Multispecific polypeptide constructs comprising constrained CD3 binding domain and receptor binding region and use methods thereof

Abstract

To provide antibodies and therapeutic agents targeting CD3 / TCR pathway.SOLUTION: Provided herein is a multispecific polypeptide construct comprising a first component comprising immunoglobulin Fc region and a second component comprising CD3 binding region, where the first and second components are coupled by a polypeptide linker, where the Fc region is positioned N-terminally relative to the CD3 binding region; one or both of the first and second components comprises at least one antigen binding domain that binds to the tumor associated antigen (TAA); and one or both of the first and second components comprises at least one co-stimulatory receptor binding region (CRBR) that binds to a co-stimulatory receptor.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority based on U.S. Provisional Application No. 62 / 702,888, filed on July 24, 2018, entitled "MULTISPECIFIC POLYPEPTIDE CONSTRUCTS CONTAINING A CONSTRAINED CD3 BINDING DOMAIN AND A CO - STIMULATORY RECEPTOR BINDING REGION AND METHODS OF USING THE SAME"; U.S. Provisional Application No. 62 / 744,641, filed on October 11, 2018, entitled "MULTISPECIFIC POLYPEPTIDE CONSTRUCTS CONTAINING A CONSTRAINED CD3 BINDING DOMAIN AND A CO - STIMULATORY RECEPTOR BINDING REGION AND METHODS OF USING THE SAME"; and U.S. Provisional Application No. 62 / 832,268, filed on April 10, 2019, entitled "MULTISPECIFIC POLYPEPTIDE CONSTRUCTS CONTAINING A CONSTRAINED CD3 BINDING DOMAIN AND A RECEPTOR BINDING REGION AND METHODS OF USING THE SAME", the contents of each of which are hereby incorporated by reference in their entireties.

[0002] Incorporation by Reference of Sequence Listing This application has been filed together with a sequence listing in electronic format. The sequence listing is provided as a file named 744952000840SeqList.TXT, created on July 23, 2019, with a size of 311 kilobytes. The information in the electronic format of the sequence listing is hereby incorporated by reference in its entirety.

[0003] Field The present invention generally relates to multispecific polypeptides that bind at least to the T cell receptors such as CD3, a second antigen, and a costimulatory or inhibitory receptor, and the multispecific polypeptides are capable of engaging CD3. In some embodiments, the multispecific polypeptide construct binds to a costimulatory receptor and provides costimulatory binding activity. In some embodiments, the multispecific polypeptide construct binds to an inhibitory receptor and blocks inhibitory activity. In some aspects, the multispecific polypeptide has restricted CD3 binding and binds or engages CD3 only when bound to a second antigen such as a tumor-associated antigen. In some embodiments, the multispecific polypeptide construct contains a cleavable linker that results in a dual effector function when cleaved. Methods of making these multispecific polypeptides and using them in a variety of therapeutic, diagnostic, and prophylactic applications are also provided.

Background Art

[0004] Background Therapeutic antibodies that cause target cell depletion generally rely on effector functions mediated through interactions with Fcγ receptors (FcγRs) and complement proteins. Effector cells expressing FcγRs are mainly of the innate immune system. T cells are not direct effector cells involved in antibody-mediated target cell depletion.

[0005] The CD3 (cluster of differentiation 3) T cell coreceptor is a multimeric protein composed of four different polypeptide chains called the ε, γ, δ, and ζ chains. The CD3 complex serves as the signaling module of the T cell receptor (TCR) by non-covalently associating with the antigen-binding a / b chains of the T cell receptor.

[0006] Direct engagement of CD3 leads to T cell activation and is thus a desirable target for a variety of therapeutic and / or diagnostic applications. Accordingly, antibodies and therapeutic agents targeting the CD3 / TCR pathway are needed.

Summary of the Invention

[0007] Summary A multispecific polypeptide construct containing a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, wherein the first and second components are coupled by a linker and the Fc region is positioned N-terminal to the CD3 binding region; one or both of the first and second components contain at least one antigen-binding domain that binds to a tumor-associated antigen (TAA); and one or both of the first and second components contain at least one costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor. Multispecific polypeptide constructs are provided herein. In some embodiments, the CD3 binding region binds to CD3 (CD3ε). In some aspects, the multispecific polypeptide construct contains at least or about or 2 antigen-binding domains and a CRBR that bind to a TAA. Exemplary constructs are described herein.

[0008] A multispecific polypeptide construct containing a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, wherein the first and second components are coupled by a linker and the Fc region is positioned N-terminal to the CD3 binding region; one or both of the first and second components contain at least one antigen-binding domain that binds to a tumor-associated antigen (TAA); and one or both of the first and second components contain at least one inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor. Multispecific polypeptide constructs are also provided herein. In some embodiments, the CD3 binding region binds to CD3 (CD3ε). In some aspects, the multispecific polypeptide construct contains at least or about or 2 antigen-binding domains and an IRBR that bind to a TAA. Exemplary constructs are described herein.

[0009] A multispecific polypeptide construct containing a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, wherein the first and second components are coupled by a linker, the Fc region is positioned N-terminal to the CD3 binding region; one or both of the first and second components contain at least one antigen-binding domain that binds to a tumor-associated antigen (TAA); one or both of the first and second components include at least one inhibitory receptor-binding region (IRBR) that binds to an inhibitory receptor; and one or both of the first and second components include at least one co-stimulatory receptor-binding region (CRBR) that binds to a co-stimulatory receptor, is also provided herein. In some embodiments, the CD3 binding region binds to CD3 (CD3ε). In some aspects, the multispecific polypeptide construct contains at least or about or 2 antigen-binding domains, a CRBR, and an IRBR that bind to a TAA. Exemplary constructs are described herein.

[0010] In some embodiments, at least one antigen-binding domain is positioned amino-terminal to the Fc region of the multispecific polypeptide construct and / or carboxy-terminal to the CD3 binding region. In some embodiments, at least one co-stimulatory 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.

[0011] In some embodiments, the first component includes a first antigen-binding domain, the second component includes a second antigen-binding domain, and each of the antigen-binding domains binds to a tumor-associated antigen (TAA). In some embodiments, the first antigen-binding domain is positioned amino-terminal to the Fc region of the multispecific construct, and the second antigen-binding domain is positioned carboxy-terminal to the CD3 binding region of the multispecific construct. In some embodiments, the first or second component further contains a co-stimulatory receptor-binding region (CRBR).

[0012] In order from the N-terminus to the C-terminus, a costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor and / or an antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker; a CD3 (CD3ε)-binding CD3-binding region; and a costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor and / or an antigen-binding domain that binds to a tumor-associated antigen (TAA). A multispecific polypeptide construct containing at least one CRBR and at least one antigen-binding domain is provided herein. In some embodiments, the multispecific polypeptide construct contains only one costimulatory receptor binding region (CRBR). In some embodiments, the multispecific polypeptide construct contains two antigen-binding domains that bind to a TAA. In some embodiments, the antigen-binding domains bind to the same tumor-associated antigen (TAA). In some embodiments, one antigen-binding domain is positioned on the amino-terminal side with respect to the Fc region, and one antigen-binding domain is positioned on the carboxy-terminal side with respect to the CD3-binding region.

[0013] A multispecific polypeptide construct containing, in order from the N-terminus to the C-terminus, an immunoglobulin Fc region; a linker; a CD3 (CD3ε)-binding CD3-binding region; and an antigen-binding domain that binds to a tumor-associated antigen (TAA) and a costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor is provided herein. A multispecific polypeptide construct containing, in order from the N-terminus to the C-terminus, an antigen-binding domain that binds to a tumor-associated antigen (TAA) and a costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor; an immunoglobulin Fc region; a linker; and a CD3 (CD3ε)-binding CD3-binding region is provided herein.

