VHH domain provided herein contains CDR1, CDR2, CDR3 contained in the VHH domain set forth in SEQ ID NO: 258, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH region amino acids set forth in SEQ ID NO: 258. In some embodiments, the DLL3 VHH domain has the amino acid sequence set forth in SEQ ID NO: 258, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acids set forth in SEQ ID NO: 258. In some embodiments, the DLL3 VHH domain is a humanized variant of the amino acid sequence set forth in SEQ ID NO: 258.

[0198] In some embodiments, the DLL3 VHH domain provided herein contains a CDR1 set forth in SEQ ID NO:327, a CDR2 set forth in SEQ ID NO:339, and a CDR3 set forth in SEQ ID NO:355.

[0199] In some embodiments, the DLL3 VHH domain provided herein contains the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 327, 339, and 355, respectively.

[0200] In some aspects, the VHH domain that binds to DLL3 comprises a VHH amino acid sequence selected from any of SEQ ID NOs: 259-263, or a VHH amino acid sequence selected from any of SEQ ID NOs: 259-263. H It comprises CDR1, CDR2, and CDR3 contained in an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the H region amino acids.

[0201] In some cases, the provided DLL3 VHH domain comprises an amino acid sequence selected from any of SEQ ID NOs: 259-263, or a VHH domain comprising any of SEQ ID NOs: 259-263. H In some embodiments, the DLL3 humanized VHH domain has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the H-region amino acids. In some embodiments, the DLL3 humanized VHH domain has an amino acid sequence set forth in any one of SEQ ID NOs:259-263.

[0202] In some embodiments, the DLL3 VHH domain provided herein contains CDR1, CDR2, CDR3 contained in the VHH domain set forth in SEQ ID NO: 264, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH region amino acids set forth in SEQ ID NO: 264. In some embodiments, the DLL3 VHH domain has the amino acid sequence set forth in SEQ ID NO: 264, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acids set forth in SEQ ID NO: 264. In some embodiments, the DLL3 VHH domain is a humanized variant of the amino acid sequence set forth in SEQ ID NO: 264.

[0203] In some embodiments, the DLL3 VHH domain provided herein contains a CDR1 set forth in SEQ ID NO:328, 329, or 456, a CDR2 set forth in SEQ ID NO:340, and a CDR3 set forth in SEQ ID NO:356.

[0204] In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 328, 340, 356, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 329, 340, 356, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 456, 340, 356, respectively.

[0205] In some aspects, the VHH domain that binds to DLL3 comprises a VHH amino acid sequence selected from any of SEQ ID NOs: 265-274, 416, 455, or 476-478, or a VHH amino acid sequence selected from any of SEQ ID NOs: 265-274, 416, 455, or 476-478. H It comprises CDR1, CDR2, and CDR3 contained in an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the H region amino acids.

[0206] In some cases, the provided DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs: 265-274, 416, 455, or 476-478, or a VHH domain selected from any of SEQ ID NOs: 265-274, 416, 455, or 476-478. H In some embodiments, the DLL3 humanized VHH domain has a sequence of amino acids set forth in any one of SEQ ID NOs:265-274, 416, 455, or 476-478.

[0207] In some embodiments, the DLL3 VHH domain provided herein contains CDR1, CDR2, CDR3 contained in the VHH domain set forth in SEQ ID NO: 275, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH region amino acids set forth in SEQ ID NO: 275. In some embodiments, the DLL3 VHH domain has an amino acid sequence set forth in SEQ ID NO: 275, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acids set forth in SEQ ID NO: 105. In some embodiments, the DLL3 VHH domain is a humanized variant of the amino acid sequence set forth in SEQ ID NO: 275.

[0208] In some embodiments, the DLL3 VHH domain provided herein contains a CDR1 set forth in SEQ ID NO:320, a CDR2 set forth in SEQ ID NO:341, and a CDR3 set forth in SEQ ID NO:357.

[0209] In some aspects, the VHH domain that binds to DLL3 comprises a VHH amino acid sequence selected from any of SEQ ID NOs: 276-279 or 479, or a VHH amino acid sequence selected from any of SEQ ID NOs: 276-279 or 479. H It comprises CDR1, CDR2, and CDR3 contained in an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the H region amino acids.

[0210] In some cases, the provided DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs: 276-279 or 479, or a VHH domain selected from any of SEQ ID NOs: 276-279 or 479.H In some embodiments, the DLL3 humanized VHH domain has a sequence of amino acids set forth in any one of SEQ ID NOs:276-279 or 479.

[0211] In some embodiments, the DLL3 VHH domain provided herein contains CDR1, CDR2, CDR3 contained in the VHH domain set forth in SEQ ID NO: 280, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH region amino acids set forth in SEQ ID NO: 280. In some embodiments, the DLL3 VHH domain has the amino acid sequence set forth in SEQ ID NO: 280, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acids set forth in SEQ ID NO: 280. In some embodiments, the DLL3 VHH domain is a humanized variant of the amino acid sequence set forth in SEQ ID NO: 280.

[0212] In some embodiments, the DLL3 VHH domain provided herein contains a CDR1 set forth in SEQ ID NO:330, a CDR2 set forth in SEQ ID NO:342, and a CDR3 set forth in SEQ ID NO:358.

[0213] In some aspects, the VHH domain that binds to DLL3 comprises a VHH amino acid sequence selected from any of SEQ ID NOs: 281-286, or a VHH amino acid sequence selected from any of SEQ ID NOs: 281-286. HIt contains CDR1, CDR2, and CDR3 contained in an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the H region amino acids.

[0214] In some cases, the provided DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs:281-286, or a VHH domain selected from any of SEQ ID NOs:281-286. H In some embodiments, the DLL3 humanized VHH domain has a sequence of amino acids set forth in any one of SEQ ID NOs:281-286.

[0215] In some embodiments, the DLL3 VHH domain provided herein contains CDR1, CDR2, CDR3 contained in the VHH domain set forth in SEQ ID NO: 287, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH region amino acids set forth in SEQ ID NO: 287. In some embodiments, the DLL3 VHH domain has the amino acid sequence set forth in SEQ ID NO: 287, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acids set forth in SEQ ID NO: 287. In some embodiments, the DLL3 VHH domain is a humanized variant of the amino acid sequence set forth in SEQ ID NO: 287.

[0216] In some embodiments, the DLL3 VHH domain provided herein contains a CDR1 set forth in SEQ ID NO:320, a CDR2 set forth in any of SEQ ID NOs:345, 346, 347, and a CDR3 set forth in any of SEQ ID NOs:359, 360, 361.

[0217] In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 320, 345, and 359, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 320, 346, and 359, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 320, 347, and 359, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 320, 345, and 360, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 320, 345, and 361, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 320, 347, and 360, respectively.

[0218] In some aspects, the VHH domain that binds to DLL3 comprises a VHH amino acid sequence selected from any of SEQ ID NOs: 288-298 or 102, or a VHH amino acid sequence selected from any of SEQ ID NOs: 288-298 or 102. HIt comprises CDR1, CDR2, and CDR3 contained in an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the H region amino acids.

[0219] In some cases, the provided DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs: 288-298 or 102, or a VHH domain selected from any of SEQ ID NOs: 288-298 or 102. H In some embodiments, the DLL3 humanized VHH domain has a sequence of amino acids set forth in any one of SEQ ID NOs:288-298 or 102.

[0220] In some embodiments, the DLL3 VHH domain provided herein contains CDR1, CDR2, CDR3 contained in the VHH domain set forth in SEQ ID NO: 299, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH region amino acids set forth in SEQ ID NO: 299. In some embodiments, the DLL3 VHH domain has the amino acid sequence set forth in SEQ ID NO: 299, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acids set forth in SEQ ID NO: 299. In some embodiments, the DLL3 VHH domain is a humanized variant of the amino acid sequence set forth in SEQ ID NO: 299.

[0221] In some embodiments, the DLL3 VHH domain provided herein contains a CDR1 set forth in SEQ ID NO:331, a CDR2 set forth in any of SEQ ID NOs:348, 349, 350, and a CDR3 set forth in SEQ ID NO:356.

[0222] In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 331, 348, and 356, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 331, 349, and 356, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 331, 350, and 356, respectively.

[0223] In some aspects, the VHH domain that binds to DLL3 comprises a VHH amino acid sequence selected from any of SEQ ID NOs: 300-305 or 480, or a VHH amino acid sequence selected from any of SEQ ID NOs: 300-305 or 480. H It comprises CDR1, CDR2, and CDR3 contained in an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the H region amino acids.

[0224] In some cases, the provided DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs: 300-305 or 480, or a VHH domain selected from any of SEQ ID NOs: 300-305 or 480. HIn some embodiments, the DLL3 humanized VHH domain has a sequence of amino acids set forth in any one of SEQ ID NOs: 300-305 or 480.

[0225] In some embodiments, the DLL3 VHH domain provided herein contains CDR1, CDR2, CDR3 contained in the VHH domain set forth in SEQ ID NO: 306, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH region amino acids set forth in SEQ ID NO: 306. In some embodiments, the DLL3 VHH domain has the amino acid sequence set forth in SEQ ID NO: 306, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 306. In some embodiments, the DLL3 VHH domain is a humanized variant of the amino acid sequence set forth in SEQ ID NO: 306.

[0226] In some embodiments, the DLL3 VHH domain provided herein contains CDR1, CDR2, CDR3 contained in the VHH domain set forth in SEQ ID NO: 507, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH region amino acids set forth in SEQ ID NO: 507. In some embodiments, the DLL3 VHH domain has the amino acid sequence set forth in SEQ ID NO: 507, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acids set forth in SEQ ID NO: 507. In some embodiments, the DLL3 VHH domain is a humanized variant of the amino acid sequence set forth in SEQ ID NO: 507.

[0227] In some embodiments, the DLL3 VHH domain provided herein contains a CDR1 set forth in SEQ ID NO:332, a CDR2 set forth in any of SEQ ID NOs:348, 349, 350, and a CDR3 set forth in SEQ ID NO:362.

[0228] In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 332, 348, and 362, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 332, 349, and 362, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 332, 350, and 362, respectively.

[0229] In some aspects, the VHH domain that binds to DLL3 comprises a VHH amino acid sequence selected from any of SEQ ID NOs: 307-313, or a VHH amino acid sequence selected from any of SEQ ID NOs: 307-313. H It comprises CDR1, CDR2, and CDR3 contained in an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the H region amino acids.

[0230] In some cases, the provided DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs: 307-313, or a VHH domain selected from any of SEQ ID NOs: 307-313. H In some embodiments, the DLL3 humanized VHH domain has a sequence of amino acids set forth in any one of SEQ ID NOs:307-313.

[0231] In some aspects, the VHH domain that binds to DLL3 comprises a VHH amino acid sequence selected from any of SEQ ID NOs: 508-514, or a VHH amino acid sequence selected from any of SEQ ID NOs: 508-514. H It comprises CDR1, CDR2, and CDR3 contained in an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the H region amino acids.

[0232] In some cases, the provided DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs: 508-514, or a VHH domain selected from any of SEQ ID NOs: 508-514. HIn some embodiments, the DLL3 humanized VHH domain has a sequence of amino acids set forth in any one of SEQ ID NOs:508-514.

[0233] In some embodiments, the DLL3 VHH domain provided herein contains CDR1, CDR2, CDR3 contained in the VHH domain set forth in SEQ ID NO: 401, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH region amino acids set forth in SEQ ID NO: 401. In some embodiments, the DLL3 VHH domain has the amino acid sequence set forth in SEQ ID NO: 401, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acids set forth in SEQ ID NO: 401. In some embodiments, the DLL3 VHH domain is a humanized variant of the amino acid sequence set forth in SEQ ID NO: 401.

[0234] In some embodiments, the DLL3 VHH domain provided herein contains a CDR1 set forth in SEQ ID NO:320, a CDR2 set forth in any of SEQ ID NOs:384, 410, 411, and a CDR3 set forth in any of SEQ ID NOs:395, 412, 413, 414, 415.

[0235] In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 320, 384, and 395, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 320, 410, and 395, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 320, 411, and 395, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 320, 384, and 412, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 320, 384, and 413, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 320, 384, and 414, respectively. In some embodiments, the DLL3 VHH domains provided herein contain the CDR1, CDR2, and CDR3 set forth in any of SEQ ID NOs: 320, 384, and 415, respectively.

[0236] In some aspects, the VHH domain that binds to DLL3 comprises a VHH amino acid sequence selected from any of SEQ ID NOs: 402-409, or a VHH amino acid sequence selected from any of SEQ ID NOs: 402-409. HIn some aspects, the VHH domain that binds to DLL3 comprises a VHH amino acid sequence selected from any of SEQ ID NOs:481-488, or a VHH amino acid sequence selected from any of SEQ ID NOs:481-488. H It comprises CDR1, CDR2, and CDR3 contained in an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the H region amino acids.

[0237] In some cases, the provided DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs: 402-409, or a VHH domain selected from any of SEQ ID NOs: 402-409. H In some embodiments, the DLL3 humanized VHH domain has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the H-region amino acids. In some embodiments, the DLL3 humanized VHH domain has a sequence of amino acids set forth in any one of SEQ ID NOs:402-409. In some cases, the provided DLL3 VHH domain has an amino acid sequence set forth in any of SEQ ID NOs:481-488, or a VHH domain selected from any of SEQ ID NOs:481-488. H In some embodiments, the DLL3 humanized VHH domain has a sequence of amino acids set forth in any one of SEQ ID NOs:481-488.

[0238] III. Fusion Proteins and Conjugates Containing DLL3-Binding Polypeptides Fusion proteins and conjugates containing a DLL3-binding polypeptide containing at least one VHH domain that specifically binds to DLL3, directly or indirectly linked to one or more additional domains or moieties, are provided herein. In some embodiments, the fusion proteins or conjugates of the present disclosure are composed of a single polypeptide. In other embodiments, the fusion proteins or conjugates of the present disclosure are composed of multiple polypeptides. In some embodiments, the DLL3-binding polypeptide of the present disclosure incorporates at least one VHH domain that specifically binds to DLL3. In some aspects, the DLL3-binding polypeptide is multivalent. In some embodiments, the DLL3-binding polypeptide contains two or more copies of the VHH domain that specifically binds to DLL3, for example, three or more, four or more, five or more, or six or more copies of the VHH domain that specifically binds to DLL3. In certain aspects, the DLL3-binding polypeptide is multispecific. For example, in some cases, the one or more additional domains can be one or more additional binding domains that bind to one or more additional antigens or proteins.

[0239] In some embodiments, the DLL3-binding polypeptide of the present disclosure comprises two or more polypeptide sequences operably linked via an amino acid linker.In some embodiments, these linkers are mainly composed of amino acids glycine and serine, and are herein referred to as GS linkers.The GS linkers of the fusion proteins of the present disclosure can be of various lengths, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length.In some embodiments, the GS linker is TIFF2025060787000014.tif16162. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of, by way of non-limiting example, and a flexible linker comprising a glycine residue, such as TIFF2025060787000015.tif10128. In some embodiments, the DLL3-binding polypeptide comprises a combination of a GS linker and a glycine linker. In some embodiments, the linker is (GGGGS)n (n is 1 to 5) (SEQ ID NO: 123); (GGGGGS)n (n is 1 to 4) (SEQ ID NO: 124); TIFF2025060787000016.tif23153. In some embodiments, the linker is a GG linker. In some embodiments, the DLL3-binding polypeptide comprises a combination of a GS linker and a glycine linker.

[0240] A.Fc fusion Provided herein is a DLL3-binding polypeptide, which is a fusion protein comprising at least one VHH domain that binds to the DLL3 provided herein and an Fc domain. In some embodiments, provided herein is a DLL3-binding polypeptide comprising one, two, three, or four VHH domains that bind to DLL3 and an Fc domain.

[0241] In some embodiments, incorporation of an immunoglobulin Fc region into a fusion protein can, in some aspects, consist of two polypeptides that together form a dimer. In some embodiments, the Fc domain mediates dimerization of the DLL3-binding polypeptide under physiological conditions, e.g., when expressed from a cell, such that a dimer is formed that doubles the number of DLL3-binding sites. For example, a DLL3-binding polypeptide comprising three VHH domains that bind to DLL3 and an Fc region is trivalent as a monomer, but the Fc region can mediate dimerization such that the DLL3-binding polypeptide exists as a hexavalent dimer under such conditions. In some embodiments, a DLL3 VHH domain is fused to an IgG Fc region; in these embodiments, the fusion protein is bivalent, with two DLL3 VHH domains per molecule. In some embodiments, two DLL3-binding domains (2x) are fused to an IgG Fc region; in these embodiments, the fusion protein is tetravalent, with four DLL3 VHH domains per molecule. In some embodiments, three DLL3 VHH domains (3x) are fused to an IgG Fc region, and in these embodiments, the fusion protein is hexavalent, with six DLL3 VHH domains per molecule.

[0242] In some embodiments, a multivalent DLL3-binding polypeptide is bivalent. In some embodiments, a bivalent DLL3-binding polypeptide of the present disclosure comprises two copies of a DLL3-binding polypeptide having the following structure: (DLL3 VHH)-linker-Fc. In some embodiments, a multivalent DLL3-binding polypeptide is tetravalent. In some embodiments, a tetravalent DLL3-binding polypeptide of the present disclosure comprises two copies of a DLL3-polypeptide having the following structure: (DLL3 VHH)-linker-(DLL3 VHH)-linker-Fc. In some embodiments, a multivalent DLL3-binding polypeptide is hexavalent. In some embodiments, a hexavalent DLL3-binding polypeptide of the present disclosure comprises two copies of a DLL3-binding polypeptide having the following structure: (DLL3 VHH)-linker-(DLL3 VHH)-linker-(DLL3 VHH)-linker-Fc.

[0243] In some cases, the CH3 domain of the Fc region can be used as a homodimerization domain, so that the resulting fusion protein is formed from two identical polypeptides. In other cases, the CH3 dimer interface region of the Fc region can be mutated to enable heterodimerization. For example, a heterodimerization domain can be incorporated into the fusion protein so that the construct is an asymmetric fusion protein.

[0244] In any of the provided embodiments, the DLL3 VHH domain can be any of those described above. In some embodiments, the DLL3 VHH domain is a humanized VHH domain that binds to DLL3.

[0245] In various embodiments, the Fc domain contained in the DLL3-binding polypeptide is a human Fc domain or is derived from a human Fc domain. In some embodiments, the fusion protein contains an immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is an IgG isotype selected from the group consisting of an IgG1 isotype, an IgG2 isotype, an IgG3 isotype, and an IgG4 subclass.

[0246] In some embodiments, the immunoglobulin Fc region or immunologically active fragment thereof is of the IgG isotype. For example, the immunoglobulin Fc region of the fusion protein is of the human IgG1 isotype, having the following amino acid sequence: TIFF2025060787000017.tif23150

[0247] In some embodiments, the immunoglobulin Fc region or immunologically active fragment thereof comprises a human IgG1 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:8.

[0248] In some embodiments where the fusion protein of the present disclosure comprises an Fc polypeptide, the Fc polypeptide is mutated or modified. In some cases, the mutations include one or more amino acid substitutions to reduce the effector function of the Fc polypeptide. Various examples of mutations to Fc polypeptides that alter, e.g., reduce, effector function are known, including any of the following: In some embodiments, references to amino acid substitutions in the Fc region are in accordance with EU numbering according to Kabat (also referred to as Kabat numbering), unless described with reference to a specific SEQ ID NO. EU numbering follows the EU index as reported in the well-known and most recently updated IMGT Scientific Chart (IMGT®, the international ImMunoGeneTics information system®, http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created May 17, 2001, last updated January 10, 2013)) and Kabat, EA et al. Sequences of Proteins of Immunological interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0249] In some embodiments, Fc regions exhibiting reduced effector function may be desirable candidates for applications in which DLL3 or CD3 binding is desirable, but certain effector functions (e.g., CDC and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the multispecific polypeptide construct and / or its truncated components lack FcγR binding (and thus likely lack ADCC activity) but retain FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96™ Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the multispecific polypeptide construct or its truncated components cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Using methods known in the art, FcRn binding and in vivo clearance / half-life determinations can also be performed (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0250] In some embodiments, the human IgG Fc region is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), e.g., Natsume et al., 2008 Cancer Res, 68(10): 3863-72; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Moore et al., 2010 mAbs, 2(2): 181-189; Lazar et al., 2006 PNAS, 103(11): 4005-4010, Shields et al., 2001 JBC, 276(9): 6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48: 152-164; Alegre et al, 1992 J Immunol, 148: 3461-3468; and amino acid modifications reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11.

[0251] Examples of mutations that enhance ADCC include modifications at Ser239 and Ile332, e.g., Ser239Asp and Ile332Glu (S239D, I332E). Examples of mutations that enhance CDC include modifications at Lys326 and Glu333. In some embodiments, the Fc region is modified at one or both of these positions, e.g., with Lys326Ala and / or Glu333Ala (K326A and E333A) using the Kabat numbering system.

[0252] In some embodiments, the Fc region of the fusion protein is altered at one or more of the following positions to reduce Fc receptor binding: Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325), Ala327 (A327), or Pro329 (P329). For example, Leu234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Pro329Ala (P329A) or Pro239Gly (P329G), Asn325Glu (N325E), or Ala327Ser (A327S). In a preferred embodiment, the modifications within the Fc region reduce binding to the Fc receptor gamma receptor while having minimal effect on binding to the neonatal Fc receptor (FcRn).

[0253] In some embodiments, the human IgG1 Fc region is modified at amino acid Asn297 (Kabat numbering) to prevent glycosylation of the fusion protein, e.g., having Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu235 (Kabat numbering) to alter Fc-receptor interactions, e.g., having Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat numbering) to alter Fc-receptor interactions, e.g., having Leu234Ala (L234A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat numbering) to alter Fc-receptor interactions, e.g., having Leu235Glu (L235E). In some embodiments, the Fc region of the fusion protein is altered at both amino acids 234 and 235, e.g., having Leu234Ala and Leu235Ala (L234A / L235A) or Leu234Val and Leu235Ala (L234V / L235A). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 297, e.g., having Leu234Ala, Leu235Ala, Asn297Ala (L234A / L235A / N297A). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 329, e.g., having Leu234Ala, Leu235Ala, Pro239Ala (L234A / L235A / P329A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Asp265 (Kabat numbering) to alter Fc-receptor interactions, e.g., has Asp265Ala (D265A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Pro329 (Kabat numbering) to alter Fc-receptor interactions, e.g., has Pro329Ala (P329A) or Pro329Gly (P329G).In some embodiments, the Fc region of the fusion protein is altered at both amino acids 265 and 329, e.g., having Asp265Ala and Pro329Ala (D265A / P329A) or Asp265Ala and Pro329Gly (D265A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 265, e.g., having Leu234Ala, Leu235Ala, Asp265Ala (L234A / L235A / D265A). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 329, e.g., having Leu234Ala, Leu235Ala, Pro329Gly (L234A / L235A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, 265, and 329, e.g., Leu234Ala, Leu235Ala, Asp265Ala, Pro329Gly (L234A / L235A / D265A / P329G). In some embodiments, the Fc region of the fusion protein is altered at Gly235 to reduce Fc receptor binding. For example, Gly235 is deleted from the fusion protein. In some embodiments, the human IgG1 Fc region is altered at amino acid Gly236 to enhance interaction with CD32A, e.g., Gly236Ala (G236A). In some embodiments, the human IgG1 Fc region lacks Lys447 (Kabat et al. 1991 Sequences of Proteins of Immunological Interest, EU Index).

[0254] In some embodiments, the Fc region of the fusion protein lacks one or more amino acids at the following positions to reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (L235). In some embodiments, the Fc region of the fusion protein lacks one or more amino acids at the following positions: Glu233 (E233), Leu234 (L234), or Leu235 (L235), and is modified at one or more of Asp265 (D265), Asn297 (N297), or Pro329 (P329) to reduce Fc receptor binding. For example, the Fc region included in the DLL3-binding polypeptide is derived from a human Fc domain and contains three amino acid deletions below the hinge corresponding to IgG1 E233, L234, and L235. In some aspects, such Fc polypeptides do not engage FcγR and are therefore referred to as "effector silent" or "effector null." For example, the Fc deletion of these three amino acids reduces complement protein C1q binding. In some embodiments, polypeptides having an Fc region with the Fc deletion of these three amino acids retain binding to FcRn and therefore have a prolonged half-life and transcytosis associated with FcRn-mediated recycling. Such modified Fc regions are referred to as "Fc xELL" or "Fc deletion" and have the following amino acid sequence: TIFF2025060787000018.tif23149

[0255] In some embodiments, the immunoglobulin Fc region or immunologically active fragment thereof comprises a human IgG1 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:9.

[0256] In some embodiments, the human IgG Fc region is modified to enhance FcRn binding. Examples of Fc mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, and Thr256Glu (M252Y, S254T, and T256E, respectively) (Kabat numbering, Dall'Acqua et al. 2006, J. Biol Chem Vol. 281(33) 23514-23524), Met428Leu and Asn434Ser (M428L and N434S) (Zalevsky et al. 2010 Nature Biotech, Vol. 28(2) 157-159), or Met252Ile, Thr256Asp, and Met428Leu (M252I, T256D, and M428L, respectively) (Kabat et al. 1991 EU Index of Sequences of Proteins of Immunological Interest).

[0257] In some embodiments, the Fc domain included in the DLL3-binding polypeptide is derived from a human Fc domain and contains the mutations M252Y and M428V, referred to herein as "Fc-YV." In some embodiments, the mutated or modified Fc polypeptide contains the following mutations using the Kabat numbering system: M252Y and M428L. In some embodiments, such mutations enhance binding to FcRn at the acidic pH of the endosome (near 6.5) while losing detectable binding at neutral pH (about 7.2), allowing for enhanced FcRn-mediated recycling and a prolonged half-life.

[0258] In specific embodiments of the multispecific polypeptide constructs provided herein, the Fc domain included in the DLL3-binding polypeptide is derived from a human Fc domain and contains a mutation that induces heterodimerization. In some embodiments, such mutations include what are referred to as "knob" and "hole" mutations. For example, an amino acid modification within the CH3 domain at Thr366, when replaced with a bulkier amino acid, such as Tyr (T366W), can preferentially pair with a second CH3 domain that has less bulky amino acids, such as Ser, Ala, and Val, at positions Thr366, Leu368, and Tyr407 (T366S / L368A / Y407V). In some embodiments, the "knob" Fc domain contains the mutation T366W. In some embodiments, the "hole" Fc domain contains the mutations T366S, L368A, and Y407V. Heterodimerization via CH3 modifications can be further stabilized by introducing a disulfide bond, for example, by changing Ser354 to Cys (S354C) and Y349 to Cys (Y349C) on opposing CH3 domains (reviewed in Carter, 2001 Journal of Immunological Methods, 248: 7-15). In some embodiments, the Fc domain used for heterodimerization contains an additional mutation, for example, the S354C mutation on the first member of the heterodimeric Fc pair, which forms an asymmetric disulfide bond with the corresponding Y349C mutation on the second member of the heterodimeric Fc pair. In some embodiments, one member of the heterodimeric Fc pair contains the modification H435R or H435K to prevent Protein A binding while maintaining FcRn binding. In some embodiments, one member of the heterodimeric Fc pair comprises the modification H435R or H435K, while the second member of the heterodimeric Fc pair is not modified at H435. In various embodiments, the hole Fc domain comprises the modification H435R or H435K (referred to as "hole-R" in some instances where the modification is H435R), while the knob Fc domain does not.In some instances, the hole-R mutation improves purification of the heterodimer over a possible homodimeric hole Fc domain.

[0259] In some embodiments, the human IgG Fc region is modified to prevent dimerization. In these embodiments, the fusion protein of the present disclosure is a monomer. For example, modification of residue Thr366 to a charged residue, such as Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu (T366K, T366R, T366D, or T366E, respectively), prevents CH3-CH3 dimerization.

[0260] In some embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG2 isotype, having the following amino acid sequence: TIFF2025060787000019.tif23149

[0261] In some embodiments, the fusion or immunologically active fragment thereof comprises a human IgG2 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:10.

[0262] In some embodiments, the human IgG2 Fc region is modified at amino acid Asn297 (e.g., to prevent glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D)). In some embodiments, the human IgG2 Fc region lacks Lys447 (Kabat et al 1991 Sequences of Proteins of Immunological Interest, EU Index).

[0263] In some embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG3 isotype, having the following amino acid sequence: TIFF2025060787000020.tif23149

[0264] In some embodiments, the antibody or immunologically active fragment thereof comprises a human IgG3 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:11.

[0265] In some embodiments, the human IgG3 Fc region is modified at amino acid Asn297 (Kabat numbering) to prevent glycosylation of the antibody, e.g., has Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human IgG3 Fc region is modified at amino acid 435 to extend half-life, e.g., has Arg435His (R435H). In some embodiments, the human IgG3 Fc region lacks Lys447 (Kabat et al. 1991 Sequences of Proteins of Immunological Interest, EU Index).

[0266] In some embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG4 isotype, having the following amino acid sequence: TIFF2025060787000021.tif23149

[0267] In some embodiments, the antibody or immunologically active fragment thereof comprises a human IgG4 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:12.

[0268] In some embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG4 isotype, having the following amino acid sequence: TIFF2025060787000022.tif23149

[0269] In some embodiments, the antibody or immunologically active fragment thereof comprises a human IgG4 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:13.

[0270] In some embodiments, the human IgG4 Fc region is modified at amino acid 235 to alter Fc-receptor interactions, e.g., has Leu235Glu (L235E). In some embodiments, the human IgG4 Fc region is modified at amino acid Asn297 (Kabat numbering) to prevent glycosylation of the antibody, e.g., has Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human IgG4 Fc region lacks Lys447 (Kabat et al. 1991 Sequences of Proteins of Immunological Interest, EU Index).

[0271] In some embodiments, the fusion protein contains a polypeptide derived from an immunoglobulin hinge region. The hinge region can be selected from any of the human IgG subclasses. For example, the fusion protein can contain In another embodiment, the fusion protein contains a modified IgG1 hinge having the sequence of TIFF2025060787000023.tif4128, in which Cys220, which forms a disulfide with the C-terminal cysteine ​​of the light chain, is mutated to serine, e.g., Cys220Ser (C220S). In another embodiment, the fusion protein contains a truncated hinge having the sequence DKTHTCPPC (SEQ ID NO:15).

[0272] In some embodiments, the fusion protein has a modified hinge from IgG4 that has been modified to prevent or reduce strand exchange, e.g., has Ser228Pro (S228P), having the sequence ESKYGPPCPPC (SEQ ID NO:16). In some embodiments, the fusion protein contains a linker polypeptide. In other embodiments, the fusion protein contains a linker and hinge polypeptide.

[0273] In some embodiments, the Fc region lacks or has reduced fucose attached to the N-linked glycan chain at N297. There are numerous ways to prevent fucosylation, including, but not limited to, production in FUT8-deficient cell lines; addition of inhibitors, such as castanospermine, to mammalian cell culture media; and metabolic engineering of the production cell line.

[0274] In some embodiments, the Fc region is engineered to eliminate recognition by pre-existing antibodies found in humans. In some embodiments, the VHH-containing polypeptides of the present disclosure are modified by mutation of position Leu11, e.g., Leu11Glu (L11E) or Leu11Lys (L11K). In other embodiments, the single domain antibodies of the present disclosure are modified by changes in the carboxy-terminal region, e.g., the terminal sequence is TIFF2025060787000024.tif4128 or a modification thereof. In some embodiments, the VHH-containing polypeptide of the disclosure is modified by a mutation at position 11 and by an alteration in the carboxy-terminal region.

[0275] In some embodiments, one or more polypeptides of the fusion protein of the present disclosure are functionally linked via amino acid linker.In some embodiments, these linkers are mainly composed of amino acid glycine and serine, and are referred to herein as GS linker.The GS linker of the fusion protein of the present disclosure can be of various lengths, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length.

[0276] In some embodiments, the GS linker is GGSGGS, i.e., In some embodiments, the linker is a flexible linker, including, but not limited to, an amino acid sequence selected from the group consisting of: In some embodiments, the linker is (GGGGS)n, where n is 1 to 5 (SEQ ID NO:123); (GGGGGS)n, where n is 1 to 4 (SEQ ID NO:124); TIFF2025060787000027.tif16170. In some embodiments, the fusion protein can include a combination of a GS linker and a glycine linker.

[0277] B. Conjugates Provided herein is a conjugate, comprising at least one VHH domain specifically binds to DLL3 provided herein and one or more additional moieties.Additional moieties can be therapeutic agents such as cytotoxic agents or detection agents.In some embodiments, said moiety can be targeting moiety, small molecule drug (non-polypeptide drug with molar mass less than 500 Daltons), toxin, cytostatic agent, cytotoxic agent, immunosuppressant, radioactive agent suitable for diagnostic purposes, radioactive metal ion for therapeutic purposes, prodrug-activating enzyme, agent for increasing biological half-life, or diagnostic or detectable agent.

[0278] In some embodiments, the conjugate is an antibody-drug conjugate (ADC, also called an immunoconjugate) comprising one or more DLL3 VHH domains provided herein conjugated to a therapeutic agent that is either cytotoxic, cytostatic, or has other effects that provide some degree of therapeutic effect. In some embodiments, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope (i.e., a radioconjugate). In some embodiments, the antibody-drug conjugates provided herein allow for targeted delivery of the drug moiety to tumors. In some cases, this can result in the death of targeted tumor cells.

[0279] In some embodiments, provided herein is a DLL3-binding conjugate comprising at least one DLL3 VHH domain provided herein conjugated to a therapeutic agent. In some embodiments, the therapeutic agent includes, for example, daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., Cancer Immunol. Immunother. 21:183-187, 1986). In some embodiments, the therapeutic agent has intracellular activity. In some embodiments, the DLL3-binding conjugate is internalized, and the therapeutic agent is a cytotoxin that blocks cellular protein synthesis, resulting in cell death. In some embodiments, the therapeutic agent is a cytotoxin comprising a polypeptide with ribosome-inactivating activity, including, for example, gelonin, bouganin, saporin, ricin, ricin A chain, bryodin, diphtheria toxin, restrictocin, pseudomonas exotoxin A, and variants thereof. In some embodiments, when the therapeutic agent is a cytotoxin comprising a polypeptide with ribosome-inactivating activity, the DLL3-binding conjugate must be internalized upon binding to the target cell in order for the protein to be cytotoxic to the cell.

[0280] In some embodiments, provided herein is a DLL3-binding conjugate comprising at least one DLL3 VHH domain provided herein conjugated to a toxin. In some embodiments, toxins include, for example, bacterial toxins such as diphtheria toxin, plant toxins such as ricin, geldanamycin (Mandler et al., J. Nat. Cancer Inst. 92(19):1573-1581(2000); Mandler et al., Bioorganic & Med. Chem. Letters 10:1025-1028(2000); Mandler et al., Bioconjugate Chem. 13:786-791(2002)), maytansinoids (EP 1391213; Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623(1996)), and calicheamicin (Lode et al., Cancer Res. 58:2928(1998); Hinman et al., Cancer Res. 53:3336-3342 (1993)). Toxins may exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

[0281] In some embodiments, provided herein is a DLL3-binding conjugate comprising at least one DLL3 VHH domain provided herein conjugated with a label that can directly or indirectly generate a detectable signal. These IgSF conjugates can be used in research or diagnostic applications, for example, in the in vivo detection of cancer. The label is preferably capable of directly or indirectly generating a detectable signal. For example, the label can be a radiopaque substance or a radioisotope, such as 3H, 14C, 32P, 35S, 123I, 125I, 131I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; an enzyme, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase; a contrast agent; or a metal ion. In some embodiments, the label is a radioactive atom, e.g., 99Tc or 123I, for scintigraphic studies, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), e.g., zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. Zirconium-89 may be complexed with various metal chelators and conjugated to antibodies, e.g., for PET imaging (WO 2011 / 056983).

[0282] DLL3-binding conjugates can be prepared using any method known in the art, see, for example, WO 2009 / 067800, WO 2011 / 133886, and U.S. Patent Application Publication No. 2014322129, which are incorporated by reference in their entireties.

[0283] In some embodiments, the binding may be covalent or non-covalent, for example, via biotin-streptavidin non-covalent interaction. In some embodiments, one, two, three, four, or five or more moieties, which may be the same or different, are conjugated, linked, or fused to the DLL3 VHH domain to form a DLL3-binding conjugate. In some embodiments, such moieties can be linked to the VHH domain using various molecular biological or chemical conjugation and linking methods known in the art. In some embodiments, linkers, such as peptide linkers, cleavable linkers, non-cleavable linkers, or linkers that facilitate conjugation reactions, can be used to link or conjugate effector moieties to variant polypeptides or immunomodulatory proteins.

[0284] In some embodiments, the DLL3 VHH domain is conjugated to one or more moieties, e.g., about 1 to about 20 drug moieties per VHH, via a linker (L). In some embodiments, the DLL3-binding conjugate comprises the following components: (VHH domain), (L) q , and (partial) m wherein the VHH domain is any of the described VHH domains capable of specifically binding to DLL3 as described; L is a linker for linking the protein or polypeptide to the moiety; m is at least 1; q is 0 or more; and the resulting DLL3-binding conjugate binds to DLL3. In certain embodiments, m is 1 to 4 and q is 0 to 8.

[0285] The linker can be composed of one or more linker components. For covalent attachment of an antibody and a drug moiety, the linker typically has two reactive functional groups, i.e., bivalent in the sense of reactivity. Bivalent linker reagents useful for linking two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups, are known, and methods for the resulting conjugates are described (Hermanson, GT (1996) Bioconjugate Techniques; Academic Press: New York, p 234-242).

[0286] Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-I carboxylate ("SMCC"), and N-succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB").

[0287] In some embodiments, the linker may comprise an amino acid residue. Exemplary amino acid linker components include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Exemplary dipeptides include valine-citrulline (vc or val-cit) and alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues comprising the amino acid linker component include naturally occurring amino acids as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker components can be designed and optimized for selectivity for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, in plasmin proteases.

[0288] Conjugates of VHH domains and cytotoxic agents can be prepared using a wide variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl substrates), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0289] Antibody-drug conjugates can be prepared by a variety of methods, such as organic chemical reactions, conditions, and reagents known to those skilled in the art. In one embodiment, the method includes: (1) reacting a nucleophilic group of a VHH domain with a bivalent linker reagent to form VHH-L via a covalent bond, followed by reaction with a drug moiety D; and (2) reacting a nucleophilic group of a drug moiety with a bivalent linker reagent to form DL via a non-covalent bond, followed by reaction with a nucleophilic group of a VHH domain.

[0290] Nucleophilic groups on antibodies, including VHH domains, include, but are not limited to, (i) N-terminal amine groups, (ii) side-chain amine groups, e.g., lysine, (iii) side-chain thiol groups, e.g., cysteine, and (iv) sugar hydroxyl or amino groups when the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and can react to form covalent bonds with electrophilic groups on linker moieties and linker reagents, including (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, e.g., haloacetamides; and (iii) aldehyde, ketone, carboxyl, and maleimide groups. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysine with 2-iminothiolane (Traut's reagent), which results in the conversion of the amine to a thiol. Reactive thiol groups may be introduced into an antibody (or fragment thereof) by introducing one, two, three, four, or more cysteine ​​residues (e.g., by preparing a mutant antibody containing one or more non-naturally occurring cysteine ​​amino acid residues).

[0291] Conjugates, e.g., antibody-drug conjugates, may be produced by modifying antibodies (e.g., VHH domains) to introduce electrophilic moieties that can react with nucleophilic substituents on linker reagents or drugs. The sugars of glycosylated antibodies can be oxidized, for example, with periodate oxidation reagents to form aldehyde or ketone groups that can be transferred to amine groups on linker reagents or drug moieties. The resulting imine Schiff base groups can form stable linkages or can be reduced, for example, with borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate moiety of a glycosylated antibody with either galactose oxidase or sodium meta-iodide can result in carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal serine or threonine residues can be reacted with sodium meta-iodide to produce an aldehyde in place of the first amino acid. Such aldehydes can react with a drug moiety or a linker nucleophile.

[0292] Similarly, nucleophilic groups on the drug moiety include, but are not limited to, (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, e.g., haloacetamides; and (iii) amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups that can react to form covalent bonds with electrophilic groups on the linker moieties and linker reagents, including aldehyde, ketone, carboxyl, and maleimide groups.

[0293] Alternatively, a fusion protein containing a VHH domain and a cytotoxic agent can be produced, for example, by recombinant techniques or peptide synthesis. The length of DNA can include regions encoding each of the two parts of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide that does not disrupt the desired properties of the conjugate.

[0294] C. Multisingularity form Provided herein is a multispecific DLL3-binding polypeptide comprising at least one VHH domain that binds to DLL3 and one or more additional binding domains. Typically, the one or more additional domains bind to a second antigen or protein other than DLL3. In some embodiments, the one or more additional domains are antibodies or antigen-binding fragments specific for the second antigen or protein. In some embodiments, the additional domain is a VHH domain.

[0295] In some embodiments, the multispecific DLL3-binding polypeptide comprises at least one VHH domain that binds to DLL3 and at least one additional binding domain that binds to a second antigen or protein. In some embodiments, the second antigen is a tumor-associated antigen (TAA) or a tumor microenvironment-associated antigen (TMEAA). In some embodiments, the second antigen is an immunomodulatory antigen, which is involved in enhancing or attenuating signaling pathways in immune cells.

[0296] In some cases, the multispecific DLL3-binding polypeptide can further comprise an Fc domain, such as any of those described above. In some embodiments, the multispecific DLL3-binding polypeptides provided herein comprise at least one VHH domain that binds to DLL3, at least one additional binding domain that binds to a second antigen or protein, and an Fc domain. In some embodiments, the Fc domain mediates dimerization of the multispecific DLL3-binding polypeptide under physiological conditions such that dimers are formed that double the number of binding sites for DLL3 and the additional antigen or protein.

[0297] Non-limiting exemplary multispecific DLL3-binding polypeptides are described below.