[0014] In some of such embodiments, the Fc region is a homodimeric Fc region. In some examples, the Fc region is the Fc region of human IgG1, human IgG2, human IgG3, or human IgG4, or an immunologically active fragment thereof. In some cases, the Fc region comprises a polypeptide having the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO:1. In some embodiments, the Fc region comprises a polypeptide having the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO:2; or the Fc region comprises a polypeptide having the amino acid sequence shown in SEQ ID NO:4, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO:4; or the Fc region comprises a polypeptide having the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO:5.

[0015] In some embodiments, the Fc region is a heterodimeric Fc region. In some embodiments, one or both Fc polypeptides of the heterodimeric Fc region optionally include at least one modification for inducing heterodimerization as compared to the polypeptide of the homodimeric Fc region or as compared to the Fc polypeptide shown in SEQ ID NO:1 or an immunologically active fragment thereof. In some embodiments, each of the Fc polypeptides of the heterodimeric Fc independently includes at least one amino acid modification. In some embodiments, each of the Fc polypeptides of the heterodimeric Fc includes a knob-into-hole modification or a charge variant for increasing the electrostatic complementarity of the polypeptide. In some cases, the amino acid modification is a knob-into-hole modification.

[0016] In some embodiments, the first Fc polypeptide of the heterodimeric Fc includes a modification selected from Thr366Ser, Leu368Ala, Tyr407Val, and combinations thereof, and the second Fc polypeptide of the heterodimeric Fc includes the modification Thr366Trp. In some embodiments, the first and second Fc polypeptides further include a modification of a non-cysteine residue to a cysteine residue, the modification of the first polypeptide being at one of positions Ser354 and Tyr349, and the modification of the second Fc polypeptide being at the other of positions Ser354 and Tyr349. In some embodiments, the amino acid modification is a charge variant for increasing the electrostatic complementarity of the polypeptide.

[0017] In 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, the modification being a substitution to an amino acid having an opposite charge to the complementary amino acid of the other polypeptide. In some embodiments, one of the first or second Fc polypeptides of the heterodimeric Fc further comprises a modification at residue Ile253. In some cases, the modification is Ile253Arg. In some instances, one of the first or second Fc polypeptides of the heterodimeric Fc further comprises a modification at residue His435. In some examples, the modification is His435Arg. In some embodiments, the Fc region comprises a polypeptide lacking Lys447.

[0018] In some embodiments, the Fc region comprises a polypeptide containing at least one modification for enhancing FcRn binding. In some examples, 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 at a position 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.

[0019] In some embodiments, the first polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any of SEQ ID NO:82, 86, 94, or 96, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any of SEQ ID NO:83, 87, 90, 92, 98, or 100. In some embodiments, the Fc region comprises a polypeptide comprising at least one amino acid modification that reduces effector function and / or reduces binding to an effector molecule selected from Fcγ receptor or C1q. In some embodiments, one or more of the amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235. In some embodiments, the first polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any of SEQ ID NO:84, 88, 95, or 97, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any of SEQ ID NO:85, 89, 91, 93, 99, or 101. In some embodiments, the Fc region comprises a polypeptide comprising at least one modification for enhancing FcγR binding. In some embodiments, the modification is a modification at Ser239 or Ile332. In some embodiments, the glycosylation of the Fc region is modified to enhance FcγR binding as compared to the unmodified Fc region. In some embodiments, the Fc region lacks fucose or has a reduced fucose content.

[0020] In some of such embodiments, the CD3 binding region is an anti-CD3 antibody or antigen-binding fragment. In some embodiments, the anti-CD3 antibody or antigen-binding fragment comprises a variable heavy chain region (VH) and a variable light chain region (VL). In some embodiments, the CD3 binding region is monovalent. In some embodiments, the CD3 binding region is a variable fragment (Fv) comprising a variable heavy chain region (VH) and a variable light chain region (VL). In some embodiments, the anti-CD3 antibody or antigen-binding fragment is not a single-chain antibody and optionally not a single-chain variable fragment (scFv). In some embodiments, the Fc is a heterodimeric Fc, and the VH and VL that constitute the anti-CD3 antibody or antigen-binding fragment are linked to the opposing polypeptides of the heterodimeric Fc.

[0021] In some embodiments, the CD3 binding region cannot or substantially cannot bind or engage CD3 unless at least one of the antigen-binding domains binds to its TAA. In some embodiments, the CD3 binding region cannot or substantially cannot bind or engage CD3 unless at least two of the antigen-binding domains bind to its TAA.

[0022] In some embodiments, the linker is a polypeptide linker. In some examples, the linker is a polypeptide up to 25 amino acids in length. In some embodiments, the linker is a polypeptide of 2 - 24 amino acids, 2 - 20 amino acids, 2 - 18 amino acids, 2 - 14 amino acids, 2 - 12 amino acids, 2 - 10 amino acids, 2 - 8 amino acids, 2 - 6 amino acids, 6 - 24 amino acids, 6 - 20 amino acids, 6 - 18 amino acids, 6 - 14 amino acids, 6 - 12 amino acids, 6 - 10 amino acids, 6 - 8 amino acids, 8 - 24 amino acids, 8 - 20 amino acids, 8 - 18 amino acids, 8 - 14 amino acids, 8 - 12 amino acids, 8 - 10 amino acids, 10 - 24 amino acids, 10 - 20 amino acids, 10 - 18 amino acids, 10 - 14 amino acids, 10 - 12 amino acids, 12 - 24 amino acids, 12 - 20 amino acids, 12 - 18 amino acids, 12 - 14 amino acids, 14 - 24 amino acids, 14 - 20 amino acids, 14 - 18 amino acids, 18 - 24 amino acids, 18 - 20 amino acids, or 20 - 24 amino acids, or about 2 - 24 amino acids, 2 - 20 amino acids, 2 - 18 amino acids, 2 - 14 amino acids, 2 - 12 amino acids, 2 - 10 amino acids, 2 - 8 amino acids, 2 - 6 amino acids, 6 - 24 amino acids, 6 - 20 amino acids, 6 - 18 amino acids, 6 - 14 amino acids, 6 - 12 amino acids, 6 - 10 amino acids, 6 - 8 amino acids, 8 - 24 amino acids, 8 - 20 amino acids, 8 - 18 amino acids, 8 - 14 amino acids, 8 - 12 amino acids, 8 - 10 amino acids, 10 - 24 amino acids, 10 - 20 amino acids, 10 - 18 amino acids, 10 - 14 amino acids, 10 - 12 amino acids, 12 - 24 amino acids, 12 - 20 amino acids, 12 - 18 amino acids, 12 - 14 amino acids, 14 - 24 amino acids, 14 - 20 amino acids, 14 - 18 amino acids, 18 - 24 amino acids, 18 - 20 amino acids, or 20 - 24 amino acids. In some embodiments, the linker is a polypeptide of 3 amino acids in length, 4 amino acids in length, 5 amino acids in length, 6 amino acids in length, 7 amino acids in length, 8 amino acids in length, 9 amino acids in length, 10 amino acids in length, 11 amino acids in length, 12 amino acids in length, 13 amino acids in length, 14 amino acids in length, 15 amino acids in length, 16 amino acids in length, 17 amino acids in length, 18 amino acids in length, 19 amino acids in length, or 20 amino acids in length. In some cases, the linker is a polypeptide of 3 - 18 amino acids in length.In some embodiments, the linker is a polypeptide that is 12 to 18 amino acids in length. In some embodiments, the linker is a polypeptide that is 15 to 18 amino acids in length.