[0298] 1. Bispecific T cell engagers In some embodiments, the DLL3-binding polypeptide is a bispecific construct that is or comprises at least one DLL3 VHH domain provided herein and at least one additional binding molecule that can bind to a surface molecule expressed on T cells. In some embodiments, the surface molecule is an activation component of T cells, such as a component of the T cell receptor complex. In certain aspects, the surface molecule is an activating T cell antigen expressed on T cells, and can induce T cell activation upon interaction with the antigen-binding molecule. For example, in some aspects, the interaction of the antigen-binding molecule with the activating T cell antigen can induce T cell activation by triggering the signal transduction cascade of the T cell receptor complex. Suitable assays for measuring T cell activation are known, including any assay for measuring or evaluating proliferation, differentiation, cytokine secretion, cytotoxic activity, and / or the expression of one or more activation markers. In some embodiments, simultaneous or near-simultaneous binding of such a DLL3-binding polypeptide to both of its targets, DLL3 expressed on a target cell and a T cell molecule (e.g., an activating T cell antigen) expressed on a T cell, can result in a transient interaction between the target cell and the T cell, thereby resulting in activation of the T cell, e.g., cytotoxic activity, and subsequent lysis of the target cell.

[0299] In some embodiments, the T surface molecule, e.g., the activating T cell antigen, is CD3 or CD2. In particular, the provided bispecific DLL3-binding polypeptides can specifically bind to an activating T cell antigen expressed on human T cells, e.g., human CD3 or human CD3. In certain aspects, the additional binding domain specific for an activating T cell antigen (e.g., CD3 or CD2) is an antibody or antigen-binding fragment. In some embodiments, the DLL3-binding polypeptide can be a bispecific antibody T cell engager comprising at least one DLL3 VHH domain that specifically binds to DLL3 and an additional binding molecule that is an antibody or antigen-binding fragment specific for an activation component of T cells (e.g., a T cell surface molecule, e.g., CD3 or CD2).

[0300] Bispecific antibody T cell engagers include tandem scFv molecules fused by a flexible linker (see, e.g., Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011); tandem scFv molecules further comprising an Fc domain composed of a first and a second subunit fused to each other and capable of stable association, e.g., via a flexible linker (WO2013026837); diabodies and derivatives thereof, including tandem diabodies (Holliger et al, Prot Eng 9, 299-305 (1996); Kipriyanov et al, J Mol Biol 293, 41-66 (1999)); dual affinity retargeting (DART) molecules, which may include diabody formats with a C-terminal disulfide bridge; or triomabs, which comprise a whole hybrid mouse / rat IgG molecule (Seimetz et al, Cancer Treat Rev 36, 458-467 (2010)). Similar formats of any of the above molecules can be made using any of the DLL3 VHH domains provided herein.

[0301] In some embodiments, the additional binding domain specific for an activating T cell antigen is an antigen-binding fragment selected from a Fab fragment, a F(ab')2 fragment, an Fv fragment, an scFv, a disulfide-stabilized Fv fragment (dsFv), an scAb, a dAb, a single-domain heavy chain antibody (VHH), or a single-domain light chain antibody. In some embodiments, the additional binding domain is monovalent for binding to an activating T cell antigen, such as CD2 or CD3.

[0302] In some embodiments, the additional binding domain is capable of binding to CD3 or the CD3 complex. The CD3 complex is a complex of at least five membrane-bound polypeptides in mature T lymphocytes that non-covalently associate with each other and with the T cell receptor. The CD3 complex includes the gamma, delta, epsilon, zeta, and eta chains (also referred to as subunits). In some embodiments, the additional binding molecule is an antibody or antigen-binding fragment capable of specifically binding to CD3 or the CD3 complex, also referred to as a CD3 binding domain. In some embodiments, the CD3 binding domain capable of binding to CD3 or the CD3 complex includes one or more copies of an anti-CD3 Fab fragment, an anti-CD3 F(ab')2 fragment, an anti-CD3 Fv fragment, an anti-CD3 scFv, an anti-CD3 dsFv, an anti-CD3 scAb, an anti-CD3 dAb, an anti-CD3 single-domain heavy chain antibody (VHH), and an anti-CD3 single-domain light chain antibody. In some embodiments, the anti-CD3 binding domain is monovalent for binding to CD3.

[0303] In some cases, the CD3 binding domain recognizes the CD3ε chain. In some embodiments, the anti-CD3ε binding domain includes one or more copies of an anti-CD3ε Fab fragment, an anti-CD3ε F(ab')2 fragment, an anti-CD3ε Fv fragment, an anti-CD3ε scFv, an anti-CD3ε dsFv, an anti-CD3ε scAb, an anti-CD3ε dAb, an anti-CD3ε single-domain heavy chain antibody (VHH), and an anti-CD3ε single-domain light chain antibody. In some embodiments, the anti-CD3ε binding domain is monovalent for binding to CD3ε.

[0304] Exemplary monoclonal antibodies against CD3 or the CD3 complex include, but are not limited to, OKT3, SP34, UCHT1 or 64.1, or antigen-binding fragments thereof (see, e.g., June, et al., J. Immunol. 136:3945-3952 (1986); Yang, et al., J. Immunol. 137:1097-1100 (1986); and Hayward, et al., Immunol. 64:87-92 (1988)). In some aspects, clustering of CD3 on T cells, for example, by immobilized or cell-localized or tethered anti-CD3 antibodies, results in T cell activation similar to T cell receptor engagement, but independent of its clonal specificity. In one embodiment, the CD3 binding domain monovalently and specifically binds to the CD3 antigen and is selected from the group consisting of OKT3 (ORTHOCLONE-OKT3™ (muromonab-CD3); humanized OKT3 (U.S. Pat. No. 7,635,475 and published international application WO2005040220); SP34 ((Pessano et al. The EMBO Journal. 4: 337-344, 1985); a humanized variant of SP34 (WO2015001085); Teplizumab™ (MGA031, Eli Lilly); an anti-CD3 binding molecule described in US2011 / 0275787; UCHT1 (Pollard et al. 1987 J Histochem Cytochem. 35(11):1329-38; WO2000041474); NI0401 (WO2007 / 033230); visilizumab (U.S. Patent No. 5,834,597); BC-3 (Anasetti et al., Transplantation 54: 844 (1992)); H2C (described in PCT Publication No. WO2008 / 119567); V9 (described in Rodrigues et al., Int J Cancer Suppl 7, 45-50 (1992) and U.S. Patent No. 6,054,297).Other anti-CD3 antibodies can also be used in the constructs provided herein, including any of those described in International Published PCT Application Nos. WO199404679, WO2008119567, WO2015095392, WO2016204966, WO2019133761; Published Patent Application Nos. US20170369563, US20180194842, US20180355038; U.S. Patent Nos. 7,728,114, 7,381,803, and 7,994,289.

[0305] In some embodiments, the CD3 binding domain comprises a variable heavy (VH) chain set forth in SEQ ID NO: 19 and / or a variable light chain set forth in SEQ ID NO: 20, or a VH and / or VL sequence having at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% identity to these sequences, and specifically binds to CD3. In some embodiments, the CD3 binding domain comprises CDRH1, CDRH2, and CDRH3 of the variable heavy (VH) chain set forth in SEQ ID NO: 19, and CDRL1, CDRL2, and CDRL3 of the variable light chain set forth in SEQ ID NO: 20. In some cases, the CD3 binding region comprises a humanized version of the VH sequence set forth in SEQ ID NO: 19 and a humanized version of the VL sequence set forth in SEQ ID NO: 20. In some embodiments, the CD3 binding region can comprise a VH domain sequence from humanized OKT3 set forth in any one of SEQ ID NOs: 21; 22; 23 and / or a VL domain sequence set forth in any one of SEQ ID NOs: 24, 25, 26, or a VH and / or VL sequence having at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% identity to these sequences, and specifically binds to CD3. In some embodiments, the CD3 binding domain is a Fab, scFv, Fv, or dsFv comprising therein any combination of the above VH and VL sequences, in particular any combination of the VH sequence set forth in any of SEQ ID NOs: 21, 22, 23 and the VL sequence set forth in any of SEQ ID NOs: 24, 25, 26.

[0306] In some embodiments, the anti-CD3ε binding domain comprises: a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO:29); At least the amino acid sequence VH CDR2 sequence containing TIFF2025060787000028.tif4128; At least the amino acid sequence VH CDR3 sequence containing TIFF2025060787000029.tif4128; At least the amino acid sequence VL CDR1 sequence containing TIFF2025060787000030.tif4128; A VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO:33); and VL CDR3 sequence containing at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 34) In some embodiments, the CD3 binding domain comprises: a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO:29); At least the amino acid sequence VH CDR2 sequence containing TIFF2025060787000031.tif4128; At least the amino acid sequence VH CDR3 sequence containing TIFF2025060787000032.tif4128; At least the amino acid sequence VL CDR1 sequence containing TIFF2025060787000033.tif4128; A VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO:33); and VL CDR3 sequence containing at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 34) are included therein, and are Fab, scFv, Fv, or dsFv.

[0307] In some embodiments, the CD3 binding domain comprises a variable heavy (VH) chain set forth in SEQ ID NO:27 and / or a variable light chain set forth in SEQ ID NO:28, or a VH and / or VL sequence having at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% identity to these sequences, and specifically binds to CD3. In some embodiments, the CD3 binding domain comprises CDRH1, CDRH2, and CDRH3 of the variable heavy (VH) chain set forth in SEQ ID NO:27, and CDRL1, CDRL2, and CDRL3 variable light chain set forth in SEQ ID NO:28. In some embodiments, the CD3 binding domain comprises CDRH1, CDRH2, and CDRH3 set forth in SEQ ID NOs:29, 30, and 31, respectively, and CDRL1, CDRL2, and CDRL3 variable light chains set forth in SEQ ID NOs:32, 33, and 34, respectively. In some cases, the CD3 binding region comprises a humanized version of the VH sequence set forth in SEQ ID NO:27 and a humanized version of the VL sequence set forth in SEQ ID NO:28. In some embodiments, the CD3 binding region can comprise a humanized VH domain sequence set forth in any one of SEQ ID NOs:35-65, 453, 454, or 460, and / or a VL domain sequence set forth in any one of SEQ ID NOs:66-84, 368, 451, or 452, or a VH and / or VL sequence having at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% identity thereto, and specifically binds to CD3. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:47 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:75.

[0308] In some embodiments, the CD3 binding domain is a Fab, scFv, Fv, or dsFv comprising therein any combination of the VH and VL sequences described above, particularly any combination of a VH sequence set forth in any of SEQ ID NOs: 35-65, 453, 454, or 460 and a VL sequence set forth in any of SEQ ID NOs: 66-84, 368, 451, or 452. In some embodiments, the anti-CD3 binding domain is a Fab, scFv, Fv, or dsFv comprising therein a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 47 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 75.

[0309] In some embodiments, the CD3 binding domain comprises a variable heavy (VH) chain set forth in SEQ ID NO:519, 520, 523, or 524. In some embodiments, the CD3 binding domain comprises a variable light (VL) chain set forth in SEQ ID NO:521, 522, 525, or 526.

[0310] The provided bispecific constructs can be made in any of several formats that include at least one DLL3 VHH domain and at least one additional domain specific for an activating T cell antigen, such as a CD3 binding domain.

[0311] In one embodiment, the bispecific construct is a bispecific single-domain antibody-linked Fab (S-Fab) comprising at least one DLL3 VHH domain as described, directly or indirectly linked to a Fab antigen-binding fragment specific for a T cell activation antigen, e.g., CD3, e.g., an anti-CD3 Fab. The Fab against the T cell activation antigen, e.g., an anti-CD3 Fab, can comprise any of the VH and VL sequences described. In some embodiments, the DLL3 VHH domain is linked to the C-terminus of the VH or VL chain of the anti-CD3 Fab. In some embodiments, the S-Fab can be further modified, for example, by conjugation with polyethylene glycol (PEG), N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer, protein (e.g., albumin), polyglutamic acid, or PAS (Pan et al. (2018) International Journal of Nanomedicine, 2018:3189-3201).

[0312] In another embodiment, the bispecific construct is an scFv-single domain antibody, in which the construct comprises at least one DLL3 VHH as described therein, directly or indirectly linked to an scFv comprising the VH and VL of an antigen-binding domain specific for a T cell activation antigen, e.g., CD3. The scFv against a T cell activation antigen, e.g., an anti-CD3 scFv, can comprise any of the VH and VL sequences described. In some embodiments, the VHH domain and the scFv are linked by a linker, such as a peptide linker. In some embodiments, the peptide linker can be a peptide linker as described herein. In some embodiments, the VHH domain and the scFv are each linked to an Fc region, e.g., the N-terminus of the Fc region, optionally via a hinge region or linker (e.g., a peptide linker). The Fc region can be any of those described herein, e.g., a human Fc region or variant thereof, e.g., a human IgG1 Fc region or variant thereof. In certain instances, the Fc region is formed by a variant Fc domain, e.g., a variant human IgG1 domain, that has been mutated or modified to promote heterodimerization, allowing different polypeptides to dimerize and form heterodimers.

[0313] In further embodiments, the CD3 binding domain is a single-domain antibody, for example, a VHH domain that specifically binds to CD3. Single-domain antibodies that bind to CD3 and contain a VHH domain are known; see, for example, published U.S. Patent Application No. US20160280795. In some embodiments, the CD3 binding domain is an anti-CD3 VHH set forth in SEQ ID NO: 85, or a sequence that exhibits at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 85 and specifically binds to CD3. In such aspects, the bispecific constructs provided herein may comprise at least one DLL3 VHH domain and at least one CD3 VHH domain. In construct formatting, in some cases, each VHH domain is linked to an Fc region, for example, the N-terminus of the Fc region, optionally via a hinge region or a linker (e.g., a peptide linker). The Fc region can be any described herein, e.g., a human Fc region or a variant thereof, e.g., a human IgG1 Fc region or a variant thereof. In certain instances, the Fc region is formed by a variant Fc domain, e.g., a variant human IgG1 domain, that has been mutated or modified to promote heterodimerization, allowing different polypeptides to dimerize and form heterodimers.

[0314] In the above embodiments, exemplary modifications of the Fc region that promote heterodimerization are known and include any of those listed below, e.g., in Table 3. In some embodiments, one Fc polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs: 103, 107, 115, 117, 440, or 446, and the other Fc polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs: 104, 108, 111, 113, 119, 121, 441, 444, or 448. In some embodiments, one Fc polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs: 105, 109, 116, 118, 442, or 447, and the other Fc polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs: 106, 110, 112, 114, 120, 122, 443, 445, or 449.

[0315] 2. Constrained CD3 Multispecific Constructs In some embodiments, the DLL3-binding polypeptide is a multispecific polypeptide construct that is a constrained T cell-engaging fusion protein. In certain aspects, the constrained multispecific constructs provided herein bind to an activating T cell antigen, such as CD3, and DLL3. The constrained multispecific polypeptide constructs provided herein include at least a first component comprising an immunoglobulin Fc region, a second component comprising one or more copies of at least one binding domain that binds to CD3 (referred to herein as an anti-CD3 binding domain or a CD3 binding domain, which are used interchangeably herein), and a linker, e.g., a polypeptide linker, connecting the first component and the second component. In the provided multispecific polypeptide constructs, one or both of the first and second components comprise at least one DLL3 VHH domain that, when engaged by binding to an antigen, substantially enables the constrained CD3-binding region to bind to CD3. Figures 3A-3E illustrate exemplary formats of constrained multispecific constructs.

[0316] In some embodiments, the constrained multispecific polypeptide constructs provided herein exist in two states with respect to their ability to bind CD3 and subsequently activate T cells: (1) an "inactive" state occurs in the absence of binding of any or all of the antigen binding domains to DLL3, whereby CD3 binding is restricted and T cell interaction is suppressed or reduced; and (2) an "active" state occurs upon antigen binding by any or all of the antigen binding domains, whereby the CD3 binding region can bind CD3 and allow T cell interaction.

[0317] In some embodiments, the Fc region is linked to the CD3-binding domain via one or more linkers. In some embodiments, the Fc region is linked to the CD3-binding region via one or more non-cleavable linkers. In some embodiments, the Fc region is linked to the CD3-binding region via a cleavable linker or one or more other labile linkers. In some embodiments, the cleavable linker is a linker that can be specifically cleaved in the presence of a protease. In some aspects, enhanced CD3 binding occurs after cleavage of the cleavable linker. In some such aspects, the "active" state can be further amplified through multiple mechanisms, including cleavage of the linker connecting the CD3-binding region and the Fc region. In some embodiments, the cleavable linker is a linker that contains a substrate recognition site for a protease. In some embodiments, where the Fc region and the CD3-binding region are linked by a cleavable linker, enhanced CD3 binding can occur after cleavage within the linker.

[0318] Furthermore, in aspects where the Fc region and the CD3-binding region are operably linked by a cleavable linker, cleavage of the linker between the Fc region and the CD3-binding region can separate the constrained multispecific polypeptide construct into a first component and a second component. Depending on the configuration of the constrained multispecific polypeptide construct, the first component and the second component may have different functionalities. In some embodiments, the Fc region is a region that exhibits one or more effector functions, such as ADCC, CDC, or ADCP functions. In such examples, the constrained multispecific polypeptide constructs of the present disclosure can be used to generate self-amplifying systems. For example, in some aspects, incorporation of a protease-cleavable linker between the Fc and CD3-binding domain components allows complete exposure of the CD3-binding domain, thereby enabling amplification of T cell activation potential. Depending on the specific linker included, the amplification step can be mediated by tumor-associated proteases or granzymes released after antigen-dependent T cell activation. When a tumor protease-cleavable linker is included, amplification is mediated by the tumor or tumor microenvironment. In contrast, when a granzyme B-cleavable linker is included, amplification can be self-mediated by T cells after antigen-dependent activation. Furthermore, when an effector-competent Fc is included in the construct, amplification can be mediated by granzymes released from NK cells, which occur through the ADCC mechanism.

[0319] The provided constrained multispecific polypeptide constructs include a configuration in which a first component comprising an Fc region is N-terminal to a second component comprising a CD3-binding region. In such embodiments, the first and second components are linked via a linker that is C-terminal to the end of the Fc region. In some embodiments, at least one DLL3 VHH domain is positioned in the amino-terminal (N-terminal) region of the multispecific polypeptide construct. In some embodiments, at least one DLL3 VHH domain is positioned in the carboxy-terminal (C-terminal) region of the multispecific polypeptide construct. In some embodiments, the constrained multispecific polypeptide construct comprises at least two DLL3 VHH domains positioned in both the N- and C-terminal regions of the multispecific polypeptide construct.

[0320] In some embodiments, the constrained multispecific polypeptide construct is a dimer, formed by covalent or non-covalent interactions between the two polypeptide chains. In some embodiments, the two polypeptide chains are covalently linked to each other, for example, by an interchain disulfide bond. In some embodiments, the Fc region mediates dimerization via an interchain disulfide bond. In certain embodiments, the constrained multispecific polypeptide construct comprises a heterodimeric Fc region, in some cases, where the polypeptide chains of the multispecific polypeptide construct are different (heterodimer). In certain examples of heterodimeric multispecific polypeptide constructs, the CD3-binding region is a two-chain polypeptide comprising a VH and a VL chain, for example, an Fv antibody fragment comprising a VH and a VL. In some embodiments, the Fv antibody fragment comprises a disulfide-stabilized anti-CD3-binding Fv fragment (dsFv).

[0321] In some embodiments, the constrained multispecific polypeptide construct is formed from or comprises two polypeptides, including a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker (e.g., a cleavable or non-cleavable linker), and a VH domain of an anti-CD3 antibody or antigen-binding fragment (e.g., Fv); and a second polypeptide comprising a second Fc polypeptide of a heterodimeric Fc region, a linker (e.g., a cleavable or non-cleavable linker), and a VL domain of an anti-CD3 antibody or antigen-binding fragment (e.g., Fv). In some embodiments, the first polypeptide comprises one or two VHH domains that bind to DLL3. In some embodiments, the second polypeptide comprises one or two VHH domains that bind to DLL3. In some embodiments, the constrained multispecific polypeptide construct comprises at least two DLL3 VHH domains. In some cases, at least one DLL3 VHH domain is located at the N-terminus of the Fc polypeptide and at least one DLL3 VHH domain is located at the C-terminus of a chain of the CD3 binding region.

[0322] In some embodiments, the first polypeptide or the second polypeptide, or both the first and second polypeptides, comprise a costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor. In some embodiments, the CRBR of the first and / or second polypeptide may be located at the N-terminus of the Fc polypeptide and / or the C-terminus of a chain of the CD3 binding region.

[0323] In some embodiments, the constrained multispecific polypeptide construct further comprises at least two VHH domains that bind to DLL3 and at least one costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor. In some embodiments, the constrained multispecific polypeptide construct comprises: (1) a first polypeptide comprising, in order from N-terminus to C-terminus: a first DLL3 VHH domain, a first Fc polypeptide of a heterodimeric Fc region, a linker (e.g., a cleavable linker), a chain (e.g., VH or VL) of an anti-CD3 antibody or antigen-binding fragment (e.g., Fv or dsFv), and a second DLL3 VHH domain; and (2) a second polypeptide comprising, in order from N-terminus to C-terminus: a second Fc polypeptide of the heterodimeric Fc region, the same linker (e.g., the same cleavable linker), the other chain (another VH or VL) of the anti-CD3 antibody or antigen-binding fragment, and a costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor.

[0324] In some embodiments, the first polypeptide or the second polypeptide, or both the first and second polypeptides, further comprise an inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor. In some embodiments, the IRBR of the first polypeptide and / or the second polypeptide may be located at the N-terminus of the Fc polypeptide and / or the C-terminus of the chain of the CD3 binding region.

[0325] In some embodiments, the constrained multispecific polypeptide construct comprises at least two VHH domains that bind to DLL3 and at least one inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor. In some embodiments, the constrained multispecific polypeptide construct comprises: (1) a first polypeptide comprising, in order from N-terminus to C-terminus: a first DLL3 VHH domain, a first Fc polypeptide of a heterodimeric Fc region, a linker (e.g., a cleavable or non-cleavable linker), a chain (e.g., VH or VL) of an anti-CD3 antibody or antigen-binding fragment (e.g., Fv or dsFv), and a second DLL3 VHH domain; and (2) a second polypeptide comprising, in order from N-terminus to C-terminus: a second Fc polypeptide of the heterodimeric Fc region, the same linker (e.g., the same cleavable linker), the other chain (another VH or VL) of the anti-CD3 antibody or antigen-binding fragment, and an inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor.

[0326] In some embodiments, at least one of the first or second polypeptides further comprises a costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor, and at least one of the first or second polypeptides further comprises an inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor. In some embodiments, the CRBR of the first and / or second polypeptide may be located at the N-terminus of the Fc polypeptide and / or the C-terminus of the chain of the CD3 binding region. In some embodiments, the IRBR of the first and / or second polypeptide may be located at the N-terminus of the Fc polypeptide and / or the C-terminus of the chain of the CD3 binding region.

[0327] In some embodiments, the constrained multispecific polypeptide construct comprises at least two VHH domains that bind to DLL3, a costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor, and an inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor. In some embodiments, the constrained multispecific polypeptide construct comprises: (1) a first polypeptide comprising, in order from N-terminus to C-terminus: a first DLL3 VHH domain, a first Fc polypeptide of a heterodimeric Fc region, a linker (e.g., a cleavable or non-cleavable linker), a chain (e.g., VH or VL) of an anti-CD3 antibody or antigen-binding fragment (e.g., Fv or dsFv), and a second DLL3 VHH domain; and (2) a second polypeptide comprising, in order from N-terminus to C-terminus: one of IRBR or CRBR, a second Fc polypeptide of the heterodimeric Fc region, the same linker (e.g., the same cleavable or non-cleavable linker), the other chain (another VH or VL) of the anti-CD3 antibody or antigen-binding fragment, and the other of IRBR or CRBR.

[0328] Each of the components of the multispecific polypeptide constructs of the present disclosure is described in more detail below.

[0329] a.DLL3 VHH antigen-binding domain The constrained multispecific polypeptide constructs of the present disclosure comprise at least one DLL3 VHH domain of any of those provided herein. In some embodiments, the DLL3 VHH domain comprises the sequence of amino acids set forth in any of SEQ ID NOs: 244-318. In some embodiments, the DLL3 VHH domain comprises the sequence of amino acids set forth in any of SEQ ID NOs: 244-318, 401-409, 416, or 455. In some embodiments, the DLL3 VHH domain comprises the sequence of amino acids set forth in any of SEQ ID NOs: 102, 244-318, 401-409, 416, 455, and 476-480-488, 507-518.

[0330] In specific embodiments, the constrained multispecific polypeptide construct contains at least two DLL3 domains, in some cases at least one DLL3 VHH domain positioned amino-terminal to the Fc polypeptide of the heterodimeric Fc, and at least one DLL3 VHH domain positioned carboxy-terminal to the VH or VL chain of the CD3 binding region.

[0331] In aspects of constrained multispecific polypeptide constructs containing at least two DLL3 VHH domains or containing two DLL3 VHH domains, each of the DLL3 VHH domains may bind to the same or overlapping epitopes on DLL3.

[0332] In aspects of constrained multispecific polypeptide constructs containing at least two DLL3 VHH domains or containing two DLL3 VHH domains, each of the DLL3 VHH domains may bind to a distinct or non-overlapping epitope on DLL3.

[0333] In some embodiments, the first and second DLL3 VHH domains bind to distinct or non-overlapping epitopes of DLL3 and / or do not compete for binding to DLL3.

[0334] In some cases, the first sdAb VHH domain comprises an amino acid sequence set forth in any of 264, 287, 299, 306, or 318, a humanized variant thereof, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to 264, 287, 299, 306, or 318, and binds to DLL3; The VHH domain comprises an amino acid sequence set forth in any of 244, 258, 275, 280, 314, 316, or 317, a humanized variant thereof, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of 244, 258, 275, 280, 314, 316, or 317, and binds to DLL3.

[0335] In some cases, the first VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs: 251, 264, 267, 268, 287, 299, 306, 314, 318, 455, 507, 517, a humanized variant thereof, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 251, 264, 267, 268, 287, 299, 306, 314, 318, 455, 507, 517, and binds DLL3; and the second VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs: 251, 264, 267, 268, 287, 299, 306, 314, 318, 455, 507, 517, a humanized variant thereof, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 251, 264, 267, 268, 287, 299, 306, 314, 318, 455, 507, 517, and binds DLL3; or a humanized variant thereof, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 244, 251, 258, 267, 275, 280, 314, 315, 316, 317, 318, 455, 515, 516, 517, 518, and binds to DLL3.

[0336] In some cases, the first sdAb VHH domain comprises the amino acid sequence set forth in SEQ ID NO:264, or a humanized variant thereof set forth in any of SEQ ID NOs:265-455, SEQ ID NO:287, or a humanized variant thereof set forth in any of SEQ ID NOs:288-298, SEQ ID NO:299, or a humanized variant thereof set forth in any of SEQ ID NOs:300-305, SEQ ID NO:306, or a humanized variant thereof set forth in any of SEQ ID NOs:307-313, or SEQ ID NO:318; and the second sdAb VHH domain comprises the amino acid sequence set forth in SEQ ID NO:244, or a humanized variant thereof set forth in any of SEQ ID NOs:245-257, SEQ ID NO:258, or a humanized variant thereof set forth in any of SEQ ID NOs:259-263, SEQ ID NO:318. SEQ ID NO:275, or a humanized variant thereof as set forth in any of SEQ ID NOs:276-279, SEQ ID NO:280, or a humanized variant thereof as set forth in any of SEQ ID NOs:281-286, SEQ ID NO:314, SEQ ID NO:316, or SEQ ID NO:317.

[0337] In some cases, the first VHH domain comprises the amino acid sequence set forth in SEQ ID NO:264, or a humanized variant thereof set forth in any of SEQ ID NOs:265-274, 416, 455, or 476-478, SEQ ID NO:287, or a humanized variant thereof set forth in any of SEQ ID NOs:102, 288-298, SEQ ID NO:299, or a humanized variant thereof set forth in any of SEQ ID NOs:300-305, or 480, SEQ ID NO:306, or a humanized variant thereof set forth in any of SEQ ID NOs:307-313, SEQ ID NO:507, or a humanized variant thereof set forth in any of SEQ ID NOs:508-514, SEQ ID NO:318, or SEQ ID NO:517; The amino acid sequence of SEQ ID NO:244, or a humanized variant thereof as set forth in any of SEQ ID NOs:245-257, SEQ ID NO:258, or a humanized variant thereof as set forth in any of SEQ ID NOs:259-263, SEQ ID NO:275, or a humanized variant thereof as set forth in any of SEQ ID NOs:276-279 or 479, SEQ ID NO:280, or a humanized variant thereof as set forth in any of SEQ ID NOs:281-286, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:515, SEQ ID NO:516, or SEQ ID NO:317.

[0338] In some embodiments, the first sdAb VHH domain and the second sdAb VHH domain are selected from the group consisting of SEQ ID NO:244 and SEQ ID NO:264; SEQ ID NO:314 and SEQ ID NO:318; SEQ ID NO:244 and SEQ ID NO:306; SEQ ID NO:314 and SEQ ID NO:306; SEQ ID NO:314 and SEQ ID NO:299; SEQ ID NO:251 and SEQ ID NO:268; SEQ ID NO:251 and SEQ ID NO:267; SEQ ID NO:275 and SEQ ID NO:318; SEQ ID NO:314 and SEQ ID NO:287; SEQ ID NO:314 and SEQ ID NO:264; SEQ ID NO:316 and SEQ ID NO:318; SEQ ID NO:317 and SEQ ID NO: NO:318; or an amino acid sequence selected from SEQ ID NO:244 and SEQ ID NO:318. In some embodiments, the first sdAb VHH domain and the second sdAb VHH domain comprise the amino acid sequences set forth in SEQ ID NO:315 and SEQ ID NO:318. In some embodiments, the first sdAb VHH domain and the second sdAb VHH domain comprise the amino acid sequences set forth in SEQ ID NO:251 and SEQ ID NO:455.

[0339] In some embodiments, the first VHH domain and the second VHH domain are selected from the group consisting of SEQ ID NO:244 and SEQ ID NO:264; SEQ ID NO:314 and SEQ ID NO:318; SEQ ID NO:314 and SEQ ID NO:517; SEQ ID NO:244 and SEQ ID NO:306; SEQ ID NO:244 and SEQ ID NO:507; SEQ ID NO:314 and SEQ ID NO:306; SEQ ID NO:314 and SEQ ID NO:507; SEQ ID NO:314 and SEQ ID NO:299; SEQ ID NO:251 and SEQ ID NO:268; SEQ ID NO:251 and SEQ ID NO:267; SEQ ID NO:275 and SEQ ID NO:318; 275 and SEQ ID NO:517; SEQ ID NO:314 and SEQ ID NO: SEQ ID NO:287; SEQ ID NO:314 and SEQ ID NO:264; SEQ ID NO:314 and SEQ ID NO:314; SEQ ID NO:315 and SEQ ID NO:264; SEQ ID NO:518 and SEQ ID NO:264; SEQ ID NO:316 and SEQ ID NO:318; SEQ ID NO:515 and SEQ ID NO:517; SEQ ID NO:318 and SEQ ID NO:318; SEQ ID NO:517 and SEQ ID NO:517; SEQ ID NO:317 and SEQ ID NO:318; SEQ ID NO:516 and SEQ ID NO:517; SEQ ID NO:251 and SEQ ID NO:455; SEQ ID NO:244 and SEQ ID NO:517; or SEQ ID NO:244 and SEQ ID NO:318. In some embodiments, the first VHH domain and the second VHH domain comprise the amino acid sequences set forth in SEQ ID NO: 315 and SEQ ID NO: 318. In some embodiments, the first VHH domain and the second VHH domain comprise the amino acid sequences set forth in SEQ ID NO: 518 and SEQ ID NO: 517.In some embodiments, the first VHH domain and the second VHH domain comprise the amino acid sequences shown in SEQ ID NO:251 and SEQ ID NO:455.

[0340] In some embodiments, the constrained multispecific polypeptide construct contains at least one DLL3 VHH domain, such as any of those provided herein, and at least one additional antigen-binding domain specific for another tumor-associated antigen (TAA). In some embodiments, the at least one additional antigen-binding domain comprises one or more copies of an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a Fv fragment, a scFv, a scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. In specific embodiments, the additional TAA antigen-binding domain is a single-chain antibody. In some examples, the single chain is an scFv, a scAb, a single-domain heavy chain antibody, or a single-domain light chain antibody. For example, in some cases, the additional TAA antigen-binding domain comprises one or more single-domain antibody (sdAb) fragments, such as a VHH fragment. H H, V NAR , manipulated V H Domain, or manipulated V K Includes domain. V H H can be generated from natural camelid heavy chain-only antibodies, from genetically modified rodents producing heavy chain-only antibodies, or from naive / synthetic camelid or humanized camelid single domain antibody libraries. NAR can be generated from cartilaginous fish heavy chain-only antibodies. Various methods, including interface engineering and specific germline family selection, have been used to generate conventional heterodimeric V H Domains and V K This has been done to generate monomeric sdAbs from the domains.

[0341] In some embodiments, the additional TAAs include 1-92-LFA-3, 5T3, alpha4 integrin, alphaV integrin, alpha4beta1 integrin, alpha4beta7 integrin, AGR2, anti-Lewis Y, apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9 (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD1 9, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD 56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132 (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), claudin 3, claudin 4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL4, DPP-4, DSG1, EDA, EDB, EG FR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE , IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL 23, IL23R, IL27 / IL27R (wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin 16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch1, Notch2, Notch3, Notch4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine-1-phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIG IT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

[0342] In some embodiments, the antigen-binding domain, such as a DLL3 VHH domain, is linked to the Fc region and / or CD3 binding region directly or indirectly via a linker. In some embodiments, the linkage is via a linker. In some embodiments, the linker is a linking peptide (LP), which may include a flexible or non-flexible linker as described. In some embodiments, the linker is TIFF2025060787000034.tif17161. In some embodiments, the linker is selected from the group consisting of, by way of non-limiting example, In some embodiments, the linker is a flexible linker comprising a glycine residue, such as TIFF2025060787000035.tif10128. In some embodiments, the linker is (GGGGS)n (n is 1 to 5) (SEQ ID NO:123); (GGGGGS)n (n is 1 to 4) (SEQ ID NO:124); TIFF2025060787000036.tif16151. In some embodiments, the linker comprises a combination of a GS linker and a glycine linker.

[0343] b.Fc region The constrained multispecific polypeptide construct comprises an immunoglobulin Fc region. Generally, the constrained multispecific polypeptide construct is a dimer formed by polypeptides each comprising an Fc. The Fc polypeptide can be any of those described above. In certain embodiments, the Fc region is formed by an Fc domain that has been mutated or modified to promote heterodimerization, allowing different polypeptides to dimerize to obtain a heterodimer. Thus, in some embodiments, the dimer is a heterodimer in which the two polypeptide chains of the multispecific polypeptide construct are different.

[0344] Various methods for promoting heterodimerization of complementary Fc polypeptides are known, e.g., Ridgway et al., Protein Eng. 9:617-621 (1996); Merchant et al., Nat. Biotechnol. 16(7): 677-81 (1998); Moore et al. (2011) MAbs, 3:546-57; Von Kreudenstein et al. MAbs, (2013) 5:646-54; Gunasekaran et al. (2010) J. Biol. Chem., 285:19637-46; Leaver-Fay et al. (2016) Structure, 24:641-51; Ha et al. (2016) Frontiers in Immunology, 7:1; Davis et al. (2010) Protein Eng Des Sel, 23:195-202; Published International PCT Application Nos. WO 1998 / 050431, WO 2009 / 089004, WO2011143545, WO 2014 / 067011, WO See, for example, US Patent Nos. 2012 / 058768, WO2018027025; Published U.S. Patent Application Nos. US20140363426, US20150307628, US20180016354, US20150239991; and U.S. Patent Nos. US5731168, US7183076, US9701759, US9605084, and US9650446. Methods for promoting heterodimerization of the Fc chain include mutagenesis of the Fc region, for example, by including a set of "knob-into-hole" mutations or by including mutations that result in electrostatic steering of the Fc in favor of attractive interactions between different polypeptide chains.For example, in some embodiments, heterodimeric Fc polypeptides contain mutations that alter the charge polarity at the Fc dimer interface, such that co-expression of electrostatically matched Fc chains favors favorable attractive interactions, thereby promoting desirable Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress undesired Fc homodimer formation (Guneskaran et al. (2010) JBC, 285: 19637-19646). When co-expressed in cells, inter-chain association is permitted, but the chains do not substantially self-associate due to charge repulsion. Another strategy for generating heterodimeric Fc involves mixing human IgG and IgA CH3 domain segments to generate complementary CH3 heterodimers, termed SEED Fc.

[0345] Methods and variants for heterodimerization also include those described in Published International PCT Application No. WO2014 / 145806, including "knob and hole" mutations (also called "skew" variants), mutations associated with "electrostatic steering" or "charge pairing," and pI variants. Heterodimer variants also include any described in U.S. Published Application Nos. US2012 / 0149876 or US2018 / 011883.

[0346] In some embodiments, both polypeptides of the Fc heterodimer contain paired or complementary amino acid modifications to promote heterodimerization. Exemplary paired amino acid modifications of polypeptides of Fc fusions are listed in Table 3.

[0347] Table 3: Amino acids that form heterodimeric Fc pairs TIFF2025060787000037.tif43133

[0348] In some embodiments, the modification comprises introducing a protuberance (knob) into the first Fc polypeptide and a cavity (hole) into the second Fc polypeptide, such that the protuberance is positionable within the cavity to facilitate complex formation of the first and second Fc-containing polypeptides. The amino acids targeted for substitution and / or modification to create the protuberance and cavity in the polypeptide are typically interface amino acids that interact or contact one or more amino acids in the interface of the second polypeptide.

[0349] In some embodiments, the first Fc polypeptide modified to contain a protrusion (hole) amino acid comprises a substitution of a native or original amino acid with an amino acid having at least one side chain that protrudes from the interface of the first Fc polypeptide and can therefore locate in a compensatory cavity (hole) in the adjacent interface of the second polypeptide. In most cases, the substituted amino acid has a larger side chain volume than the original amino acid residue. Those skilled in the art will understand how to determine and / or evaluate the properties of amino acid residues to identify amino acid residues that are ideal replacement amino acids for creating protrusions. In some embodiments, the replacement residues for forming protrusions are naturally occurring amino acid residues, such as arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). In some examples, the original residues identified for substitution are amino acid residues with small side chains, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0350] In some embodiments, the second Fc polypeptide to be modified to contain a cavity (hole) comprises a substitution of a native or original amino acid with an amino acid having at least one side chain that recesses the second polypeptide interface and can accommodate a corresponding protrusion from the first polypeptide interface. In most cases, the substituted amino acid has a smaller side chain volume than the original amino acid residue. Those skilled in the art will understand how to determine and / or evaluate the properties of amino acid residues to identify amino acid residues that are ideal replacement residues for forming a cavity. Generally, replacement residues for forming a cavity are naturally occurring amino acids, including, for example, alanine (A), serine (S), threonine (T), and valine (V). In some examples, the original amino acid identified for substitution is an amino acid with a large side chain, such as tyrosine, arginine, phenylalanine, or tryptophan.

[0351] The CH3 interface of human IgG1, for example, comprises 16 residues on each domain located in four antiparallel β-strands that bury 1090 Å from each surface (see, e.g., Deisenhofer et al. (1981) Biochemistry, 20:2361-2370; Miller et al., (1990) J Mol. Biol., 216, 965-973; Ridgway et al., (1996) Prot. Engin., 9: 617-621; U.S. Pat. No. 5,731,168). The modification of CH3 domain to create protrusion or cavity is described in, for example, U.S. Patent No. 5,731,168; International Patent Application No. WO98 / 50431 and WO2005 / 063816; and Ridgway et al., (1996) Prot.Engin., 9: 617-621.In some cases, the modification of CH3 domain to create protrusion and cavity typically targets the residue located in the two central antiparallel β-strands.The purpose is to minimize the risk that the protrusion created can be accommodated by protruding into the surrounding solvent rather than being accommodated by the compensatory cavity in the partner CH3 domain.

[0352] For example, in some embodiments, a heterodimeric Fc comprises a polypeptide having an amino acid modification in the CH3 domain at Thr366, which, when substituted with a bulkier amino acid, e.g., Try (T366W), can preferentially pair with a second CH3 domain having less bulky amino acids at Thr366, Leu368, and Tyr407, e.g., Ser, Ala, and Val (T366S / L368A / Y407V). Heterodimerization due to CH3 modifications can be further stabilized by introducing disulfide bonds, e.g., by changing Ser354 to Cys (S354C) and Tyr349 to Cys (Y349C) on the opposing CH3 domain (Reviewed in Carter, 2001 Journal of Immunological Methods, 248: 7-15).

[0353] In certain embodiments, the multispecific polypeptide construct comprises a first Fc and a second Fc capable of mediating Fc heterodimerization, comprising a first Fc polypeptide comprising the mutations T366W and S354C and a second Fc polypeptide comprising the mutations T366S, L368A, Y407V, and Y349C. In some embodiments, the first Fc polypeptide is selected from an Fc polypeptide comprising the sequence set forth in SEQ ID NO:440 or 446, and the second Fc polypeptide is selected from an Fc polypeptide comprising the sequence set forth in SEQ ID NO:441, 444, or 448. In some embodiments, the first Fc polypeptide is or comprises the sequence of amino acids set forth in any of SEQ ID NO:103, 107, 115, or 117, and the second Fc polypeptide is or comprises the sequence of amino acids set forth in any of SEQ ID NO:104, 108, 111, 113, 119, or 121.

[0354] In some embodiments, the Fc polypeptides exhibit characteristics that result in Fc-mediated effector function. In particular examples, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:440, and the second Fc polypeptide is or comprises SEQ ID NO:441 or 444. In some embodiments, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:103, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:104 or 111. In some embodiments, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:107, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:108 or 113. The first and second Fc polypeptides may be formatted into either polypeptide chain of the construct.

[0355] In some embodiments, one or both of the first and second Fc polypeptides can further comprise one or more amino acid mutations to further reduce one or more Fc effector functions (e.g., reduced Fc receptor binding). Exemplary mutations for reducing Fc effector function include any of those described. In some embodiments, the modification can be a deletion of one or more of positions Glu233 (E233), Leu234 (L234), or Leu235 (L235), e.g., a deletion of amino acids Glu233 (E233), Leu234 (L234), and Leu235 (L235). In some embodiments, the first Fc polypeptide is selected from an Fc polypeptide comprising the sequence set forth in SEQ ID NO:442 or 447, and the second Fc polypeptide is selected from an Fc polypeptide comprising the sequence set forth in SEQ ID NO:443, 445, or 449. In some embodiments, the first Fc polypeptide is or comprises the sequence of amino acids set forth in any of SEQ ID NOs: 105, 109, 116, or 118, and the second Fc polypeptide is or comprises the sequence of amino acids set forth in any of SEQ ID NOs: 106, 110, 112, 114, 120, or 122.