[0023] In some embodiments, the linker is a non-cleavable linker. In some embodiments, the non-cleavable linker does not contain a substrate recognition site that is specifically recognized by a protease for cleavage. In some embodiments, the non-cleavable linker includes GS, GGS, GGGGS (SEQ ID NO:149), GGGGGGGS (SEQ ID NO:135), and combinations thereof. In some embodiments, the non-cleavable linker includes (GGS)n where n is from 1 to 10. In some embodiments, the non-cleavable linker includes (GGGGS)n (SEQ ID NO:173) where n is from 1 to 10. In some embodiments, the non-cleavable linker includes (GGGGGS)n (SEQ ID NO:172) where n is from 1 to 4. In some embodiments, the non-cleavable linker includes GGS. In some embodiments, the non-cleavable linker includes GGGGS (SEQ ID NO:149). In some embodiments, the non-cleavable linker includes GGGGGGGS (SEQ ID NO:135). In some embodiments, the non-cleavable linker includes (GGS)2 (SEQ ID NO:10). In some embodiments, the non-cleavable linker includes GGSGGSGGS (SEQ ID NO:11). In some embodiments, the non-cleavable linker includes GGSGGSGGSGGS (SEQ ID NO:12). In some embodiments, the non-cleavable linker includes TIFF2025098077000001.tif4128. In some embodiments, the non-cleavable linker includes TIFF2025098077000002.tif4128. In some embodiments, the non-cleavable linker includes TIFF2025098077000003.tif4128. In some embodiments, the non-cleavable linker includes It includes TIFF2025098077000004.tif4128.

[0024] In some embodiments, the linker is a cleavable linker.

[0025] A multispecific polypeptide construct containing a first component comprising a heterodimeric Fc region and 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 the opposing polypeptides of the heterodimeric Fc; the first and second components are coupled by a cleavable linker, and the heterodimeric Fc region is positioned N-terminal to the anti-CD3 antibody; one or both of the first and second components comprise at least one antigen-binding domain that binds to a tumor-associated antigen (TAA); one or both of the first and second components comprise at least one costimulatory receptor-binding region (CRBR) that binds to a costimulatory receptor. Multispecific polypeptide constructs are provided herein. In some embodiments, the binding of the CD3-binding region to CD3 is substantially reduced compared to the cleaved state when the multispecific polypeptide construct is in the uncleaved state. In some embodiments, in the cleaved state, the first and second components are not linked.

[0026] In some embodiments, the cleavable linker is a polypeptide that functions as a substrate for a protease. In some embodiments, the protease is produced by immune effector cells, by tumors, or by cells present in the tumor microenvironment. In some embodiments, the protease is produced by immune effector cells, and the immune effector cells are activated T cells, natural killer (NK) cells, or NK T cells. In some embodiments, the protease is selected from the group consisting of matriptase, matrix metalloprotease (MMP), granzyme B, and combinations thereof. In some embodiments, the protease is granzyme B.

[0027] In some embodiments, the cleavable linker comprises the general formula P4 P3 P2 P1↓P1' (SEQ ID NO:150) (where P4 is amino acid I, L, Y, M, F, V, or A; P3 is amino acid A, G, S, V, E, D, Q, N, or Y; P2 is amino acid H, P, A, V, G, S, or T; P1 is amino acid D or E; P1' is amino acid I, L, Y, M, F, V, T, S, G, or A). In some embodiments, the cleavable linker comprises the general formula P4 P3 P2 P1↓P1' (SEQ ID NO:151) (where P4 is amino acid I or L; P3 is amino acid E; P2 is amino acid P or A; P1 is amino acid D; P1' is amino acid I, V, T, S, or G). In some embodiments, the cleavable linker comprises the amino acid sequences IEPDI (SEQ ID NO:136), LEPDG (SEQ ID NO:152), LEADT (SEQ ID NO:137), IEPDG (SEQ ID NO:138), IEPDV (SEQ ID NO:139), IEPDS (SEQ ID NO:140), IEPDT (SEQ ID NO:141), or LEADG (SEQ ID NO:153). In some embodiments, the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:22, 105 - 112, 136 - 141, 148, 150 - 153. In some examples, the cleavable linker comprises the amino acid sequence shown in SEQ ID NO:105.

[0028] In some embodiments, the protease is matriptase. In some embodiments, the cleavable linker comprises the sequence P1QAR↓(A / V)(SEQ ID NO:154) (where P1 is any amino acid); or the cleavable linker comprises the sequence RQAR(A / V)(SEQ ID NO:155). In some examples, the cleavable linker comprises the sequence RQARV(SEQ ID NO:156). In some embodiments, the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:23, 154 - 156. In some embodiments, the protease is MMP. In some embodiments, the MMP is MMP-2. In some examples, the cleavable linker contains the general formula P3 P2 P1↓P1' (SEQ ID NO:157) (where P3 is P, V, or A; P2 is Q or D; P1 is A or N; P1' is L, I, or M). In some embodiments, the cleavable linker contains the general formula P3 P2 P1↓P1' (SEQ ID NO:158) (where P3 is P; P2 is Q or D; P1 is A or N; P1' is L or I). In some cases, the cleavable linker comprises the sequence PAGL(SEQ ID NO:24). In some embodiments, the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:22 - 31, 104 - 114, 117 - 118, 136 - 144, 148, 150 - 158.

[0029] In some embodiments, the multispecific polypeptide construct comprises a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and a VH domain or a VL domain of an anti-CD3 antibody or antigen-binding fragment; and a second polypeptide comprising a second Fc polypeptide of the heterodimeric Fc region, a linker, optionally the same linker as present in the first polypeptide, and the other of the VH domain or VL domain of the anti-CD3 antibody or antigen-binding fragment, wherein one or both of the first and second components comprise at least one antigen-binding domain that binds to a tumor-associated antigen (TAA), and one or both of the first and second components comprise at least one costimulatory receptor-binding region (CRBR) that binds to a costimulatory receptor, and the multispecific polypeptide construct contains at least one CRBR and at least one antigen-binding domain.

[0030] In some embodiments, only one of the first or second polypeptides comprises at least one antigen-binding domain that binds to a TAA. In some embodiments, the at least one antigen-binding domain is positioned amino-terminal to an Fc region of one of the first or second polypeptides of the multispecific polypeptide construct and / or carboxy-terminal to the CD3-binding region. In some embodiments, the at least one antigen-binding domain is positioned amino-terminal to the Fc region of the multispecific construct and a second antigen-binding domain is positioned carboxy-terminal to the CD3-binding region of the multispecific construct. In some embodiments, only one of the first or second polypeptides comprises at least one costimulatory receptor-binding region (CRBR) that binds to a costimulatory receptor. In some embodiments, the costimulatory receptor-binding region (CRBR) is positioned amino-terminal to an Fc region of one of the first or second polypeptides of the multispecific polypeptide construct or carboxy-terminal to the CD3-binding region.

[0031] In some embodiments, the first polypeptide contains, in order from the N-terminus to the C-terminus, a first antigen-binding domain that binds to a tumor-associated antigen (TAA), a first Fc polypeptide of a heterodimeric Fc region, a linker, the VL or VH of an anti-CD3 antibody or antigen-binding fragment, and a second antigen-binding domain that binds to a tumor-associated antigen (TAA); the second polypeptide contains, in order from the N-terminus to the C-terminus, a second Fc polypeptide of a heterodimeric Fc region, a linker, optionally the same linker as present in the first polypeptide, the other of the VH or VL of an anti-CD3 antibody or antigen-binding fragment, and a co-stimulatory receptor-binding region (CRBR) that binds to a co-stimulatory receptor.

[0032] In some embodiments, the antigen-binding domain, or each of the antigen-binding domains, independently contains an extracellular domain of a native cognate binding partner of the TAA or a binding fragment thereof, or a variant thereof that exhibits binding activity with the TAA. In some embodiments, the antigen-binding domain, or each of the antigen-binding domains, independently is an antibody or an antigen-binding fragment thereof selected from the group consisting of a Fab fragment, an 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.