[0356] In a particular example, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:442, and the second Fc polypeptide is or comprises SEQ ID NO:443 or 445. In some embodiments, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:105, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:106 or 112. In some embodiments, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:109, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:110 or 114. The first and second Fc polypeptides may be formatted into either polypeptide chain of the construct.

[0357] In some embodiments, the first Fc polypeptide or the second Fc polypeptide further comprises the mutation M252Y and / or M428V. In a particular example, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:446, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:448. In some embodiments, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:115, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:119. In some embodiments, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:117, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:121. In other examples, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:447, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:449. In some embodiments, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO: 116, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO: 120. In some embodiments, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO: 118, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO: 122. The first and second Fc polypeptides may be formatted into either polypeptide chain of the construct.

[0358] Further examples of variants that can help promote heterodimerization include: S364K / E357Q and L368D / K370S; L368D / K370S and S364K; L368E / K370S and S364K; T411T / E360E / Q362E and D401K; L368D / K370S and S364K / E357L, K370S and S364K / E357Q, and T and any combination or pair of conformational variants (e.g., asymmetric variants) of a first and a second Fc polypeptide from among the following: 366S / L368A / Y407V and T366W, or 366S / L368A / Y407V / Y349C and T366W / S354C, where each pair represents a mutation in the first and second Fc polypeptide. In certain embodiments, the provided constructs comprise first and second Fc polypeptides comprising the pair of mutations L368D / K370S and S364K and E357Q.

[0359] Another mechanism that can be used to generate heterodimers is sometimes referred to as "electrostatic steering," as described in Gunasekaran et al., J. Biol. Chem. 285(25):19637 (2010). This is also sometimes referred to herein as "charge pairing." In this embodiment, electrostatics are used to skew formation toward heterodimerization. As one skilled in the art will appreciate, these may also have an effect on pI and therefore purification, and thus, in some cases, may also be considered pI variants. However, because they were generated to force heterodimerization and not used as a purification tool, they are classified as "stereovariants." In one embodiment, the first Fc polypeptide may contain the mutations D221E / P228E / L368E, and the second Fc polypeptide may contain the mutations D221R / P228R / K409R. In another embodiment, the first Fc polypeptide may comprise the mutations C220E / P228E / 368E and the second Fc polypeptide may comprise the mutations C220R / E224R / P228R / K409R.

[0360] In some embodiments, heterodimerization can be promoted by pI variants. In some aspects, pI variants can include those that increase the pI of the protein (basic changes). In other aspects, pI variants can include those that decrease the pI of the protein (acidic changes). In some cases, all combinations of these variants can be performed, including combinations in which one Fc polypeptide can be wild-type or a variant that does not exhibit a pI significantly different from the wild-type, and the other Fc polypeptide can be either more basic or more acidic. Alternatively, each Fc polypeptide can be altered, one more basic and one more acidic. In some embodiments, at least one Fc polypeptide is a negative pI variant Fc containing the mutations Q295E / N384D / Q418E / N421D.

[0361] In some embodiments, a combination of stereo-heterodimerization variants (eg, knobs and holes) and pI or charge pair variants can be used.

[0362] In certain embodiments, the provided construct comprises (a) a first Fc polypeptide comprising the asymmetric variant S364K / E357Q; and (b) a second Fc polypeptide comprising the asymmetric variant L368D / K370S and pI variants N208D / Q295E / N384D / Q418E / N421D. In some embodiments, one or both of the first and second polypeptides can further comprise mutations that reduce Fc effector activity, such as the exemplary mutations E233P / L234V / L235A / G236del / S267K. Examples of such first and second Fc polypeptides capable of mediating Fc heterodimerization include the sequences set forth in SEQ ID NOs:472 and 473. The first and second Fc polypeptides can be formatted into either polypeptide chain of the construct.

[0363] The resulting constrained multispecific polypeptide construct can be purified by any suitable method, such as affinity chromatography on a protein A or protein G column. When two nucleic acid molecules encoding different polypeptides are transformed into cells, homo- and heterodimers are formed. Expression conditions can be adjusted to favor heterodimer formation over homodimer formation.

[0364] Techniques for recovering heterodimers from homodimers based on the differential affinities of the heterodimers for affinity reagents are known. In some aspects, such techniques involve designing a heterodimer such that one of the Fc polypeptide chains does not bind to the affinity reagent Protein A. In some cases, one of the polypeptide chains may contain one or more amino acid substitutions that suppress or reduce affinity for the Protein A reagent in one of the polypeptides of the Fc heterodimer, see, e.g., WO2017134440, WO2010151792, Jendeberg et al. (Jendeberg et al., (1997) J. Immunol. Methods, 201(1): 25-34. In some of these embodiments, the Fc region may be modified at the Protein A binding site on one member of the heterodimer to prevent Protein A binding, thereby allowing for more efficient purification of the heterodimeric fusion protein. An exemplary modification within this binding site is Ile253, e.g., Ile253Arg (I253R). In some embodiments, the modification may be H435R or H435R / Y436F. In some embodiments, the Fc polypeptide of the Fc heterodimer may contain a modification such that it is capable of binding to Protein A but not Protein G (pA+ / pG-). An exemplary pA+ / pG- amino acid modification comprises an Fc containing serine at position 428, serine at position 434, and optionally a histidine at position 436 relative to human IgG1, or these residues at the corresponding positions in human IgG2, 3, or 4. In some aspects, one IgG Such amino acid modifications at positions 428, 434, and optionally 436 in the Fc polypeptide reduce or prevent Protein G binding and enhance protein purification.

[0365] In some embodiments, any of these modifications that confer different affinities to affinity reagents can be combined with one or more of the other amino acid modifications described above. For example, the I253R modification can be combined with either the T366S / L368A / Y407V modification or the T366W modification. The T366S / L368A / Y407V modified Fc, like the T336W modified Fc, is capable of forming homodimers due to the absence of a sterically closed dimerization interface. Thus, in some embodiments, the I253R modification is combined with the T366S / L368A / Y407V modified Fc, which precludes the purification of any homodimeric Fc that may have formed. A similar modification can be utilized by combining T366S / L368A / Y407V and H453R.

[0366] In some embodiments, the Fc region of the heterodimeric molecule may additionally comprise one or more other Fc mutations, such as any described above. In some embodiments, the heterodimeric molecule comprises an Fc region with a mutation that reduces effector function. In some embodiments, the Fc region is altered to result in reduced Fc-mediated effector function, e.g., through reduced Fc receptor binding, e.g., through binding to FcγR but generally not FcRn.

[0367] In some embodiments, the Fc region is mutated at one or more of the following positions to reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (L235). The one or more mutations may include E233P, L234V, and / or L235A.

[0368] In certain embodiments, mutations in the Fc region to reduce Fc effector function, e.g., via reduced Fc receptor binding to FcγR, include G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K ... Contains mutations from any of the following: 236del / S267K / A327G or E233P / L234V / L235A / G236del, D265A / P329A, D265A / P329G, D265A / N297A, L234V / L235A / D265A, L234V / L235A / N297A, L234V / L235A / P329A, or L234V / L235A / P329G.

[0369] In some embodiments, one Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NO:440 (e.g., SEQ ID NO:103 or 107), 446 (e.g., SEQ ID NO:115 or 117), and the other Fc polypeptide of the heterodimeric Fc contains the amino acid sequence set forth in any of SEQ ID NO:441 (e.g., SEQ ID NO:104 or 108), 444 (e.g., SEQ ID NO:111 or 113), 448 (e.g., SEQ ID NO:119 or 121). In some embodiments, one Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NO:442 (SEQ ID NO:105 or 109), 447 (e.g., SEQ ID NO:116 or 118), and the other Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NO:443 (e.g., SEQ ID NO:106 or 110), 445 (e.g., SEQ ID NO:112 or 114), 449 (e.g., SEQ ID NO:120 or 122).

[0370] In some embodiments, the Fc region of the provided multispecific polypeptide construct exhibits one or more effector functions. In some cases, the Fc region can mediate Fc-mediated effector functions, such as ADCC (e.g., release of granzyme B by NK cells), ADCP, and / or CDC. Generally, the Fc region is responsible for effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in addition to antigen binding, which is the primary function of immunoglobulins. In addition, the FcRn sequence present in the Fc region plays a role in regulating serum IgG levels by increasing in vivo half-life through conjugation to the in vivo FcRn receptor. In some embodiments in which the multispecific polypeptide construct comprises a cleavable linker, cleavage of the linker can generate two components, each with biological activity: a CD3-binding region capable of binding to and engaging CD3 on T cells, which in some aspects may also comprise a CRBR for inducing a costimulatory signal on T cells and / or an IRBR for inducing an inhibitory signal on T cells; and an Fc region linked to a DLL3 VHH domain that can exhibit target-specific effector function. In certain embodiments provided herein, the multispecific polypeptide construct comprises a non-cleavable linker and, in some aspects, may not exhibit independent Fc-mediated effector function.

[0371] In some embodiments, the Fc region comprises an Fc polypeptide that is mutated or modified to alter one or more effector functions. Thus, in some cases, one or more of the effector functions, e.g., ADCC, ADCP, and / or CDC, may be altered, e.g., reduced or enhanced, in an Fc for use with a provided constrained multispecific polypeptide construct. Exemplary mutations that reduce effector function include any of those described above.

[0372] In some embodiments, an IgG1 Fc polypeptide or variant thereof, such as any described below, can be made with the G1 m1 or G1 m3 allotype. In some embodiments, the Fc region can include amino acids of the human G1 m1 allotype, such as residues containing Asp (D) and Leu (L) at positions 356 and 358 as set forth in SEQ ID NO:8. In some cases, the Fc polypeptide can include amino acid substitutions E356D and M358L to reconstitute residues of the G1 m1 allotype. In other embodiments, the Fc region can include amino acids of the human G1 m3 allotype, such as residues Glu (E) and Met (M) at positions 356 and 358 (EU numbering) as set forth in SEQ ID NOs:472 and 473. In some cases, the Fc polypeptide can include amino acid substitutions D356E and L358M to reconstitute residues of the G1 m3 allotype.

[0373] c CD3 binding domain The constrained multispecific polypeptide construct comprises one or more copies of an anti-CD3 binding domain. The anti-CD3 binding domain of the present disclosure activates T cells through engagement of CD3 or a member of the CD3 complex on T cells. In a preferred embodiment, the anti-CD3 binding domain of the present disclosure specifically binds to the epsilon chain of CD3, also known as CD3ε. The anti-CD3ε binding domain of the present disclosure activates T cells through engagement of CD3ε on T cells. The anti-CD3 binding domain of the present disclosure agonizes, stimulates, activates, and / or otherwise enhances CD3-mediated T cell activation. The biological activity of CD3 includes, for example, T cell activation and other signaling through the interaction between CD3 and the antigen-binding subunit of the T cell receptor (TCR). For example, the anti-CD3 binding domain of the present disclosure activates T cells fully or partially through engagement of CD3ε on T cells by partially or fully modulating, e.g., agonizing, stimulating, activating, or otherwise enhancing, CD3-mediated T cell activation.

[0374] The CD3-binding domain can be any of those described above. In certain embodiments, the CD3-binding domain is an Fv antibody fragment that binds to CD3ε (herein referred to as an anti-CD3ε Fv fragment). In some embodiments, the anti-CD3ε Fv antibody fragment is a disulfide-stabilized anti-CD3-binding Fv fragment (dsFv). In some embodiments, the anti-CD3-binding domain is monovalent for binding to CD3.

[0375] In some embodiments, the CD3 binding region is an Fv antibody fragment, such as any described, comprising a variable heavy chain (Hv, also referred to as VH) and a variable light chain (Lv, also referred to as VL). In aspects of such embodiments, the immunoglobulin Fc region is a heterodimeric Fc region, such as any described, comprising two different Fc polypeptides that allow heterodimeric association between both polypeptides of the Fc heterodimer. In such embodiments, the variable heavy chain (VH) and variable light chain (VL) of the CD3 binding region are linked to opposing chains of the heterodimeric Fc.

[0376] In some embodiments, the CD3 binding region is an Fv or dsFv of SP34 (Pessano et al. The EMBO Journal. 4: 337-344, 1985) or a humanized variant of SP34 (WO2015001085).

[0377] In some embodiments, the anti-CD3ε binding domain is an Fv, e.g., a dsFv fragment, that comprises a combination of a heavy chain variable amino acid sequence and a light chain variable amino acid sequence. a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO:29); At least the amino acid sequence VH CDR2 sequence containing TIFF2025060787000038.tif4128; At least the amino acid sequence VH CDR3 sequence containing TIFF2025060787000039.tif4128; At least the amino acid sequence VL CDR1 sequence containing TIFF2025060787000040.tif4128; a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO:33); and VL CDR3 sequence containing at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 34) In some embodiments, the anti-CD3ε binding domain is an Fv, e.g., a dsFv fragment, comprising a heavy chain variable amino acid sequence selected from the group of SEQ ID NOs: 35-65 and a light chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 66-84 or 368.

[0378] In some embodiments, the anti-CD3ε binding domain comprises: a VH CDR1 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence TYAMN (SEQ ID NO:29); Amino acid sequence a VH CDR2 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to TIFF2025060787000041.tif4128; Amino acid sequence a VH CDR3 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to TIFF2025060787000042.tif4128; Amino acid sequence a VL CDR1 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to TIFF2025060787000043.tif4128; a VL CDR2 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence GTNKRAP (SEQ ID NO:33); and a VL CDR3 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence ALWYSNLWV (SEQ ID NO:34) Includes.

[0379] In some embodiments, the anti-CD3ε binding domain comprises: a VH CDR1 sequence comprising at least the amino acid sequence GFTFNTYAMN (SEQ ID NO:461); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATY (SEQ ID NO:462); At least the amino acid sequence VH CDR3 sequence containing TIFF2025060787000044.tif4128; At least the amino acid sequence VL CDR1 sequence containing TIFF2025060787000045.tif4128; a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO:33); and VL CDR3 sequence containing at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 34) Includes.

[0380] In some embodiments, the anti-CD3 epsilon binding domain comprises a VH CDR1 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence GFTFNTYAMN (SEQ ID NO:461); a VH CDR2 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence RIRSKYNNYATY (SEQ ID NO:462); an amino acid sequence VH CDR3 sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to TIFF2025060787000046.tif4128, amino acid sequences TIFF2025060787000047.tif4128; a VL CDR2 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence GTNKRAP (SEQ ID NO:33); and a VL CDR3 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence ALWYSNLWV (SEQ ID NO:34).

[0381] In some embodiments, the anti-CD3ε binding domain comprises a VH CDR1 sequence comprising at least the amino acid sequence GFTFNTYAMN (SEQ ID NO:461); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATY (SEQ ID NO:462); VH CDR3 sequence containing at least TIFF2025060787000048.tif4128, amino acid sequence TIFF2025060787000049.tif4128; a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO:469); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNHWV (SEQ ID NO:464).

[0382] In some embodiments, the anti-CD3 epsilon binding domain comprises a VH CDR1 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence GFTFNTYAMN (SEQ ID NO:461); a VH CDR2 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence RIRSKYNNYATY (SEQ ID NO:462); an amino acid sequence VH CDR3 sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to TIFF2025060787000050.tif4128, amino acid sequences TIFF2025060787000051.tif4128; a VL CDR2 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence GTNKRAP (SEQ ID NO:469); and a VL CDR3 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence ALWYSNHWV (SEQ ID NO:464).

[0383] In some embodiments, the anti-CD3ε binding domain comprises a VH CDR1 sequence comprising at least the amino acid sequence GFTFSTYAMN (SEQ ID NO:466); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATY (SEQ ID NO:467); VH CDR3 sequence containing at least TIFF2025060787000052.tif4128, amino acid sequence TIFF2025060787000053.tif4128; a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO:469); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNHWV (SEQ ID NO:464).

[0384] In some embodiments, the anti-CD3 epsilon binding domain comprises a VH CDR1 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence GFTFSTYAMN (SEQ ID NO:466); a VH CDR2 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence RIRSKYNNYATY (SEQ ID NO:467); an amino acid sequence VH CDR3 sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to TIFF2025060787000054.tif4128, amino acid sequences TIFF2025060787000055.tif4128; a VL CDR2 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence GTNKRAP (SEQ ID NO:469); and a VL CDR3 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence ALWYSNHWV (SEQ ID NO:464).

[0385] In some embodiments, the anti-CD3ε binding domain comprises a CDR3 comprising at least the amino acids VLWYSNRWV (SEQ ID NO:465). In some embodiments, the anti-CD3ε binding domain comprises a CDR3 with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acids VLWYSNRWV (SEQ ID NO:465).

[0386] In some embodiments, the anti-CD3ε binding domain comprises one or more copies of an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, an scFv, a scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. In some embodiments, the anti-CD3ε binding domain comprises an Fv antibody fragment (herein referred to as an anti-CD3ε Fv fragment) that binds to CD3ε. In some embodiments, the anti-CD3ε Fv antibody fragment is a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv). In some embodiments, the anti-CD3 binding domain is monovalent for binding to CD3.

[0387] In some embodiments, the CD3 binding region is not a single chain antibody, e.g., in some aspects, the CD3 binding region is not a single chain variable fragment (scFv).

[0388] In some embodiments, the CD3 binding region is an Fv antibody fragment comprising a variable heavy chain (Hv, also referred to as VH) and a variable light chain (Lv, also referred to as VL), such as any of those described. In aspects of such embodiments, the immunoglobulin Fc region is a heterodimeric Fc region comprising two different Fc polypeptides that allow heterodimeric association between both polypeptides of the Fc heterodimer, such as any of those described in Section III.C.2.b. In such embodiments, the variable heavy chain (VH) and variable light chain (VL) of the CD3 binding region are linked to opposing chains of the heterodimeric Fc.

[0389] In some embodiments, the anti-CD3ε binding domain comprises a combination of a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence comprising an amino acid sequence selected from the group of SEQ ID NOs: 27, 28, 35-84, 368, 451-454, and 460. In some embodiments, the anti-CD3ε binding domain comprises a combination of a heavy chain variable region amino acid sequence selected from the group of SEQ ID NOs: 27, 35-65, 453, 454, and 460 and a light chain variable region amino acid sequence comprising an amino acid sequence selected from the group of SEQ ID NOs: 28, 66-84, 368, 451, and 452.

[0390] In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence and a light chain variable amino acid sequence. In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence and a light chain variable amino acid sequence comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-84, 368, 451-454, and 460. In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence and a light chain variable amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-84, 368, 451-454, and 460. In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence selected from the group of SEQ ID NOs: 35-65, 453, 454, and 460 and a light chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 66-84, 368, 451, and 452. In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising a heavy chain variable amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-65, 453, 454, and 460 in combination with a light chain variable amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-84, 368, 451, and 452.

[0391] In some embodiments, the anti-CD3 epsilon binding domain is an Fv or dsFv fragment comprising a heavy chain variable amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-65, 453, 454, and 460, and an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-84 or 368, 451, and 452. In some embodiments, the anti-CD3 binding domain is an Fv or dsFv containing a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 47 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 75. In some embodiments, the anti-CD3 binding domain is an Fv or dsFv containing a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:47 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:368.

[0392] In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence and a light chain variable amino acid sequence. In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence and a light chain variable amino acid sequence comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 35-84, 368, 451-454, 460. In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence and a light chain variable amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 35-84, 368, 451-454, 460. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a heavy chain variable amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 35-65, 453, 454, and 460 in combination with a light chain variable amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 66-84, 368, 451, and 452. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of a heavy chain variable amino acid sequence selected from the group of SEQ ID NOs: 27, 35-65, 453, 454, and 460 and a light chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 66-84, 368, 451, and 452.

[0393] In some embodiments, the anti-CD3ε Fv antibody fragment comprises a heavy chain variable amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 35-46, 48-50, 453, 454, and 460 in combination with a light chain variable amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 66, 68-74, 76, 78, 80, 368, 451, and 452. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of a heavy chain variable amino acid sequence selected from the group of SEQ ID NOs: 27, 35-46, 48-50, 454, 454, and 460 and a light chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 66, 68-74, 76, 78, 80, 451, and 452.

[0394] In some embodiments, the anti-CD3ε Fv antibody fragment comprises a heavy chain variable amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 35-46, 48-50, 453, 454, and 460 in combination with a light chain variable amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 66, 68-74, 76, 78, 80, 368, 451, and 452. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of a heavy chain variable amino acid sequence selected from the group of SEQ ID NOs: 27, 35-46, 48-50, 454, 454, and 460 and a light chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 66, 68-74, 76, 78, 80, 368, 451, and 452.

[0395] In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO:27, and a variable light chain (VL) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO:28. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:27. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:28. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:27 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:28.

[0396] In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 453. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 451. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:453, and a variable light chain (VL) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO:451. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:453. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:451. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:453 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:451.

[0397] In a specific embodiment, Fv is V H -V LThe heterodimer is a disulfide-stabilized Fv fragment (dsFv) stabilized by an interch...

Claims

1. A DLL3-binding polypeptide construct comprising at least one heavy chain-only variable domain (DLL3 VHH domain) that specifically binds DLL3 and one or more additional binding domains that bind to a target other than DLL3.

2. The at least one DLL3 VHH domain comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, and 456; a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 384, 410, and 411; and The DLL3-binding polypeptide construct of claim 1, which comprises a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of NOs:354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 395, and 412-415, and binds to DLL3.

3. a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, and 456; a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 384, 410, and 411; and At least one heavy chain-only variable domain (DLL3 VHH domain) that specifically binds to DLL3, comprising a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of NO:354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 395, and 412-415, and that binds to DLL3.

2. A DLL3-binding polypeptide construct comprising:

4. The DLL3-binding polypeptide construct of any one of claims 1 to 3, wherein said DLL3 is human DLL3.

5. The DLL3-binding polypeptide construct of any one of claims 1 to 4, wherein said at least one DLL3 VHH domain is humanized.

6. The DLL3-binding polypeptide construct of any one of claims 1, 2, 4, and 5, wherein the one or more additional binding domains bind to an activating receptor on an immune cell.

7. The DLL3-binding polypeptide construct of claim 6, wherein the immune cell is a T cell.

8. 8. The DLL3-binding polypeptide construct of claim 6 or claim 7, wherein the activating receptor is CD3 (CD3ε).

9. 9. The DLL3-binding polypeptide construct of claim 8, which is bispecific for DLL3 and CD3.

10. The DLL3-binding polypeptide construct of claim 9, wherein the immune cell is a natural killer (NK) cell.

11. 11. The DLL3-binding polypeptide construct of claim 6 or claim 10, wherein the activating receptor is CD16 (CD16a).

12. 12. The DLL3-binding polypeptide construct of claim 11, which is bispecific for DLL3 and CD16a.

13. 6. The DLL3-binding polypeptide construct of any one of claims 1, 2, 4, and 5, wherein said one or more additional binding domains bind to a cytokine receptor.

14. The DLL3-binding polypeptide construct of any one of claims 1, 2, and 4 to 13, wherein said one or more additional binding domains comprise an antibody or an antigen-binding fragment thereof.

15. 15. The DLL3-binding polypeptide construct of any one of claims 1, 2, and 4 to 14, wherein said one or more additional binding domains are monovalent.

16. 16. The DLL3-binding polypeptide construct of claim 14 or claim 15, wherein the antibody or antigen-binding fragment thereof is an Fv, a disulfide-stabilized Fv (dsFv), a scFv, a Fab, a single domain antibody (sdAb), a VNAR, or a VHH.

17. 14. The DLL3-binding polypeptide construct of claim 13, wherein the one or more additional binding domains are a cytokine or a truncated fragment or variant thereof capable of binding to a cytokine receptor.

18. 18. The DLL3-binding polypeptide construct of claim 17, wherein the cytokine is an interferon or a truncated fragment or variant of an interferon.

19. 19. The DLL3-binding polypeptide construct of claim 18, wherein the interferon is a type I interferon or a type II interferon, a truncated fragment or variant of a type I interferon, or a truncated fragment or variant of a type II interferon.

20. The type I interferon is IFNα or IFNβ, or a truncated fragment or variant thereof; or The type II interferon is IFNγ or a truncated fragment or variant thereof; 20. The DLL3-binding polypeptide construct of claim 19.

21. The DLL3-binding polypeptide construct of any one of claims 1 to 20, wherein the polypeptide comprises an immunoglobulin Fc region.

22. The DLL3-binding polypeptide construct of any one of claims 1, 2 and 4 to 21, wherein the polypeptide construct comprises an immunoglobulin Fc region linking the at least one VHH domain and the one or more additional binding domains.

23. 23. The DLL3-binding polypeptide construct of any one of claims 1 to 22, which is a dimer.

24. 24. The DLL3-binding polypeptide construct of any one of claims 21 to 23, wherein the Fc region is a homodimeric Fc region.

25. 25. The DLL3-binding polypeptide construct of any one of claims 21 to 24, wherein the Fc region comprises a sequence of amino acids set forth in any of SEQ ID NOs:8, 10, 11, 12, or 13, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:8, 10, 11, 12, or 13.

26. The DLL3-binding polypeptide construct of any one of claims 21 to 24, wherein the Fc region is human IgG1.

27. 27. The DLL3-binding polypeptide construct of claim 26, wherein the Fc region comprises an amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:

8.

28. 24. The DLL3-binding polypeptide construct of any one of claims 21 to 23, wherein the Fc region is a heterodimeric Fc region.

29. 29. The DLL3-binding polypeptide construct of any one of claims 21 to 28, wherein the Fc region exhibits an effector function.

30. The Fc region is One or more amino acid modifications that reduce effector function and / or reduce binding to an effector molecule selected from Fcγ receptors or C1q.

30. The DLL3-binding polypeptide construct of any one of claims 21 to 29, comprising a polypeptide comprising:

31. 31. The DLL3-binding polypeptide construct of claim 30, wherein the one or more amino acid modifications are a deletion of one or more of Glu233, Leu234, or Leu235.

32. 32. The DLL3-binding polypeptide construct of claim 30 or claim 31, wherein the Fc region comprises a sequence of amino acids set forth in SEQ ID NO:9 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:

9.

33. The at least one DLL3 VHH domain is selected from the group consisting of a VHH domain sequence shown in any of SEQ ID NOs: 102, 244-275, 277-300, 302-305, 314, 401, 416, 455, 476-480-488, and 507-518, or a VHH domain sequence shown in any of SEQ ID NOs:

33. The DLL3-binding polypeptide construct of any one of claims 1 to 32, comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of NOs:102, 244-275, 277-300, 302-305, 314, 401, 416, 455, 476-480-488, and 507-518, and which binds to DLL3.

34. 34. The DLL3-binding polypeptide construct of any one of claims 1 to 33, wherein the at least one DLL3 VHH domain comprises (i) the sequence shown in SEQ ID NO:244, (ii) a humanized variant of SEQ ID NO:244, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:244, and binds to DLL3.

35. A DLL3-binding polypeptide according to any one of claims 1 to 34, wherein the at least one DLL3 VHH domain comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:319, 320, 321, 322, 323, 324, 325, and 326; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:336, 337, and 338; and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:354, and binds to DLL3.

36. 36. The DLL3-binding polypeptide construct of any one of claims 1 to 35, wherein said at least one DLL3 VHH domain comprises the CDR1, CDR2 and CDR3 as shown in SEQ ID NOs:319, 336 and 354, respectively; SEQ ID NOs:319, 337 and 354, respectively; SEQ ID NOs:319, 338 and 354, respectively; SEQ ID NOs:320, 338 and 354, respectively; SEQ ID NOs:321, 338 and 354, respectively; SEQ ID NOs:322, 338 and 354, respectively; SEQ ID NOs:323, 338 and 354, respectively; SEQ ID NOs:324, 338 and 354, respectively; SEQ ID NOs:325, 338 and 354, respectively; or SEQ ID NOs:326, 338 and 354, respectively, and binds to DLL3.

37. 37. The DLL3-binding polypeptide construct of any one of claims 1 to 36, wherein the at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any of SEQ ID NOs: 245-257, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 245-257, and binds to DLL3.

38. 38. The DLL3-binding polypeptide construct of any one of claims 1 to 37, wherein said at least one DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs:245 to 257 and binds to DLL3.

39. 34. The DLL3-binding polypeptide construct of any one of claims 1 to 33, wherein the at least one DLL3 VHH domain comprises (i) the sequence shown in SEQ ID NO:258, (ii) a humanized variant of SEQ ID NO:258, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:258, and binds to DLL3.

40. A DLL3-binding polypeptide according to any one of claims 1 to 33 and 39, wherein the at least one DLL3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:327; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:339; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:355, and binds to DLL3.

41. A DLL3-binding polypeptide construct according to any one of claims 1 to 33, 39 and 40, wherein said at least one DLL3 VHH domain comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NOs:327, 339 and 335, respectively, and binds to DLL3.

42. 42. The DLL3-binding polypeptide construct of any one of claims 1 to 33 and 39 to 41, wherein the at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any of SEQ ID NOs: 259-263, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 259-263, and binds to DLL3.

43. 43. The DLL3-binding polypeptide construct of any one of claims 1 to 33 and 39 to 42, wherein the at least one DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs:259 to 263 and binds to DLL3.

44. 34. The DLL3-binding polypeptide construct of any one of claims 1 to 33, wherein the at least one DLL3 VHH domain comprises (i) the sequence shown in SEQ ID NO:264, (ii) a humanized variant of SEQ ID NO:264, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:264, and binds to DLL3.

45. A DLL3-binding polypeptide construct according to any one of claims 1 to 33 and 44, wherein the at least one DLL3 VHH domain comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO:328, 329, or 456; a CDR2 comprising the amino acid sequence shown in SEQ ID NO:340; and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:356, and binds to DLL3.

46. A DLL3-binding polypeptide construct according to any one of claims 1 to 33, 44 and 45, wherein the at least one DLL3 VHH domain comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NOs:328, 340 and 356, respectively; SEQ ID NOs:329, 340 and 356, respectively, or SEQ ID NOs:456, 340 and 356, respectively, and binds to DLL3.

47. 47. The DLL3-binding polypeptide construct of any one of claims 1 to 33 and claims 44 to 46, wherein the at least one DLL3 VHH domain comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 265-274, 416, 455, or 476-478, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 265-274, 416, 455, or 476-478, and binds to DLL3.

48. A DLL3-binding polypeptide construct according to any one of claims 1 to 33 and claims 44 to 47, wherein the at least one DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs: 265-274, 416, 455, or 476-478, and binds to DLL3.

49. 34. The DLL3-binding polypeptide construct of any one of claims 1 to 33, wherein the at least one DLL3 VHH domain comprises (i) the sequence shown in SEQ ID NO:275, (ii) a humanized variant of SEQ ID NO:275, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:275, and binds to DLL3.

50. 50. A DLL3-binding polypeptide construct according to any one of claims 1 to 33 and 49, wherein said at least one DLL3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:320; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:341; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:357, and binds to DLL3.

51. 51. The DLL3-binding polypeptide construct of any one of claims 1 to 33, 49 and 50, wherein the at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any of SEQ ID NOs: 277-279 and 479, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 277-279 and 479, and binds to DLL3.

52. 52. The DLL3-binding polypeptide construct of any one of claims 1 to 33 and 49 to 51, wherein the at least one DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs: 277 to 279 and 479 and binds to DLL3.

53. 34. The DLL3-binding polypeptide construct of any one of claims 1 to 33, wherein the at least one DLL3 VHH domain comprises (i) the sequence shown in SEQ ID NO:280, (ii) a humanized variant of SEQ ID NO:280, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:280, and binds to DLL3.

54. A DLL3-binding polypeptide construct according to any one of claims 1 to 33 and 53, wherein the at least one DLL3 VHH domain comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO:330; a CDR2 comprising the amino acid sequence shown in SEQ ID NO:342; and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:358, and binds to DLL3.

55. 55. The DLL3-binding polypeptide construct of any one of claims 1 to 33, 53 and 54, wherein the at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any of SEQ ID NOs:281-286, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:281-286, and binds to DLL3.

56. 56. The DLL3-binding polypeptide construct of any one of claims 1 to 33 and 53 to 55, wherein said at least one DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs:281 to 286 and binds to DLL3.

57. 34. The DLL3-binding polypeptide construct of any one of claims 1 to 33, wherein the at least one DLL3 VHH domain comprises (i) the sequence shown in SEQ ID NO:287, (ii) a humanized variant of SEQ ID NO:287, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:287, and binds to DLL3.

58. The DLL3-binding polypeptide construct of any one of claims 1 to 33 and 57, wherein the at least one DLL3 VHH domain comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO:320; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:345, 346, and 347; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:359, 360, and 361, and binds to DLL3.

59. 59. The DLL3-binding polypeptide construct of any one of claims 1 to 33 and 57 to 58, wherein the at least one DLL3 VHH domain comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NOs:320, 345 and 359, respectively; SEQ ID NOs:320, 346 and 359, respectively; SEQ ID NOs:320, 347 and 359, respectively; SEQ ID NOs:320, 345 and 360, respectively; SEQ ID NOs:320, 345 and 361, respectively; or SEQ ID NOs:320, 347 and 360, respectively, and binds to DLL3.

60. 60. The DLL3-binding polypeptide construct of any one of claims 1 to 33 and 57 to 59, wherein the at least one DLL3 VHH domain comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 288-298 or 102, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 288-298 or 102, and binds to DLL3.

61. 61. A DLL3-binding polypeptide construct according to any one of claims 1 to 33 and 57 to 60, wherein said at least one DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs: 288 to 298 or 102 and binds to DLL3.

62. 34. The DLL3-binding polypeptide construct of any one of claims 1 to 33, wherein the at least one DLL3 VHH domain comprises (i) the sequence shown in SEQ ID NO:299, (ii) a humanized variant of SEQ ID NO:299, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:299, and binds to DLL3.

63. A DLL3-binding polypeptide according to any one of claims 1 to 33 and 62, wherein the at least one DLL3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:331; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:348, 349, and 350; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:356, and binds to DLL3.

64. A DLL3-binding polypeptide construct according to any one of claims 1 to 33 and 62 to 63, wherein the at least one DLL3 VHH domain comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NOs:331, 348 and 356, respectively; SEQ ID NOs:331, 349 and 356, respectively; or SEQ ID NOs:331, 350 and 356, respectively, and binds to DLL3.

65. 65. The DLL3-binding polypeptide construct of any one of claims 1 to 33 and 62 to 64, wherein the at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any of SEQ ID NOs: 300, 302-305, and 480, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 300, 302-305, and 480, and binds to DLL3.

66. 66. A DLL3-binding polypeptide construct according to any one of claims 1 to 33 and 62 to 65, wherein said at least one DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs: 300, 302-305, and 480, and binds to DLL3.

67. 34. The DLL3-binding polypeptide construct of any one of claims 1 to 33, wherein the at least one DLL3 VHH domain comprises (i) the sequence shown in SEQ ID NO:507, (ii) a humanized variant of SEQ ID NO:507, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:507, and binds to DLL3.

68. 68. The DLL3-binding polypeptide construct of any one of claims 1 to 33 and 67, wherein the at least one DLL3 VHH domain comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO:332; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:348, 349, and 350; and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:362, and binds to DLL3.

69. The DLL3-binding polypeptide construct of any one of claims 1 to 33 and 67 to 68, wherein the at least one DLL3 VHH domain comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 332, 348, and 362, respectively; SEQ ID NOs: 332, 349, and 362, respectively; or SEQ ID NOs: 332, 350, and 362.

70. 70. The DLL3-binding polypeptide construct of any one of claims 1 to 33 and 67 to 69, wherein the at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any of SEQ ID NOs:508-514, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:508-514, and binds to DLL3.

71. 71. A DLL3-binding polypeptide construct according to any one of claims 1 to 33 and 67 to 70, wherein said at least one DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs:508 to 514 and binds to DLL3.

72. 34. The DLL3-binding polypeptide construct of any one of claims 1 to 33, wherein the at least one DLL3 VHH domain comprises (i) the sequence shown in SEQ ID NO:401, (ii) a humanized variant of SEQ ID NO:401, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:401, and binds to DLL3.

73. A DLL3-binding polypeptide construct according to any one of claims 1 to 33 and 72, wherein the at least one DLL3 VHH domain comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO:320; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:384, 410, and 411; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:395, 412, 413, 414, and 415, and binds to DLL3.

74. DLL3-binding polypeptide construct according to any one of claims 1 to 33 and 72 to 73, wherein said at least one DLL3 VHH domain comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NOs:320, 384 and 395, respectively; SEQ ID NOs:320, 410 and 395, respectively; SEQ ID NOs:320, 411 and 395, respectively; SEQ ID NOs:320, 384 and 412, respectively; SEQ ID NOs:320, 384 and 413, respectively; SEQ ID NOs:320, 384 and 414, respectively; or SEQ ID NOs:320, 384 and 415, respectively, and binds to DLL3.

75. 75. The DLL3-binding polypeptide construct of any one of claims 1 to 33 and 72 to 74, wherein the at least one DLL3 VHH domain comprises a sequence of amino acids set forth in any of SEQ ID NOs: 481-488, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 481-488, and binds to DLL3.

76. DLL3-binding polypeptide construct according to any one of claims 1 to 33 and 72 to 75, wherein said at least one DLL3 VHH domain comprises an amino acid sequence set forth in any of SEQ ID NOs: 481 to 488 and binds to DLL3.

77. 34. The DLL3-binding polypeptide construct of any one of claims 1 to 33, wherein the at least one DLL3 VHH domain comprises (i) a sequence as set forth in SEQ ID NO:314, 518, 515, 516, or 517, (ii) a humanized variant of SEQ ID NO:314, 518, 515, 516, or 517, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NO:314, 518, 515, 516, or 517, and binds to DLL3.

78. 80. The DLL3-binding polypeptide construct of any one of claims 1 to 33 and 77, wherein said at least one DLL3 VHH domain comprises CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 333, 351 and 363, respectively; 334, 352 and 364, respectively; 320, 353 and 365, respectively; 334, 339 and 366, respectively; or 335, 348 and 367, respectively, and binds to DLL3.

79. 80. The DLL3-binding polypeptide construct of any one of claims 1 to 33, 77 and 78, wherein said at least one DLL3 VHH domain is set forth in SEQ ID NO: 314, 518, 515, 516 or 517 and binds to DLL3.

80. 1. A multispecific polypeptide construct comprising: (a) a first component comprising a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide; and (b) a second component comprising an anti-CD3 antibody or antigen-binding fragment comprising a variable heavy chain region (VH) and a variable light chain region (VL), the VH and VL constituting the anti-CD3 antibody or antigen-binding fragment are linked to opposing polypeptides of a heterodimeric Fc; the first and second components are coupled by a linker, and the heterodimeric Fc region is positioned N-terminal to the anti-CD3 antibody; one or both of the first and second components comprises at least one antigen-binding domain that comprises a VHH domain that specifically binds to DLL3 (DLL3 VHH domain); The multispecific polypeptide construct.

81. at least (i) a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and a VH or VL domain of an anti-CD3 antibody or antigen-binding fragment; and (ii) a second polypeptide comprising a second Fc polypeptide of a heterodimeric Fc region, a linker, optionally the same linker as present in the first polypeptide, and the other of the VH or VL domain of the anti-CD3 antibody or antigen-binding fragment.

81. The multispecific polypeptide construct of claim 80, comprising:

82. 82. The multispecific polypeptide construct of claim 80 or claim 81, wherein one or both of the first and second Fc polypeptides of the heterodimeric Fc region comprises at least one modification that induces heterodimerization compared to a polypeptide of a homodimeric Fc region, and optionally compared to an Fc polypeptide as set forth in SEQ ID NO:8 or an immunologically active fragment thereof.

83. 83. The multispecific polypeptide construct of claim 82, wherein each of the first and second Fc polypeptides of the heterodimeric Fc region independently comprises at least one amino acid modification.

84. 84. The multispecific polypeptide construct of claim 83, wherein each of the first and second Fc polypeptides of the heterodimeric Fc region comprises a knob-into-hole modification or a charge mutation that increases electrostatic complementarity of the polypeptides.

85. The multispecific polypeptide construct of claim 84, wherein said amino acid modification is a knob-into-hole modification.

86. 86. The multispecific polypeptide construct of any one of claims 80 to 85, wherein said first Fc polypeptide of said heterodimeric Fc region comprises an alteration selected from among Thr366Ser, Leu368Ala, Tyr407Val, and a combination thereof, and said second Fc polypeptide of said heterodimeric Fc region comprises the alteration Thr366Trp.

87. 87. The multispecific polypeptide construct of claim 86, wherein said first and said second Fc polypeptides further comprise a modification of a non-cysteine ​​residue to a cysteine ​​residue, said modification of said first Fc polypeptide being at one of positions Ser354 and Tyr349 and said modification of said second Fc polypeptide being at the other of positions Ser354 and Tyr349.

88. 85. The multispecific polypeptide construct of any one of claims 80 to 84, wherein said amino acid modification is a charge mutation that increases electrostatic complementarity of said polypeptide.

89. 89. The multispecific polypeptide construct of any one of claims 80 to 84 and 88, wherein said first and / or said second Fc polypeptide, or each of said first and said second Fc polypeptide, comprises a modification in a complementary position, which modification is a substitution with an amino acid having an opposite charge to the complementary amino acid of the other polypeptide.

90. 70. The multispecific polypeptide construct of any one of claims 60 to 69, wherein one of the first or second Fc polypeptides of said heterodimeric Fc region further comprises a modification at residue Ile253.

91. 91. The multispecific polypeptide construct of claim 90, wherein said modification is Ile253Arg.

92. 92. The multispecific polypeptide construct of any one of claims 80 to 91, wherein one of the first or second Fc polypeptides of said heterodimeric Fc region further comprises a modification at residue His435.

93. The multispecific polypeptide construct of claim 92, wherein the modification is His435Arg.

94. 94. The multispecific polypeptide construct of any one of claims 80 to 93, wherein said Fc region comprises a polypeptide lacking Lys447.

95. 95. The multispecific polypeptide construct of any one of claims 80 to 94, wherein said Fc region comprises a polypeptide comprising at least one modification to enhance FcRn binding.

96. 96. The multispecific polypeptide construct of claim 95, wherein the modification is at a position selected from the group consisting of Met252, Ser254, Thr256, Met428, Asn434, and combinations thereof.

97. 97. The multispecific polypeptide construct of claim 96, wherein the modification is selected from the group consisting of Met252Y, Ser254T, Thr256E, Met428L, Met428V, Asn434S, and combinations thereof.