[0033] In some embodiments, at least one costimulatory receptor binding region (CRBR) is, or comprises, the extracellular domain or a binding fragment thereof of a native cognate binding partner of a costimulatory receptor, or variants thereof that exhibit binding activity to the 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 Fab fragments, F(ab')2 fragments, Fv fragments, scFv, scAb, dAb, single-domain heavy-chain antibodies, and single-domain light-chain antibodies. In some embodiments, the antibody or an antigen-binding fragment thereof is an Fv, scFv, Fab, single-domain antibody (sdAb), VNAR, or VHH. In some examples, the antibody or an antigen-binding fragment thereof is an sdAb. In some embodiments, the sdAb is a human sdAb or a humanized sdAb. In some embodiments, the sdAb is a VHH, VNAR, a modified VH domain, or a modified VK domain. In some embodiments, the antibody or an antigen-binding fragment thereof is an scFv. In some embodiments, the antibody or an antigen-binding fragment thereof is a Fab.

[0034] In some embodiments, the multispecific polypeptide construct comprises a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and the VH domain of an anti-CD3 antibody or antigen-binding fragment; a second polypeptide comprising a second Fc polypeptide of a heterodimeric Fc region, a linker, and the VL domain of an anti-CD3 antibody or antigen-binding fragment, a third polypeptide containing the VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen, and a fourth polypeptide containing the VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a costimulatory receptor, wherein the first and / or second polypeptide further contains (1) the other of the VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen, and (2) the other of the VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a costimulatory receptor.

[0035] In some embodiments, the multispecific polypeptide construct comprises a first polypeptide containing a first Fc polypeptide of a heterodimeric Fc region, a linker, and the VH domain of an anti-CD3 antibody or antigen-binding fragment; a second polypeptide containing a second Fc polypeptide of the heterodimeric Fc region, a linker, and the VL domain of an anti-CD3 antibody or antigen-binding fragment; and a third polypeptide containing the VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a costimulatory receptor, wherein the first and / or second polypeptide further contains the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment that binds to the costimulatory receptor, and the first and / or second polypeptide further contains at least one antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0036] In some embodiments, the multispecific polypeptide construct comprises a first polypeptide containing a first Fc polypeptide of a heterodimeric Fc region, a linker, and the VH domain of an anti-CD3 antibody or antigen-binding fragment; a second polypeptide containing a second Fc polypeptide of the heterodimeric Fc region, a linker, and the VL domain of an anti-CD3 antibody or antigen-binding fragment, and a third polypeptide containing the VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen, wherein the first and / or second polypeptide further contains the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment that binds to the tumor-associated antigen, and the first and / or second polypeptide further contains at least one costimulatory receptor-binding region (CRBR) that binds to a costimulatory receptor.

[0037] In some embodiments, only one of the first or second polypeptides contains the other of VH-CH1 (Fd) or VL-CL of the Fab antibody fragment. In some embodiments, both the first and second polypeptides contain the other of VH-CH1 (Fd) or VL-CL of the Fab antibody fragment. In some embodiments, the other of VH-CH1 (Fd) or VL-CL of the Fab antibody fragment is positioned on the amino-terminal side with respect to the Fc region of one of the first or second polypeptides of the multispecific polypeptide construct and / or on the carboxy-terminal side with respect to the CD3 binding region. In some embodiments, the other of VH-CH1 (Fd) or VL-CL of the Fab antibody fragment is positioned on the amino-terminal side with respect to the Fc region of the first or second polypeptide and on the carboxy-terminal side with respect to the CD3 binding region of the other of the first or second polypeptides.

[0038] In some of such embodiments, the antigen-binding domain, or each of the antigen-binding domains independently, is 1-92-LFA-3, 5T4, α4 integrin, αV integrin, α4β1 integrin, α4β7 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, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, 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, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor α (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIt binds to tumor antigens selected from IIb / 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, IL23, IL23R, IL27 / IL27R (wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged1, Jagged2, 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, TIGIT, 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.

[0039] In some embodiments, the antigen-binding domain comprises at least a first antigen-binding domain and a second antigen-binding domain, and the first antigen-binding domain and the second antigen-binding domain bind to the same TAA. In some embodiments, the first antigen-binding domain and the second antigen-binding domain bind to different epitopes of the same TAA. In some embodiments, the first antigen-binding domain and the second antigen-binding domain bind to the same epitope of the same TAA. In some embodiments, the antigen-binding domain comprises at least a first antigen-binding domain and a second antigen-binding domain, and the first antigen-binding domain and the second antigen-binding domain bind to different TAAs.

[0040] In some embodiments, the co-stimulatory receptor binding region (CRBR) comprises at least a first CRBR and a second CRBR, and the first CRBR and the second CRBR bind to the same co-stimulatory receptor. In some embodiments, the first co-stimulatory receptor binding region (CRBR) and the second CRBR bind to different epitopes of the same co-stimulatory receptor. In some embodiments, the first co-stimulatory receptor binding region (CRBR) and the second CRBR bind to the same epitope of the same co-stimulatory receptor. In some embodiments, the co-stimulatory receptor binding region (CRBR) comprises at least a first CRBR and a second CRBR. In some embodiments, the first CRBR and the second CRBR bind to different co-stimulatory receptors.

[0041] In some embodiments, at least one co-stimulatory receptor binding region (CRBR) binds to a co-stimulatory receptor selected from 41BB (CD137), OX40 (CD134), CD27, glucocorticoid-induced 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 co-stimulatory receptor binding region (CRBR) binds to a co-stimulatory receptor selected from 41BB (CD137), OX40 (CD134), and glucocorticoid-induced TNFR-related protein (GITR).

[0042] In any of the provided aspects, at least one inhibitory receptor binding region (IRBR) is an antibody or an antigen-binding fragment thereof selected from the group consisting of a Fab fragment, an 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. In some aspects, the antibody or antigen-binding fragment thereof is an Fv, scFv, Fab, single-domain antibody (sdAb), VNAR, or VHH. In some aspects, the antibody or antigen-binding fragment is an sdAb. In some aspects, the sdAb is a human sdAb or a humanized sdAb. In some aspects, the sdAb is a VHH, VNAR, modified VH domain, or modified VK domain. In some aspects, the antibody or antigen-binding fragment thereof is a scFv. In some aspects, the antibody or antigen-binding fragment thereof is a Fab. In any of the aspects provided herein that include a construct containing at least one inhibitory receptor binding domain, the inhibitory receptor is selected from PD-1, CTLA-4, TIGIT, VISTA, or TIM3 as expressed in T cells, e.g., human T cells. In some aspects, the T cell is an activated T cell. In any of the provided aspects, at least one inhibitory receptor binding region (IRBR) binds to PD-1.

[0043] In some embodiments, the multispecific polypeptide construct contains a first linker peptide (LP1) between the first antigen-binding domain and the Fc region. In some embodiments, the multispecific polypeptide construct contains a second linker peptide (LP2) between the CD3-binding region and the second antigen-binding domain. In some embodiments, the multispecific polypeptide construct contains a first linker peptide (LP1) between the first costimulatory receptor-binding region (CRBR) and the Fc region. In some embodiments, the multispecific polypeptide construct contains a second linker peptide (LP2) between the CD3-binding region and the second costimulatory receptor-binding region (CRBR). In some embodiments, the multispecific polypeptide construct includes a first linker peptide (LP1) between the antigen-binding domain or costimulatory receptor-binding region and the Fc region and a second linker peptide (LP2) between the CD3-binding region and the antigen-binding domain or CRBR, and has a structural arrangement of the first antigen-binding domain or CRBR - LP1 - Fc region - linker - CD3-binding region - LP2 - the second antigen-binding domain or CRBR from the N-terminus to the C-terminus.

[0044] In some embodiments, the linker is a cleavable linker. In some embodiments, the two linker peptides are not identical to each other. In some embodiments, LP1 or LP2 is independently a peptide about 1 to 20 amino acids in length. In some embodiments, LP1 or LP2 is independently a Gly-Ser linker or GGS shown in SEQ ID NOs: 10 to 13, 119, 135, 147, 149, or contains a peptide containing the same.

[0045] In some embodiments, the anti-CD3 antibody or antigen-binding fragment is an Fv antibody fragment. In some embodiments, the Fv antibody fragment includes a disulfide-stabilized anti-CD3 binding Fv (dsFv). In some embodiments, the anti-CD3 antibody or antigen-binding fragment includes a VH CDR1 containing the amino acid sequence TYAMN (SEQ ID NO: 16); VH CD2 including TIFF2025098077000005.tif4128; VH CDR3 including the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:18), VL CDR1 including the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:19); VL CDR2 including the amino acid sequence GTNKRAP (SEQ ID NO:20); and VL CDR3 including the amino acid sequence ALWYSNLWV (SEQ ID NO:21).

[0046] In some examples, the anti-CD3 Fv comprises a VH having the amino acid sequence of any one of SEQ ID NO:14 and 32 - 62, or a sequence showing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO:14 and 32 - 62; and a VL having the amino acid sequence of any one of SEQ ID NO:15 and 63 - 81, or a sequence showing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO:15 and 63 - 81.

[0047] In some embodiments, the anti-CD3 antibody or antigen-binding fragment is an Fv. In some embodiments, the anti-CD3 Fv has an amino acid sequence of any one of SEQ ID NO: 14, 32-43, 45-47, 48, and 287, or a VH having a sequence showing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 14, 32-43, 45-47, 48, and 287; and a VL having an amino acid sequence of any one of SEQ ID NO: 15, 63, 65-71, 73, 75, 77, and 288, or a sequence showing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 15, 63, 65-71, 73, 75, 77, and 288. In some embodiments, the anti-CD3 Fv comprises the amino acid sequence of SEQ ID NO: 14 and the amino acid sequence of SEQ ID NO: 15. In other cases, the anti-CD3 Fv comprises the amino acid sequence of SEQ ID NO: 287 and the amino acid sequence of SEQ ID NO: 288.

[0048] In some embodiments, the VH chain region and the VL chain region of the CD3-binding domain each independently comprise at least one amino acid modification. In some embodiments, at least one amino acid modification of the VH chain region and the VL chain region of the CD3-binding domain increases the stability of the CD3-binding domain. In some embodiments, at least one amino acid modification of the VH chain region and the VL chain region of the CD3-binding domain increases the ability of the CD3-binding domain to bind to CD3. In some embodiments, at least one amino acid modification of the VH chain region and the VL chain region of the CD3-binding domain increases the stability of the CD3-binding domain by creating a disulfide linkage between the VH chain region and the VL chain region.

[0049] In some embodiments, the CD3 binding region has a disulfide-stabilized bond between the VH region and the VL region. In some embodiments, the anti-CD3 antibody or antigen-binding fragment is a disulfide-stabilized Fv (dsFv). In some embodiments, the disulfide-stabilized anti-CD3 Fv comprises an anti-CD3 VH containing a mutation to Cys at position 44 and an anti-CD3 VL containing a mutation to Cys at position 100, according to Kabat numbering. In some embodiments, the disulfide-stabilized anti-CD3 Fv comprises an anti-CD3 VH containing the mutation G44C and an anti-CD3 VL containing the mutation G100C, according to Kabat numbering. In some embodiments, the disulfide-stabilized anti-CD3 Fv comprises an anti-CD3 VH containing a mutation to Cys at position 105 and an anti-CD3 VL containing a mutation to Cys at position 43, according to Kabat numbering. In some embodiments, the anti-CD3 dsFv comprises a VH having an amino acid sequence of any one of SEQ ID NO: 44, 49-62, 290, and 311, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NO: 44, 49-62, 290, and 311; and a VL having an amino acid sequence of any one of SEQ ID NO: 64, 72, 74, 76, 78-81, 241, and 289, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NO: 64, 72, 74, 76, 78-81, 241, and 289. In some embodiments, the anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO: 44 and the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO: 44 and the amino acid sequence of SEQ ID NO: 241. In some embodiments, its anti-CD3ε binding domain comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 290 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 289.In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 311 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 289.

[0050] In some of such embodiments, the multispecific polypeptide construct is conjugated to an agent. In some embodiments, the agent is a therapeutic agent, an anti-tumor agent, a toxin or a fragment thereof, a detectable moiety, or a diagnostic agent. In some embodiments, the agent is conjugated to the multispecific polypeptide construct via a linker.

[0051] Polynucleotides encoding any of the provided multispecific polypeptide constructs are provided herein. Polynucleotides encoding any of the provided polypeptide chains of any of the provided multispecific polypeptide constructs are provided herein. A polynucleotide containing a first nucleic acid sequence encoding a first polypeptide of any of the provided multispecific constructs and a second nucleic acid sequence encoding any of the provided second polypeptides of the multispecific construct, 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, is provided herein. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter. In some embodiments, the multispecific polypeptide construct contains a third polypeptide chain, and the polynucleotide further contains a third nucleic acid encoding the third polypeptide of the multispecific construct. In some embodiments, the third nucleic acid is separated from the first and / or second polypeptides by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, and / or the third nucleic acid sequence is operably linked to the same promoter as the first and / or second nucleic acid sequences. In some embodiments, the nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping is selected from T2A, P2A, E2A, or F2A.

[0052] Vectors containing any of the provided polynucleotides are provided herein. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector or a eukaryotic vector, and optionally, the eukaryotic vector is a mammalian vector.

[0053] Cells containing any of the polynucleotides or the provided polynucleotides, or any of the vectors or the provided vectors are provided herein. In some embodiments, the cells are recombinant or isolated. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are HEK293 cells or CHO cells.

[0054] A method for producing a multispecific polypeptide construct is provided herein, which includes the step of introducing into a cell any of the polynucleotides or the provided polynucleotides, or any of the vectors or the provided vectors, and culturing the cell under conditions in which a multispecific polypeptide construct is produced.

[0055] A method for producing a multispecific polypeptide construct is provided herein, which includes culturing any of the provided cells under conditions in which a multispecific polypeptide is produced by the cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are HEK293 cells or CHO cells. In some embodiments, the method further includes isolating or purifying the multispecific polypeptide construct from the cells. In some embodiments, the multispecific polypeptide construct is a heterodimer.

[0056] Multispecific polypeptide constructs produced by any of the provided methods are provided herein.

[0057] Pharmaceutical compositions containing any of the provided multispecific polypeptide constructs and a pharmaceutically acceptable carrier are provided herein. In some embodiments, the pharmaceutical composition is sterile.

[0058] A method of stimulating or inducing an immune response comprising contacting a target cell and a T cell with any of the provided multispecific polypeptide constructs or pharmaceutical compositions, wherein the target cell expresses a tumor-associated antigen recognized by the multispecific polypeptide construct, is provided herein. In some embodiments, the target cell is a tumor cell expressing a tumor-associated antigen (TAA). In some embodiments, the multispecific polypeptide construct comprises a cleavable linker that functions as a substrate for a protease, and induction or stimulation of the immune response is increased in the presence of the protease. In some embodiments, the protease is produced by immune effector cells, by a tumor, or by cells present in the tumor microenvironment. In some embodiments, the protease is produced by immune effector cells, and the immune effector cells are activated T cells, natural killer (NK) cells, or NK T cells. In some embodiments, the immune effector cells are in the vicinity of cells expressing an antigen. In some embodiments, the protease is produced by a tumor in the vicinity of cells expressing TAA in a tissue and / or by a tumor co-localized with TAA in a tissue, and when the multispecific polypeptide construct is exposed to the protease, the protease cleaves the cleavable linker within the multispecific polypeptide construct.