98. The multispecific polypeptide construct of claim 96, wherein the modifications are at positions Met252 and Met428.

99. The multispecific polypeptide construct of claim 98, wherein said modifications are Met252Y and Met428L.

100. The multispecific polypeptide construct of claim 76, wherein said modifications are Met252Y and Met428V.

101. 101. The multispecific polypeptide construct of any one of claims 80 to 100, wherein said first Fc polypeptide of said heterodimeric Fc region comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 103, 107, 115, or 117, and said second Fc polypeptide of said heterodimeric Fc region comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 104, 108, 111, 113, 119, or 121.

102. The Fc region is At least one amino acid modification that reduces effector function and / or reduces binding to an effector molecule selected from Fcγ receptors or C1q.

102. The multispecific polypeptide construct of any one of claims 1 to 101, comprising a polypeptide comprising:

103. The multispecific polypeptide construct of claim 102, wherein said one or more amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235.

104. 104. The multispecific polypeptide construct of any one of claims 80 to 103, wherein said first Fc polypeptide of said heterodimeric Fc region comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 105, 109, 116, or 118, and said second Fc polypeptide of said heterodimeric Fc region comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 106, 110, 112, 114, 120, or 122.

105. 105. The multispecific polypeptide construct of any one of claims 80 to 104, wherein said anti-CD3 antibody or antigen-binding fragment is monovalent.

106. 106. The multispecific polypeptide construct of any of claims 80 to 105, wherein said anti-CD3 antibody or antigen-binding fragment is not a single chain antibody, and optionally is not a single chain variable fragment (scFv).

107. 107. The multispecific polypeptide construct of any of claims 80 to 106, wherein said anti-CD3 antibody or antigen-binding fragment is an Fv antibody fragment.

108. The multispecific polypeptide construct of claim 107, wherein said Fv antibody fragment comprises a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv).

109. the anti-CD3 antibody or antigen-binding fragment thereof VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO:29); Amino acid sequence VH CDR2 comprising: Amino acid sequence VH CDR3 comprising: Amino acid sequence VL CDR1 comprising: VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO:33); and VL CDR3 containing the amino acid sequence ALWYSNLWV (SEQ ID NO:34) 109. The multispecific polypeptide construct of any one of claims 80 to 108, comprising:

110. the anti-CD3 antibody or antigen-binding fragment thereof a VH having an amino acid sequence of any of SEQ ID NOs:35-65, or a sequence exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:35-65; and VL having an amino acid sequence of any of SEQ ID NOs: 66-84 and 368, or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 66-84 and 368.

110. The multispecific polypeptide construct of any one of claims 80 to 109, comprising:

111. 111. The multispecific polypeptide construct of any one of claims 80 to 110, wherein said anti-CD3 antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO:47 and the amino acid sequence of SEQ ID NO:

75.

112. 111. The multispecific polypeptide construct of any one of claims 80 to 110, wherein said anti-CD3 antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO:47 and the amino acid sequence of SEQ ID NO:

368.

113. 113. The multispecific polypeptide construct of any of claims 80 to 112, wherein said at least one DLL3 VHH domain is positioned amino-terminal to the Fc region of the multispecific construct and / or carboxy-terminal to the CD3 binding region of the multispecific construct.

114. 114. The multispecific polypeptide construct of any one of claims 80 to 113, comprising a first DLL3 VHH domain that specifically binds to DLL3 and a second DLL3 VHH domain that specifically binds to DLL3.

115. 115. The multispecific polypeptide construct of claim 114, wherein the first or the second DLL3 VHH domain is positioned amino-terminal to the Fc region of the multispecific construct and the other of the first or the second DLL3 VHH domain is positioned carboxy-terminal to the CD3 binding region of the multispecific construct.

116. the first component comprises, in order from N-terminus to C-terminus, a first DLL3 VHH domain that binds DLL3, the first Fc polypeptide of the heterodimeric Fc region, the linker, a VH or VL domain of the anti-CD3 antibody or antigen-binding fragment, and a second DLL3 VHH domain that binds DLL3; the second polypeptide comprises, in order from N-terminus to C-terminus, the second Fc polypeptide of the heterodimeric Fc region, the linker, optionally the same linker as is present in the first component, and the other of the VH or VL domain of the anti-CD3 antibody or antigen-binding fragment.

116. A multispecific polypeptide construct according to claim 114 or claim 115.

117. 117. The multispecific polypeptide construct of any one of claims 114 to 116, wherein said first and said second DLL3 VHH domains are identical.

118. 117. The multispecific polypeptide construct of any one of claims 114 to 116, wherein said first and said second DLL3 VHH domains are different.

119. The multispecific polypeptide construct of claim 118, wherein the first and second DLL3 VHH domains bind to distinct or non-overlapping epitopes of DLL3 and / or do not compete for binding to DLL3.

120. the first VHH domain comprises an amino acid sequence set forth in any of 251, 264, 267, 268, 287, 299, 507, 314, 517, or 455, a humanized variant thereof, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of 251, 264, 267, 268, 287, 299, 507, 314, 517, or 455, and binds DLL3; the second VHH domain comprises an amino acid sequence set forth in any of 244, 251, 258, 267, 275, 280, 314, 518, 515, 516, 517, 455, or a humanized variant thereof, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of 244, 251, 258, 267, 275, 280, 314, 518, 515, 516, 517, 455, and binds DLL3; 120. The multispecific polypeptide construct of claim 119.

121. the first VHH domain and the second VHH domain are selected from the group consisting of SEQ ID NO:244 and SEQ ID NO:264; SEQ ID NO:314 and SEQ ID NO:517; SEQ ID NO:244 and SEQ ID NO:507; SEQ ID NO:314 and SEQ ID NO:507; SEQ ID NO:314 and SEQ ID NO:314; SEQ ID NO:314 and SEQ ID NO:299; SEQ ID NO:518 and SEQ ID NO:264; SEQ ID NO:251 and SEQ ID NO:268; SEQ ID NO:251 and SEQ ID NO:267; SEQ ID NO:275 and SEQ ID NO:517; SEQ ID NO:314 and SEQ ID NO:287; SEQ ID NO:314 and SEQ ID NO:264; SEQ ID NO:515 and SEQ ID 121. The multispecific polypeptide construct of claim 119 or 120, comprising an amino acid sequence selected from SEQ ID NO:517; SEQ ID NO:516 and SEQ ID NO:517; SEQ ID NO:517 and SEQ ID NO:517; SEQ ID NO:251 and SEQ ID NO:455; or SEQ ID NO:244 and SEQ ID NO:

517.

122. The at least one DLL3 VHH domain, or the first and second DLL3 VHH domains, each independently have a VHH domain sequence as set forth in any of SEQ ID NOs: 102, 244-275, 277-300, 302-305, 314, 401, 416, 455, 476-488, or 507-518, or a VHH domain sequence as set forth in any of SEQ ID NOs:

122. The multispecific polypeptide construct of any one of claims 80-121, comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of NOs: 102, 244-275, 277-300, 302-305, 314, 401, 416, 455, 476-488, or 507-518, and which binds to DLL3.

123. 123. The multispecific polypeptide construct of any of claims 80-122, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises (i) the sequence set forth in SEQ ID NO:244, (ii) a humanized variant of SEQ ID NO:244, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:244, and binds to DLL3.

124. 124. The multispecific polypeptide construct of any one of claims 80 to 123, wherein each of the at least one DLL3 VHH domain, or the first and second DLL3 VHH domains, independently, comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 319, 320, 321, 322, 323, 324, 325, and 326; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 336, 337, and 338; and a CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 354, and binds to DLL3.

125. wherein the at least one DLL3 VHH domain, or each of the first and second DLL3 VHH domains, is independently selected from SEQ ID NOs:319, 336, and 354, respectively; SEQ ID NOs:319, 337, and 354, respectively; SEQ ID NOs:319, 338, and 354, respectively; SEQ ID NOs:320, 338, and 354, respectively; SEQ ID NOs:321, 338, and 354, respectively; SEQ ID NOs:322, 338, and 354, respectively; SEQ ID NOs:323, 338, and 354, respectively; SEQ ID NOs:324, 338, and 354, respectively; SEQ ID NOs:325, 338, and 354, respectively; or SEQ ID 15. The multispecific construct of any one of claims 80 to 124, comprising CDR1, CDR2 and CDR3 as set forth in NOs:326, 338 and 354, and binding to DLL3.

126. 126. The multispecific polypeptide construct of any of claims 80-125, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises a sequence of amino acids as set forth in any of SEQ ID NOs:245-257, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:245-257, and binds DLL3.

127. 127. The multispecific polypeptide construct of any one of claims 80-126, wherein said at least one DLL3 VHH domain, or each of said first and said second DLL3 VHH domains, independently, comprises a sequence of amino acids set forth in any of SEQ ID NOs:245-257, and binds to DLL3.

128. 123. The multispecific polypeptide construct of any of claims 80-122, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises (i) the sequence set forth in SEQ ID NO:258, (ii) a humanized variant of SEQ ID NO:258, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:258, and binds to DLL3.

129. 129. The multispecific polypeptide construct of any one of claims 80 to 122 and 128, wherein each of the at least one DLL3 VHH domain, or the first and second DLL3 VHH domains, independently comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:327; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:339; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:355, and binds to DLL3.

130. 130. The multispecific polypeptide construct of any one of claims 80 to 122, 128 and 129, wherein each of the at least one DLL3 VHH domain, or the first and second DLL3 VHH domains, independently comprises CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:327, 339 and 335, respectively, and binds to DLL3.

131. 131. The multispecific polypeptide construct of any one of claims 80-122 and 128-130, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises a sequence of amino acids as set forth in any of SEQ ID NOs:259-263, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:259-263, and binds to DLL3.

132. 132. The multispecific polypeptide construct of any one of claims 80-122 and 128-131, wherein said at least one DLL3 VHH domain, or each of said first and said second DLL3 VHH domains, independently, comprises a sequence of amino acids set forth in any of SEQ ID NOs:259-263, and binds to DLL3.

133. 123. The multispecific polypeptide construct of any of claims 80-122, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises (i) the sequence set forth in SEQ ID NO:264, (ii) a humanized variant of SEQ ID NO:264, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:264, and binds to DLL3.

134. 134. The multispecific polypeptide construct of any one of claims 80 to 122 and 133, wherein said at least one DLL3 VHH domain, or each of said first and second VHH domains, independently comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:328, 329, or 456; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:340; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:356, and binds to DLL3.

135. 15. The multispecific polypeptide construct of any one of claims 80 to 122, 133 and 134, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently comprises CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 328, 340 and 356, respectively; SEQ ID NOs: 329, 340 and 356, respectively; or SEQ ID NOs: 456, 340 and 356, respectively, and binds to DLL3.

136. 136. The multispecific polypeptide construct of any one of claims 80-122 and claims 133-135, wherein said at least one DLL3 VHH domain or each of said first and second DLL3 VHH domains independently comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 265-274, 416, 455, or 476-478, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 265-274, 416, 455, or 476-478, and binds DLL3.

137. 137. The multispecific polypeptide construct of any one of claims 80-122 and claims 133-136, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises a sequence of amino acids set forth in any of SEQ ID NOs: 265-274, 416, 455, or 476-478, and binds to DLL3.

138. 123. The multispecific polypeptide construct of any of claims 80-122, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises (i) a sequence as set forth in SEQ ID NO:275, (ii) a humanized variant of SEQ ID NO:275, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:275, and binds to DLL3.

139. 139. The multispecific polypeptide construct of any one of claims 80 to 122 and 138, wherein said at least one DLL3 VHH domain, or each of said first and second VHH domains, independently, comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:320; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:341; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:357, and binds to DLL3.

140. 140. The multispecific polypeptide construct of any one of claims 80-122, 138 and 139, wherein said at least one DLL3 VHH domain, or each of said first and second VHH domains, independently, comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 277-279 and 479, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 277-279 and 479, and binds DLL3.

141. 141. The multispecific polypeptide construct of any one of claims 80-122 and 138-140, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises a sequence of amino acids set forth in any of SEQ ID NOs: 277-279 and 479, and binds to DLL3.

142. 123. The multispecific polypeptide construct of any of claims 80-122, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises (i) the sequence set forth in SEQ ID NO:280, (ii) a humanized variant of SEQ ID NO:110, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:280, and binds to DLL3.

143. 143. The multispecific polypeptide construct of any one of claims 80 to 122 and 142, wherein each of the at least one DLL3 VHH domain, or the first and second DLL3 VHH domains, independently comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:330; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:342; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:358, and binds to DLL3.

144. 144. The multispecific polypeptide construct of any one of claims 80-122, 142 and 143, wherein said at least one DLL3 VHH domain, or each of said first and second VHH domains, independently, comprises a sequence of amino acids as set forth in any of SEQ ID NOs:281-286, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:281-286, and binds DLL3.

145. 145. The multispecific polypeptide construct of any one of claims 80-122 and 142-144, wherein said at least one DLL3 VHH domain, or each of said first and said second DLL3 VHH domains, independently, comprises a sequence of amino acids set forth in any of SEQ ID NOs:281-286, and binds to DLL3.

146. 123. The multispecific polypeptide construct of any of claims 80-122, wherein each of said at least one DLL3 VHH domain, or said first and said second DLL3 VHH domains, independently, comprises (i) the sequence set forth in SEQ ID NO:287, (ii) a humanized variant of SEQ ID NO:287, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:287, and binds to DLL3.

147. 152. The multispecific polypeptide construct of any one of claims 80 to 122 and 146, wherein each of the at least one DLL3 VHH domain, or the first and second DLL3 VHH domains, independently, comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:320; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:345, 346, and 347; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:359, 360, and 361, and binds to DLL3.

148. 152. The multispecific polypeptide construct of any one of claims 80 to 122, 146 and 147, wherein said at least one DLL3 VHH domain comprises CDR1, CDR2 and CDR3 as depicted in SEQ ID NOs:320, 345 and 359, respectively; SEQ ID NOs:320, 346 and 359, respectively; SEQ ID NOs:320, 347 and 359, respectively; SEQ ID NOs:320, 345 and 360, respectively; SEQ ID NOs:320, 345 and 361, respectively; or SEQ ID NOs:320, 347 and 360, respectively, and binds to DLL3.

149. 149. The multispecific polypeptide construct of any one of claims 80-122 and 146-148, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 288-298 or 102, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 288-298 or 102, and binds DLL3.

150. 150. The multispecific polypeptide construct of any one of claims 80-122 and 146-149, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises a sequence of amino acids set forth in any of SEQ ID NOs: 288-298 or 102, and binds to DLL3.

151. 123. The multispecific polypeptide construct of any of claims 80-122, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises (i) a sequence as set forth in SEQ ID NO:299, (ii) a humanized variant of SEQ ID NO:299, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:299, and binds to DLL3.

152. 152. The multispecific polypeptide construct of any one of claims 80 to 122 and 151, wherein each of the at least one DLL3 VHH domain, or the first and second DLL3 VHH domains, independently, comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:331; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:348, 349, and 350; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:356, and binds to DLL3.

153. The multispecific polypeptide construct of any one of claims 80 to 122, 151 and 152, wherein said at least one DLL3 VHH domain comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NOs:331, 348 and 356, respectively; SEQ ID NOs:331, 349 and 356, respectively; or SEQ ID NOs:331, 350 and 356, respectively, and binds to DLL3.

154. 154. The multispecific polypeptide construct of any one of claims 80-122 and 151-153, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 300, 302-305, and 480, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 300, 302-305, and 480, and binds DLL3.

155. 155. The multispecific polypeptide construct of any one of claims 80-122 and 151-154, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises a sequence of amino acids set forth in any of SEQ ID NOs: 300, 302-305, and 480, and binds to DLL3.

156. 123. The multispecific polypeptide construct of any of claims 80-122, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises (i) the sequence set forth in SEQ ID NO:507, (ii) a humanized variant of SEQ ID NO:507, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:507, and binds to DLL3.

157. 157. The multispecific polypeptide construct of any one of claims 80 to 122 and 156, wherein each of the at least one DLL3 VHH domain, or the first and second DLL3 VHH domains, independently comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:332; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:348, 349, and 350; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:362, and binds to DLL3.

158. The multispecific polypeptide construct of any one of claims 80 to 122, 156 and 157, wherein said at least one DLL3 VHH domain comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 332, 348 and 362, respectively; SEQ ID NOs: 332, 349 and 362, respectively; or SEQ ID NOs: 332, 350 and 362, respectively, and binds to DLL3.

159. 159. The multispecific polypeptide construct of any one of claims 80-122 and 156-158, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises a sequence of amino acids as set forth in any of SEQ ID NOs:508-514, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:508-514, and binds DLL3.

160. 160. The multispecific polypeptide construct of any one of claims 80-122 and 156-159, wherein said at least one DLL3 VHH domain, or each of said first and said second DLL3 VHH domains, independently, comprises a sequence of amino acids set forth in any of SEQ ID NOs:508-514, and binds to DLL3.

161. 123. The multispecific polypeptide construct of any of claims 80-122, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises (i) the sequence as set forth in SEQ ID NO:401, (ii) a humanized variant of SEQ ID NO:401, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:401, and binds to DLL3.

162. 162. The multispecific polypeptide construct of any one of claims 80 to 122 and 161, wherein each of the at least one DLL3 VHH domain, or the first and second DLL3 VHH domains, independently, comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO:320; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:384, 410, and 411; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:395, 412, 413, 414, and 415, and binds to DLL3.

163. 163. The multispecific polypeptide construct of any one of claims 80 to 122, 161 and 162, wherein said at least one DLL3 VHH domain comprises CDR1, CDR2 and CDR3 as depicted in SEQ ID NOs:320, 384, and 395, respectively; SEQ ID NOs:320, 410, and 395, respectively; SEQ ID NOs:320, 411, and 395, respectively; SEQ ID NOs:320, 384, and 412, respectively; SEQ ID NOs:320, 384, and 413, respectively; SEQ ID NOs:320, 384, and 414, respectively; or SEQ ID NOs:320, 384, and 415, respectively, and binds to DLL3.

164. 164. The multispecific polypeptide construct of any one of claims 80-122 and 161-163, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 481-488, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 481-488, and binds DLL3.

165. 165. The multispecific polypeptide construct of any one of claims 80-122 and 161-164, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises a sequence of amino acids set forth in any of SEQ ID NOs:481-488, and binds to DLL3.

166. 123. The multispecific polypeptide construct of any one of claims 80-122, wherein said at least one DLL3 VHH domain, or each of said first and second VHH domains, independently, comprises (i) a sequence as set forth in SEQ ID NO:314, 518, 515, 516, or 517, (ii) a humanized variant of SEQ ID NO:314, 518, 515, 516, or 517, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NO:314, 518, 515, 516, or 517, and binds DLL3.

167. 167. The multispecific polypeptide construct of any one of claims 80 to 122 and 166, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, comprises CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 333, 351, and 363, respectively; 334, 352, and 364, respectively; 320, 353, and 365, respectively; 334, 339, and 366, respectively; or 335, 348, and 367, respectively, and binds to DLL3.

168. 168. The multispecific polypeptide construct of any one of claims 80-122, 166 and 167, wherein said at least one DLL3 VHH domain, or each of said first and second DLL3 VHH domains, independently, is set forth in SEQ ID NO:314, 518, 515, 516, or 517 and binds to DLL3.

169. 169. The multispecific polypeptide construct of any one of claims 80-168, wherein one or both of said first and second components comprises at least one costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor.

170. 170. The multispecific polypeptide construct of claim 169, wherein said at least one costimulatory receptor binding region (CRBR) is positioned amino-terminal to said Fc region of said multispecific polypeptide construct and / or carboxy-terminal to said CD3 binding region of said multispecific polypeptide construct.

171. 171. The multispecific polypeptide construct of claim 169 or claim 170, which comprises only one costimulatory receptor binding region (CRBR).

172. the first component comprises, in order from N-terminus to C-terminus, a first DLL3 VHH domain that binds DLL3, the first Fc polypeptide of the heterodimeric Fc region, the linker, a VH or VL domain of the anti-CD3 antibody or antigen-binding fragment, and a second DLL3 VHH domain that binds DLL3; the second component comprises a CRBR and comprises, in order from N-terminus to C-terminus, the second Fc polypeptide of the heterodimeric Fc region, the linker, optionally the same linker as present in the first component, the other of the VH or VL domains of the anti-CD3 antibody or antigen-binding fragment, wherein the CRBR is positioned amino-terminal to the Fc region of the second component or carboxy-terminal to the anti-CD3 antibody or antigen-binding fragment of the second component.

172. The multispecific polypeptide construct of any one of claims 169-171.

173. The at least one costimulatory receptor binding region (CRBR) is The extracellular domain of the natural cognate binding partner of said costimulatory receptor or a binding fragment thereof, or a variant thereof that exhibits binding activity with said costimulatory receptor.

173. The multispecific polypeptide construct of any of claims 169-172, which is or comprises:

174. 174. The multispecific polypeptide construct of any of claims 169-173, wherein said at least one costimulatory receptor binding region (CRBR) is an antibody or an antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

175. The multispecific polypeptide construct of claim 174, wherein the antibody or antigen-binding fragment thereof is an Fv, scFv, Fab, single domain antibody (VHH domain), VNAR, or VHH.