[0059] In some embodiments, the protease is selected from the group consisting of matriptase, matrix metalloprotease (MMP), granzyme B, and combinations thereof. In some embodiments, the protease is granzyme B. In some embodiments, the contacting is performed ex vivo or in vitro. In some embodiments, the contacting is performed in vivo in a subject.

[0060] Provided herein is a method of stimulating or inducing an immune response in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of any of the provided multispecific conjugates or pharmaceutical compositions. In some embodiments, the method increases cellular immunity. In some embodiments, the method increases T cell activity. In some embodiments, the method increases cytotoxic T lymphocyte (CTL) activity. In some embodiments, the immune response against a tumor or cancer is increased. In some embodiments, the method treats a disease or condition in the subject.

[0061] Provided herein is a method of treating a disease or condition in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of any of the provided multispecific conjugates or any of the provided pharmaceutical compositions. In some embodiments, the disease or condition is a tumor or cancer. In some embodiments, the subject is human.

[0062] One of ordinary skill in the art will recognize that the antibodies of the present disclosure have a variety of uses. For example, the proteins of the present disclosure are used as therapeutic agents for a variety of disorders. The antibodies of the present disclosure are used as reagents in diagnostic kits or as diagnostic tools, or these antibodies may be used in competitive assays to generate therapeutic reagents. [Invention 1001] A multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, wherein the first and second components are coupled by a linker and the Fc region is positioned N-terminal to the CD3 binding region; wherein one or both of the first and second components comprise at least one antigen binding domain that binds to a tumor associated antigen (TAA); wherein one or both of the first and second components comprise at least one costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor. Multispecific polypeptide construct. [Invention 1002] A multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, wherein the first and second components are coupled by a linker and the Fc region is positioned N-terminal to the CD3 binding region; wherein one or both of the first and second components comprise at least one antigen-binding domain that binds to a tumor-associated antigen (TAA); wherein one or both of the first and second components comprise at least one inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor, Multispecific polypeptide construct. [Invention 1003] A multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, wherein the first and second components are coupled by a linker and the Fc region is positioned N-terminal to the CD3 binding region; wherein one or both of the first and second components comprise at least one antigen-binding domain that binds to a tumor-associated antigen (TAA); wherein one or both of the first and second components comprise at least one co-stimulatory receptor binding region (CRBR) that binds to a co-stimulatory receptor; wherein one or both of the first and second components comprise at least one inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor, Multispecific polypeptide construct. [Invention 1004] The multispecific polypeptide construct according to any one of Inventions 1001 to 1003, wherein the CD3 binding region binds to CD3 (CD3ε). [Invention 1005] Any of the multispecific polypeptide constructs of the present invention 1001 to 1004, wherein at least one antigen-binding domain is positioned on the amino-terminal side with respect to the Fc region of the multispecific polypeptide construct and / or on the carboxy-terminal side with respect to the CD3-binding region. [The present invention 1006] Any of the multispecific polypeptide constructs of the present invention 1001 to 1005, wherein the first component contains a first antigen-binding domain, the second component contains a second antigen-binding domain, and each of the antigen-binding domains binds to a tumor-associated antigen (TAA). [The present invention 1007] The multispecific polypeptide construct of the present invention 1006, wherein the first antigen-binding domain is positioned on the amino-terminal side with respect to the Fc region of the multispecific construct, and the second antigen-binding domain is positioned on the carboxy-terminal side with respect to the CD3-binding region of the multispecific construct. [The present invention 1008] Any of the multispecific polypeptide constructs of the present invention 1001 and 1003 to 1007, wherein at least one co-stimulatory receptor-binding region (CRBR) is positioned on the amino-terminal side with respect to the Fc region of the multispecific polypeptide construct and / or on the carboxy-terminal side with respect to the CD3-binding region. [The present invention 1009] Any of the multispecific polypeptide constructs of the present invention 1001 and 1003 to 1008, wherein at least one co-stimulatory receptor-binding region (CRBR) is positioned on the carboxy-terminal side with respect to the CD3-binding region of the multispecific polypeptide construct. [The present invention 1010] Any of the multispecific polypeptide constructs of the present invention 1002 to 1007, wherein at least one inhibitory receptor-binding region (IRBR) is positioned on the amino-terminal side with respect to the Fc region of the multispecific polypeptide construct and / or on the carboxy-terminal side with respect to the CD3-binding region. [The present invention 1011] A multispecific polypeptide construct according to any one of inventions 1002 to 1007 and 1010, wherein at least one inhibitory receptor binding region (IRBR) is positioned on the carboxy-terminal side with respect to the CD3 binding region of the multispecific polypeptide construct. [Invention 1012] A multispecific polypeptide construct according to any one of inventions 1001 to 1011, wherein the first component comprises a first antigen-binding domain and the second component comprises a second antigen-binding domain, and each of the antigen-binding domains binds to a tumor-associated antigen (TAA). [Invention 1013] A multispecific polypeptide construct according to invention 1012, wherein the first antigen-binding domain is positioned on the amino-terminal side with respect to the Fc region of the multispecific construct, and the second antigen-binding domain is positioned on the carboxy-terminal side with respect to the CD3 binding region of the multispecific construct. [Invention 1014] A multispecific polypeptide construct according to invention 1012 or invention 1013, wherein the first or second component further comprises a costimulatory receptor binding region (CRBR). [Invention 1015] A multispecific polypeptide construct according to invention 1012 or invention 1013, wherein the first or second component further comprises an inhibitory receptor binding region (IRBR). [Invention 1016] In order from the N-terminus to the C-terminus, a costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor and / or an antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker; a CD3 binding region that binds to CD3 (CD3ε); and a costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor and / or an antigen-binding domain that binds to a tumor-associated antigen (TAA) A multispecific polypeptide construct comprising: a multispecific polypeptide construct comprising at least one CRBR and at least one antigen-binding domain. [Invention 1017] The multispecific polypeptide construct of Invention 1016, which contains only one costimulatory receptor binding region (CRBR). [Invention 1018] In order from the N-terminus to the C-terminus, An inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor and / or an antigen-binding domain that binds to a tumor-associated antigen (TAA); An immunoglobulin Fc region; A linker; A CD3 binding region that binds to CD3 (CD3ε); and An inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor and / or an antigen-binding domain that binds to a tumor-associated antigen (TAA) A multispecific polypeptide construct comprising: A multispecific polypeptide construct comprising at least one IRBR and at least one antigen-binding domain. [Invention 1019] The multispecific polypeptide construct of Invention 1018, which contains only one inhibitory receptor binding region (IRBR). [Invention 1020] In order from the N-terminus to the C-terminus, One of an inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor or a costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor, and / or an antigen-binding domain that binds to a tumor-associated antigen (TAA); An immunoglobulin Fc region; A linker; A CD3 binding region that binds to CD3 (CD3ε); and The other of IRBR or CRBR, and / or an antigen-binding domain that binds to a tumor-associated antigen (TAA) A multispecific polypeptide construct comprising: A multispecific polypeptide construct comprising at least one IRBR, at least one CRBR, and at least one antigen-binding domain. [Invention 1021] A multispecific polypeptide construct according to any one of inventions 1016 to 1020, comprising two antigen-binding domains that bind to TAA. [Invention 1022] A multispecific polypeptide construct according to invention 1021, wherein the antigen-binding domains bind to the same tumor-associated antigen (TAA). [Invention 1023] A multispecific polypeptide construct according to invention 1021 or invention 1022, wherein one antigen-binding domain is positioned on the amino-terminal side with respect to the Fc region, and one antigen-binding domain is positioned on the carboxy-terminal side with respect to the CD3-binding region. [Invention 1024] In order from the N-terminus to the C-terminus, an immunoglobulin Fc region; a linker; a CD3-binding region that binds to CD3 (CD3ε); and an antigen-binding domain that binds to a tumor-associated antigen (TAA) and a co-stimulatory receptor-binding region (CRBR) that binds to a co-stimulatory receptor A multispecific polypeptide construct comprising. [Invention 1025] In order from the N-terminus to the C-terminus, an antigen-binding domain that binds to a tumor-associated antigen (TAA) and a co-stimulatory receptor-binding region (CRBR) that binds to a co-stimulatory receptor; an immunoglobulin Fc region; a linker; and a CD3-binding region that binds to CD3 (CD3ε) A multispecific polypeptide construct comprising. [Invention 1026] In order from the N-terminus to the C-terminus, an immunoglobulin Fc region; a linker; a CD3-binding region that binds to CD3 (CD3ε); and an antigen-binding domain that binds to a tumor-associated antigen (TAA) and an inhibitory receptor-binding region (IRBR) that binds to an inhibitory receptor A multispecific polypeptide construct comprising. [Invention 1027] In order from the N-terminus to the C-terminus, an antigen-binding domain that binds to a tumor-associated antigen (TAA) and an inhibitory receptor-binding region (IRBR) that binds to an inhibitory receptor; an immunoglobulin Fc region; a linker; and a CD3-binding region that binds to CD3 (CD3ε) A multispecific polypeptide construct comprising. [Invention 1028] A multispecific polypeptide construct according to any one of Inventions 1001 to 1027, wherein the Fc region is a homodimeric Fc region. [Invention 1029] A multispecific polypeptide construct according to any one of Inventions 1001 to 1028, wherein the Fc region is the Fc region of human IgG1, human IgG2, human IgG3, or human IgG4, or an immunologically active fragment thereof. [Invention 1030] A multispecific polypeptide construct according to any one of Inventions 1001 to 1029, wherein the Fc region comprises a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 1. [Invention 1031] Does the Fc region comprise a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 2; Does the Fc region comprise a polypeptide having the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 4; or The Fc region comprises a polypeptide comprising the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO:5. Any of the multispecific polypeptide constructs of the present invention 1001 to 1029. [The present invention 1032] Any of the multispecific polypeptide constructs of the present invention 1001 to 1023, 1029 to 1031, wherein the Fc region is a heterodimeric Fc region. [The present invention 1033] Any of the multispecific polypeptide constructs of the present invention 1032, wherein one or both of the Fc polypeptides of the heterodimeric Fc region optionally comprise at least one modification for inducing heterodimerization compared to the polypeptide of the homodimeric Fc region or compared to the Fc polypeptide shown in SEQ ID NO:1 or an immunologically active fragment thereof. [The present invention 1034] Any of the multispecific polypeptide constructs of the present invention 1033, wherein each of the Fc polypeptides of the heterodimeric Fc independently comprises at least one amino acid modification. [The present invention 1035] Any of the multispecific polypeptide constructs of the present invention 1034, wherein each of the Fc polypeptides of the heterodimeric Fc comprises a knob-into-hole modification or a charge variation for increasing the electrostatic complementarity of the polypeptide. [The present invention 1036] Any of the multispecific polypeptide constructs of the present invention 1035, wherein the amino acid modification is a knob-into-hole modification. [The present invention 1037] Any of the multispecific polypeptide constructs of the present invention 1032 to 1036, wherein the CD3 binding region comprises a variable heavy chain region (VH) and a variable light chain region (VL), the VL is on the C-terminal side with respect to the first Fc polypeptide of the heterodimeric Fc region, the VH is on the C-terminal side with respect to the second Fc polypeptide of the heterodimeric Fc region, the first Fc polypeptide comprises a hole mutation, and the second Fc polypeptide comprises a knob mutation. [The present invention 1038] The first Fc polypeptide of the heterodimeric Fc contains a modification selected from Thr366Ser, Leu368Ala, Tyr407Val, and combinations thereof, and the second Fc polypeptide of the heterodimeric Fc contains the modified Thr366Trp. Any of the multispecific polypeptide constructs of the present inventions 1032 to 1037. [The present invention 1039] The first and second Fc polypeptides further contain a modification of a non-cysteine residue to a cysteine residue, the modification of the first 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. The multispecific polypeptide construct of the present invention 1038. [The present invention 1040] The multispecific polypeptide construct of the present invention 1035, wherein the amino acid modification is a charge mutation for increasing the electrostatic complementarity of the polypeptide. [The present invention 1041] The first and / or second Fc polypeptide or each of the first and second Fc polypeptides contains a modification at a complementary position, and the modification is a substitution with an amino acid having a charge opposite to that of the complementary amino acid of the other polypeptide. Any of the multispecific polypeptide constructs of the present inventions 1032 to 1035 and 1040. [The present invention 1042] One of the first or second Fc polypeptides of the heterodimeric Fc further contains a modification at residue Ile253. Any of the multispecific polypeptide constructs of the present inventions 1033 to 1041. [The present invention 1043] The multispecific polypeptide construct of the present invention 1042, wherein the modification is Ile253Arg. [The present invention 1044] One of the first or second Fc polypeptides of the heterodimeric Fc further contains a modification at residue His435. Any of the multispecific polypeptide constructs of the present inventions 1033 to 1043. [The present invention 1045] The multispecific polypeptide construct of the 1044th invention of the present invention, wherein the modification is His435Arg. [The 1046th invention of the present invention] The multispecific polypeptide construct of any one of the 1001st to 1045th inventions of the present invention, wherein the Fc region comprises a polypeptide lacking Lys447. [The 1047th invention of the present invention] The multispecific polypeptide construct of any one of the 1001st to 1046th inventions of the present invention, wherein the Fc region comprises a polypeptide comprising at least one modification for enhancing FcRn binding. [The 1048th invention of the present invention] The multispecific polypeptide construct of the 1047th invention of the present invention, wherein the modification is at a position selected from the group consisting of Met252, Ser254, Thr256, Met428, Asn434, and combinations thereof. [The 1049th invention of the present invention] The multispecific polypeptide construct of the 1048th invention of the present invention, wherein the modification is at a position selected from the group consisting of Met252Y, Ser254T, Thr256E, Met428L, Met428V, Asn434S, and combinations thereof. [The 1050th invention of the present invention] The multispecific polypeptide construct of the 1048th invention of the present invention, wherein the modification is at position Met252 and position Met428. [The 1051st invention of the present invention] The multispecific polypeptide construct of the 1050th invention of the present invention, wherein the modification is Met252Y and Met428L. [The 1052nd invention of the present invention] The multispecific polypeptide construct of the 1050th invention of the present invention, wherein the modification is Met252Y and Met428V. [The 1053rd invention of the present invention] The multispecific polypeptide