176. 176. The multispecific polypeptide construct of claim 174 or claim 175, wherein said antibody or antigen-binding fragment is a VHH domain.

177. The multispecific polypeptide construct of claim 176, wherein said VHH domain is a human VHH domain or a humanized VHH domain.

178. 178. The multispecific polypeptide construct of any of claims 169-177, wherein said at least one costimulatory receptor binding region (CRBR) binds to a costimulatory receptor selected from among 41BB (CD137), OX40 (CD134), CD27, Glucocorticoid-inducible TNFR-related protein (GITR), CD28, ICOS, CD40, B-cell activating factor receptor (BAFF-R), B-cell maturation antigen (BCMA), Transmembrane activator and CAML interactor (TACI), and NKG2D.

179. 179. The multispecific polypeptide construct of any one of claims 169-178, wherein said at least one costimulatory receptor binding region (CRBR) binds to a costimulatory receptor selected from among 41BB (CD137), OX40 (CD134), and glucocorticoid-inducible TNFR-related protein (GITR).

180. 180. The multispecific polypeptide construct of any one of claims 169-179, wherein said at least one costimulatory receptor binding region (CRBR) comprises a sequence of amino acids as set forth in SEQ ID NO:210, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence as set forth in SEQ ID NO:210, and binds to 4-1BB.

181. 181. The multispecific polypeptide construct of any one of claims 80 to 180, wherein one or both of said first and second components comprises at least one inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor.

182. 182. The multispecific polypeptide construct of claim 181, wherein said at least one inhibitory receptor binding region (IRBR) is positioned amino-terminal to said Fc region of said multispecific polypeptide construct and / or carboxy-terminal to said CD3 binding region of said multispecific polypeptide construct.

183. 183. The multispecific polypeptide construct of claim 181 or claim 182, comprising only one inhibitory receptor binding region (IRBR).

184. the first component comprises, in order from N-terminus to C-terminus, a first DLL3 VHH domain that binds DLL3, the first Fc polypeptide of the heterodimeric Fc region, the linker, a VH or VL domain of the anti-CD3 antibody or antigen-binding fragment, and a second DLL3 VHH domain that binds DLL3; the second component comprises an IRBR and comprises, in order from N-terminus to C-terminus, the second Fc polypeptide of the heterodimeric Fc region, the linker, optionally the same linker as present in the first component, the other of the VH or VL domains of the anti-CD3 antibody or antigen-binding fragment, wherein the IRBR is positioned amino-terminal to the Fc region of the second component or carboxy-terminal to the anti-CD3 antibody or antigen-binding fragment of the second component.

184. The multispecific polypeptide construct of any one of claims 181 to 183.

185. The at least one IRBR is The extracellular domain of the natural cognate binding partner of said inhibitory receptor or a binding fragment thereof, or a variant thereof that exhibits binding activity with said inhibitory receptor.

185. The multispecific polypeptide construct of any of claims 181-184, which is or comprises:

186. 186. The multispecific polypeptide construct of any of claims 181-185, wherein said at least one IRBR is an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

187. The multispecific polypeptide construct of claim 186, wherein the antibody or antigen-binding fragment thereof is an Fv, scFv, Fab, single domain antibody (VHH domain), VNAR, or VHH.

188. The multispecific polypeptide construct of claim 186 or claim 187, wherein said antibody or antigen-binding fragment is a VHH domain.

189. The multispecific polypeptide construct of claim 188, wherein said VHH domain is a human VHH domain or a humanized VHH domain.

190. The multispecific polypeptide construct of any of claims 181-189, wherein said at least one IRBR binds to an inhibitory receptor selected from among PD-1, CTLA-4, TIGIT, VISTA, and TIM3.

191. The multispecific polypeptide construct of any of claims 181-189, wherein said at least one IRBR binds to PD-1.

192. the first component comprises, in order from N-terminus to C-terminus, a first DLL3 VHH domain that binds DLL3, the first Fc polypeptide of the heterodimeric Fc region, the linker, a VH or VL domain of the anti-CD3 antibody or antigen-binding fragment, and a second DLL3 VHH domain that binds DLL3; the second component comprises, in order from N-terminus to C-terminus, one of the IRBR or the CRBR, the second Fc polypeptide of the heterodimeric Fc region, the linker, optionally the same linker as is present in the first component, the other of the VH or VL domains of the anti-CD3 antibody or antigen-binding fragment, and the other of the CRBR or the IRBR.

192. The multispecific polypeptide construct of any one of claims 181-191.

193. 193. The multispecific polypeptide construct of any one of claims 80-192, wherein said linker is a peptide or polypeptide linker, optionally said linker being 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

194. The multispecific polypeptide construct of any one of claims 80 to 193, wherein said linker is a non-cleavable linker.

195. The non-cleavable linker is and combinations thereof.

196. The linker has the sequence 196. The multispecific polypeptide construct of any one of claims 80 to 195, which is or comprises:

197. The multispecific polypeptide construct of any one of claims 80 to 193, wherein said linker is a cleavable linker.

198. 198. The multispecific polypeptide construct of claim 197, wherein said cleavable linker is a polypeptide that functions as a substrate for a protease.

199. The multispecific polypeptide construct of claim 198, wherein said protease is produced by an immune effector cell, by a tumor, or by a cell present in the tumor microenvironment.

200. 200. The multispecific polypeptide construct of claim 198 or claim 199, wherein said protease is produced by an immune effector cell, said immune effector cell being an activated T cell, a natural killer (NK) cell, or a NK T cell.

201. 201. The multispecific polypeptide construct of any one of claims 198-200, wherein said protease is selected from among matriptase, matrix metalloprotease (MMP), granzyme B, and combinations thereof.

202. The multispecific polypeptide construct of claim 201, wherein said protease is granzyme B.

203. The cleavable linker has the amino acid sequence 203. The multispecific polypeptide construct of any of claims 198-202, comprising:

204. a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, and 456; a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 384, 410, and 411; and Complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of NO:354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 395, and 412-415 An isolated single domain antibody that binds to DLL3, comprising:

205. 205. The isolated single domain antibody of claim 204, comprising an amino acid sequence set forth in any of SEQ ID NOs: 102, 244-275, 277-300, 302-305, 314, 401, 416, 455, 476-480-488, and 507-518, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 102, 244-275, 277-300, 302-305, 314, 401, 416, 455, 476-480-488, and 507-518, and which binds to DLL3.

206. 206. The isolated single domain antibody of claim 204 or claim 205, wherein the single domain antibody comprises (i) the sequence set forth in SEQ ID NO:244, (ii) a humanized variant of SEQ ID NO:244, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:244, and binds to DLL3.

207. The isolated single domain antibody of any one of claims 204 to 206, wherein the sdAb comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:319, 320, 321, 322, 323, 324, 325, and 326; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:336, 337, and 338; and a CDR3 comprising the amino acid sequence depicted in SEQ ID NO:354, and binds to DLL3.

208. 208. The isolated single domain antibody of any one of claims 204 to 207, wherein said sdAb comprises the CDR1, CDR2 and CDR3 as depicted in SEQ ID NOs:319, 336, and 354, respectively; SEQ ID NOs:319, 337, and 354, respectively; SEQ ID NOs:319, 338, and 354, respectively; SEQ ID NOs:320, 338, and 354, respectively; SEQ ID NOs:321, 338, and 354, respectively; SEQ ID NOs:322, 338, and 354, respectively; SEQ ID NOs:323, 338, and 354, respectively; SEQ ID NOs:324, 338, and 354, respectively; SEQ ID NOs:325, 338, and 354, respectively; or SEQ ID NOs:326, 338, and 354, respectively, and binds to DLL3.

209. 209. The isolated single domain antibody of any one of claims 204-208, wherein said sdAb comprises a sequence of amino acids as set forth in any of SEQ ID NOs:245-257, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:245-257, and binds to DLL3.

210. The isolated single domain antibody of any one of claims 204-209, wherein said sdAb comprises a sequence of amino acids set forth in any of SEQ ID NOs:245-257 and binds to DLL3.

211. 206. The isolated single domain antibody of claim 204 or claim 205, wherein the sdAb comprises (i) the sequence set forth in SEQ ID NO:258, (ii) a humanized variant of SEQ ID NO:258, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:258, and binds to DLL3.

212. The isolated single domain antibody of any one of claims 204, 205 and 211, wherein the sdAb comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:327; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:339; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:355, and binds to DLL3.

213. The isolated single domain antibody of any one of claims 204, 205, 211 and 212, wherein the sdAb comprises CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:327, 339 and 335, respectively, and binds to DLL3.

214. The isolated single domain antibody of any one of claims 204, 205, and 211-213, wherein said sdAb comprises a sequence of amino acids as set forth in any of SEQ ID NOs:259-263, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:259-263, and binds to DLL3.

215. The isolated single domain antibody of any one of claims 204, 205, and 211-214, wherein said sdAb comprises a sequence of amino acids set forth in any of SEQ ID NOs:259-263 and binds to DLL3.

216. 206. The isolated single domain antibody of claim 204 or claim 205, wherein the sdAb comprises (i) the sequence set forth in SEQ ID NO:264, (ii) a humanized variant of SEQ ID NO:264, or (iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:264, and binds to DLL3.

217. The isolated single domain antibody of claim 204, claim 205, or claim 216, wherein the sdAb comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:328, 329, or 456; a CDR2 comprising an amino acid sequence set forth in SEQ ID NO:340; and a CDR3 comprising an amino acid sequence set forth in SEQ ID NO:356, and binds to DLL3.

218. The isolated single domain antibody of any one of claims 204, 205, and 216-217, wherein said sdAb comprises CDR1, CDR2, and CDR3 as set forth in SEQ ID NOs:328, 340, and 356, respectively; or SEQ ID NOs:329, 340, and 356, respectively; and binds to DLL3.

219. 209. The isolated single domain antibody of any one of claims 204, 205, and 216-218, wherein said sdAb comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 265-274, 416, 455, or 476-478, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 265-274, 416, 455, or 476-478, and binds to DLL3.

220. The isolated single domain antibody of any one of claims 204, 205, and 216-219, wherein said at least one DLL3 sdAb comprises a sequence of amino acids set forth in any of SEQ ID NOs:265-274, 416, 455, or 476-478, and binds to DLL3.

221. 206. The isolated single domain antibody of claim 204 or claim 205, wherein the sdAb comprises (i) the sequence set forth in SEQ ID NO:275, (ii) a humanized variant of SEQ ID NO:275, or (iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:275, and binds to DLL3.

222. The isolated single domain antibody of any one of claims 204, 205, or 221, wherein the sdAb comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:320; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:341; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:357, and binds to DLL3.

223. The isolated single domain antibody of any one of claims 204, 205, and 221-222, wherein said sdAb comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 277-279 and 479, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 277-279 and 479, and binds to DLL3.

224. The isolated single domain antibody of any one of claims 204, 205, and 221-223, wherein said sdAb comprises a sequence of amino acids set forth in any of SEQ ID NOs: 277-279 and 479.

225. 206. The isolated single domain antibody of claim 204 or claim 205, wherein the sdAb comprises (i) the sequence set forth in SEQ ID NO:280, (ii) a humanized variant of SEQ ID NO:280, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:280, and binds to DLL3.

226. The isolated single domain antibody of any one of claims 204, 205 and 225, wherein the sdAb comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:330; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:342; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:358, and binds to DLL3.

227. The isolated single domain antibody of any one of claims 204, 205, and 225-226, wherein said sdAb comprises a sequence of amino acids as set forth in any of SEQ ID NOs:281-286, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:281-286, and binds to DLL3.

228. The isolated single domain antibody of any one of claims 204, 205, and 225-227, wherein said sdAb comprises a sequence of amino acids set forth in any of SEQ ID NOs:281-286 and binds to DLL3.

229. 206. The isolated single domain antibody of claim 204 or claim 205, wherein the sdAb comprises (i) the sequence set forth in SEQ ID NO:287, (ii) a humanized variant of SEQ ID NO:287, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:287, and binds to DLL3.

230. The isolated single domain antibody of any one of claims 204, 205 and 229, wherein the sdAb comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO:320; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:345, 346, and 347; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:359, 360, and 361, and binds to DLL3.

231. 231. The isolated single domain antibody of any one of claims 204, 205 and 229-230, wherein said sdAb comprises the CDR1, CDR2 and CDR3 as depicted in SEQ ID NOs:320, 345, and 359, respectively; SEQ ID NOs:320, 346, and 359, respectively; SEQ ID NOs:320, 347, and 359, respectively; SEQ ID NOs:320, 345, and 360, respectively; SEQ ID NOs:320, 345, and 361, respectively; or SEQ ID NOs:320, 347, and 360, respectively, and binds to DLL3.

232. The isolated single domain antibody of any one of claims 204, 205, and 229-231, wherein said sdAb comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 288-298 or 102, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 288-298 or 102, and binds to DLL3.

233. The isolated single domain antibody of any one of claims 204, 205, and 229-232, wherein the sdAb comprises a sequence of amino acids set forth in any of SEQ ID NOs:288-298 or 102, and binds to DLL3.

234. 206. The isolated single domain antibody of claim 204 or claim 205, wherein the sdAb comprises (i) the sequence set forth in SEQ ID NO:299, (ii) a humanized variant of SEQ ID NO:299, or (iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:299, and binds to DLL3.

235. The isolated single domain antibody of any one of claims 204, 205 and 234, wherein the sdAb comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:331; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:348, 349, and 350; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:356, and binds to DLL3.

236. The isolated single domain antibody of any one of claims 204, 205, and 234-235, wherein the sdAb comprises CDR1, CDR2, and CDR3 as set forth in SEQ ID NOs:331, 348, and 356, respectively; SEQ ID NOs:331, 349, and 356, respectively; or SEQ ID NOs:331, 350, and 356, respectively, and binds to DLL3.

237. 237. The isolated single domain antibody of any one of claims 204, 205, and 234-236, wherein said sdAb comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 300, 302-305, and 480, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 300, 302-305, and 480, and binds to DLL3.

238. The isolated single domain antibody of any one of claims 204, 205, and 234-237, wherein said sdAb comprises a sequence of amino acids set forth in any of SEQ ID NOs:300, 302-305, and 480, and binds to DLL3.

239. 206. The isolated single domain antibody of claim 204 or claim 205, wherein the sdAb comprises (i) the sequence set forth in SEQ ID NO:507, (ii) a humanized variant of SEQ ID NO:507, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:507, and binds to DLL3.

240. The isolated single domain antibody of any one of claims 204, 205 and 239, wherein the sdAb comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:332; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:348, 349, and 350; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:362, and binds to DLL3.

241. The isolated single domain antibody of any one of claims 204, 205, and 239-240, wherein the sdAb comprises CDR1, CDR2, and CDR3 as set forth in SEQ ID NOs:332, 348, and 362, respectively; SEQ ID NOs:332, 349, and 362, respectively; or SEQ ID NOs:332, 350, and 362, respectively, and binds to DLL3.

242. The isolated single domain antibody of any one of claims 204, 205, and 239-241, wherein said sdAb comprises a sequence of amino acids as set forth in any of SEQ ID NOs:508-514, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:508-514, and binds to DLL3.

243. The isolated single domain antibody of any one of claims 204, 205, and 239-242, wherein said sdAb comprises a sequence of amino acids set forth in any of SEQ ID NOs:508-514 and binds to DLL3.

244. 206. The isolated single domain antibody of claim 204 or claim 205, wherein the sdAb comprises (i) the sequence shown in SEQ ID NO:401, (ii) a humanized variant of SEQ ID NO:401, or (iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:401, and binds to DLL3.

245. The isolated single domain antibody of any one of claims 204, 205 and 244, wherein the sdAb comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO:320; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:384, 410, and 411; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:395, 412, 413, 414, and 415, and binds to DLL3.

246. 320, 384, and 415, respectively; SEQ ID NOs:320, 410, and 395, respectively; SEQ ID NOs:320, 411, and 395, respectively; SEQ ID NOs:320, 384, and 412, respectively; SEQ ID NOs:320, 384, and 413, respectively; SEQ ID NOs:320, 384, and 414, respectively; or SEQ ID NOs:320, 384, and 415, respectively, and binds to DLL3.

247. The isolated single domain antibody of any one of claims 204, 205, and 244-246, wherein said sdAb comprises a sequence of amino acids as set forth in any of SEQ ID NOs:481-488, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:481-488, and binds to DLL3.

248. The isolated single domain antibody of any one of claims 204, 205, and 244-247, wherein said sdAb comprises a sequence of amino acids set forth in any of SEQ ID NOs:481-488 and binds to DLL3.

249. 206. The isolated single domain antibody of claim 204 or claim 205, wherein the sdAb comprises (i) a sequence as set forth in SEQ ID NO:314, 518, 515, 516, or 517, (ii) a humanized variant of SEQ ID NO:314, 518, 515, 516, or 517, or (iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NO:314, 518, 515, 516, or 517, and binds to DLL3.

250. The isolated single domain antibody of any one of claims 204, 205 and 249, wherein said at least one DLL3 sdAb comprises CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:333, 351 and 363, respectively; 334, 352 and 364, respectively; 320, 353 and 365, respectively; 334, 339 and 366, respectively; or 335, 348 and 367, respectively, and binds to DLL3.

251. A polynucleotide encoding the DLL3-binding polypeptide construct of any one of claims 1 to 79.

252. A polynucleotide encoding the multispecific polypeptide construct of any one of claims 80 to 203.

253. 204. A polynucleotide comprising a first nucleic acid sequence encoding a first polypeptide of a multispecific construct according to any one of claims 80 to 203 and a second nucleic acid sequence encoding a second polypeptide of said multispecific construct, wherein said first and second nucleic acid sequences are separated by an internal ribosome entry site (IRES) or by a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping.

254. 254. The polynucleotide of claim 253, wherein said first nucleic acid sequence and said second nucleic acid sequence are operably linked to the same promoter.

255. 255. The polynucleotide of claim 254, wherein said nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping is selected from T2A, P2A, E2A, or F2A.

256. A polynucleotide encoding the single domain antibody of any one of claims 204-250.

257. A vector comprising the polynucleotide of any one of claims 251 to 256.

258. The vector of claim 257 which is an expression vector.

259. The vector of claim 257 or claim 258, which is a viral vector or a eukaryotic vector, optionally wherein the eukaryotic vector is a mammalian vector.

260. 259. A cell comprising one or more polynucleotides of any one of claims 251-256 or one or more vectors of any one of claims 257-259.

261. 261. The cell of claim 260, which is recombinant or isolated.

262. The cell of claim 261, which is a mammalian cell.

263. 259. A method of producing a polypeptide comprising the steps of introducing into a cell one or more polynucleotides of any one of claims 251-256 or one or more vectors of any one of claims 257-259 and culturing said cell under conditions to produce the multispecific polypeptide construct.

264. The method of claim 263, further comprising the step of isolating or purifying said polypeptide from said cell.

265. 265. A polypeptide produced by the method of claim 263 or claim 264.

266. An extracellular domain comprising the single domain antibody of any one of claims 204-250; A transmembrane domain; and Intracellular signaling domains An engineered immune cell comprising a chimeric antigen receptor comprising:

267. The engineered immune cell of claim 266, wherein said cell is a lymphocyte.

268. The engineered immune cell of claim 266 or claim 267, wherein said cell is a T cell or a natural killer (NK) cell.

269. The engineered immune cell of any one of claims 266-268, wherein said intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM) signaling domain.

270. The engineered immune cell of any one of claims 266-269, wherein said intracellular signaling domain is or comprises a CD3 zeta signaling domain, optionally a human CD3 zeta signaling domain.

271. The engineered immune cell of claim 269 or claim 270, wherein said intracellular signaling domain further comprises a signaling domain of a costimulatory molecule.

272. The engineered immune cell of claim 271, wherein said costimulatory molecule is CD28, ICOS, 41BB, or OX40, optionally human CD28, human ICOS, human 41BB, or human OX40.

273. 272. A pharmaceutical composition comprising a DLL3-binding polypeptide construct according to any one of claims 1 to 79, a multispecific polypeptide construct according to any one of claims 80 to 203, a single domain antibody according to any one of claims 204 to 250, or an engineered immune cell according to any one of claims 266 to 272.

274. 274. The pharmaceutical composition of claim 273, comprising a pharma- ceutically acceptable carrier.

275. The pharmaceutical composition of claim 273 or claim 274, which is sterile.

276. A method of stimulating or inducing an immune response in a subject comprising administering to a subject in need thereof a DLL3 binding polypeptide construct of any one of claims 1-79, a multispecific polypeptide construct of any one of claims 80-203, a single domain antibody of any one of claims 204-250, or an engineered immune cell of any one of claims 266-272, or a pharmaceutical composition of any one of claims 273-275.

277. The method of claim 276, wherein the immune response against a tumor or cancer, optionally a tumor or cancer that expresses DLL3, is increased.

278. The method of claim 276 or claim 277, wherein a disease or condition in the subject is treated.

279. A method of treating a disease or condition in a subject comprising administering to a subject in need thereof a therapeutically effective amount of a DLL3-binding polypeptide construct of any one of claims 1-79, a multispecific polypeptide construct of any one of claims 80-203, a single domain antibody of any one of claims 204-250, or an engineered immune cell of any one of claims 266-272, or a pharmaceutical composition of any one of claims 273-275.

280. The method of claim 278 or claim 279, wherein the disease or condition is a tumor or cancer.

281. The method of any one of claims 276-277, wherein the subject is a human.

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