construct of any one of the 1032nd to 1052nd inventions of the present invention, wherein the first polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NO: 82, 86, 94, or 96, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NO: 83, 87, 90, 92, 98, or 100. [The present invention 1054] The first polypeptide of the heterodimeric Fc contains the amino acid sequence shown in any of SEQ ID NO: 291, 293, 297, or 298, and the second polypeptide of the heterodimeric Fc contains the amino acid sequence shown in any of SEQ ID NO: 292, 294, 295, 296, 299, or 300, a multispecific polypeptide construct of any of the present inventions 1032 - 1053. [The present invention 1055] The Fc region A polypeptide containing at least one amino acid modification that reduces effector function and / or reduces binding to an effector molecule selected from Fcγ receptor or C1q A multispecific polypeptide construct of any of the present inventions 1001 - 1054. [The present invention 1056] The multispecific polypeptide construct of the present invention 1055, wherein one or more amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235. [The present invention 1057] The first polypeptide of the heterodimeric Fc contains the amino acid sequence shown in any of SEQ ID NO: 84, 88, 95, or 97, and the second polypeptide of the heterodimeric Fc contains the amino acid sequence shown in any of SEQ ID NO: 85, 89, 91, 93, 99, or 101, a multispecific polypeptide construct of any of the present inventions 1032 - 1056. [The present invention 1058] The first polypeptide of the heterodimeric Fc contains the amino acid sequence shown in any of SEQ ID NO: 291, 293, 297, or 298, and the second polypeptide of the heterodimeric Fc contains the amino acid sequence shown in any of SEQ ID NO: 292, 294, 295, 296, 299, or 300, a multispecific polypeptide construct of any of the present inventions 1032 - 1056. [The present invention 1059] A multispecific polypeptide construct according to any one of the present inventions 1001 to 1054, comprising a polypeptide in which the Fc region comprises at least one modification for enhancing FcγR binding. [Present Invention 1060] A multispecific polypeptide construct according to Present Invention 1059, wherein the modification is a modification at Ser239 or Ile332. [Present Invention 1061] A multispecific polypeptide construct according to any one of the present inventions 1001 to 1054 and 1059, wherein the glycosylation of the Fc region is modified to enhance FcγR binding as compared to the unmodified Fc region. [Present Invention 1062] A multispecific polypeptide construct according to Present Invention 1061, wherein the Fc region lacks fucose or has a reduced fucose content. [Present Invention 1063] A multispecific polypeptide construct according to any one of the present inventions 1001 to 1062, wherein the CD3 binding region is an anti-CD3 antibody or an antigen-binding fragment. [Present Invention 1064] A multispecific polypeptide construct according to Present Invention 1063, wherein the anti-CD3 antibody or antigen-binding fragment comprises a variable heavy chain region (VH) and a variable light chain region (VL). [Present Invention 1065] A multispecific construct according to Present Invention 1064, wherein the VL of the anti-CD3 antibody or antigen-binding fragment is linked to the first Fc polypeptide of a heterodimeric Fc comprising a modification selected from Thr366Ser, Leu368Ala, Tyr407Val, and combinations thereof, and the VH of the anti-CD3 antibody or antigen-binding fragment is linked to the second Fc polypeptide of a heterodimeric Fc comprising the modified Thr366Trp. [Present Invention 1066] A multispecific polypeptide construct according to any one of the present inventions 1001 to 1065, wherein the CD3 binding region is monovalent. [Present Invention 1067] A multispecific polypeptide construct according to any one of the present inventions 1001 to 1066, wherein the CD3 binding region is a variable fragment (Fv) comprising a variable heavy chain region (VH) and a variable light chain region (VL). [The present invention 1068] A multispecific polypeptide construct according to any one of the present inventions 1063 to 1066, wherein the anti-CD3 antibody or antigen-binding fragment is not a single-chain antibody and optionally not a single-chain variable fragment (scFv). [The present invention 1069] A multispecific polypeptide construct according to any one of the present inventions 1064 to 1068, wherein the Fc is a heterodimeric Fc, and the VH and VL constituting the anti-CD3 antibody or antigen-binding fragment are linked to the opposing polypeptides of the heterodimeric Fc. [The present invention 1070] A multispecific polypeptide construct according to any one of the present inventions 1001 to 1069, wherein the CD3-binding region cannot or substantially cannot bind or engage CD3 unless at least one of the antigen-binding domains binds to its TAA. [The present invention 1071] A multispecific polypeptide construct according to any one of the present inventions 1001 to 1070, wherein the CD3-binding region cannot or substantially cannot bind or engage CD3 unless at least two of the antigen-binding domains bind to its TAA. [The present invention 1072] A multispecific polypeptide construct according to any one of the present inventions 1001 to 1071, wherein the linker is a polypeptide linker. [The present invention 1073] The multispecific polypeptide construct of the present invention 1072, wherein the linker is a polypeptide having a maximum length of 25 amino acids. [The present invention 1074] The linker is a polypeptide of 2 to 24 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to 24 amino acids, 6 to 20 amino acids, 6 to 18 amino acids, 6 to 14 amino acids, 6 to 12 amino acids, 6 to 10 amino acids, 6 to 8 amino acids, 8 to 24 amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14 amino acids, 8 to 12 amino acids, 8 to 10 amino acids, 10 to 24 amino acids, 10 to 20 amino acids, 10 to 18 amino acids, 10 to 14 amino acids, 10 to 12 amino acids, 12 to 24 amino acids, 12 to 20 amino acids, 12 to 18 amino acids, 12 to 14 amino acids, 14 to 24 amino acids, 14 to 20 amino acids, 14 to 18 amino acids, 18 to 24 amino acids, 18 to 20 amino acids, or 20 to 24 amino acids, or about 2 to 24 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to 24 amino acids, 6 to 20 amino acids, 6 to 18 amino acids, 6 to 14 amino acids, 6 to 12 amino acids, 6 to 10 amino acids, 6 to 8 amino acids, 8 to 24 amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14 amino acids, 8 to 12 amino acids, 8 to 10 amino acids, 10 to 24 amino acids, 10 to 20 amino acids, 10 to 18 amino acids, 10 to 14 amino acids, 10 to 12 amino acids, 12 to 24 amino acids, 12 to 20 amino acids, 12 to 18 amino acids, 12 to 14 amino acids, 14 to 24 amino acids, 14 to 20 amino acids, 14 to 18 amino acids, 18 to 24 amino acids, 18 to 20 amino acids, or 20 to 24 amino acids, of the multispecific polypeptide construct of the present invention 1072 or the present invention 1073. [The present invention 1075] The linker is a polypeptide of 3 amino acids in length, 4 amino acids in length, 5 amino acids in length, 6 amino acids in length, 7 amino acids in length, 8 amino acids in length, 9 amino acids in length, 10 amino acids in length, 11 amino acids in length, 12 amino acids in length, 13 amino acids in length, 14 amino acids in length, 15 amino acids in length, 16 amino acids in length, 17 amino acids in length, 18 amino acids in length, 19 amino acids in length, or 20 amino acids in length, of any of the multispecific polypeptide constructs of the present invention 1072 to 1074. [The present invention 1076] The linker is a polypeptide that is 3 to 18 amino acids in length, and any of the multispecific polypeptide constructs of the present invention Nos. 1072 to 1075. [The present invention 1077] The linker is a polypeptide that is 12 to 18 amino acids in length, and any of the multispecific polypeptide constructs of the present invention Nos. 1072 to 1075. [The present invention 1078] The linker is a polypeptide that is 15 to 18 amino acids in length, and any of the multispecific polypeptide constructs of the present invention Nos. 1072 to 1075. [The present invention 1079] The linker is a non-cleavable linker, and any of the multispecific polypeptide constructs of the present invention Nos. 1001 to 1078. [The present invention 1080] The non-cleavable linker does not contain a substrate recognition site that is specifically recognized by a protease for cleavage, and the multispecific polypeptide construct of the present invention 1079. [The present invention 1081] The non-cleavable linker includes GS, GGS, GGGGS (SEQ ID NO: 149), GGGGGGGS (SEQ ID NO: 135), and combinations thereof, and the multispecific polypeptide construct of the present invention 1079 or the present invention 1080. [The present invention 1082] The non-cleavable linker includes (GGS)n, where n is 1 to 10, and any of the multispecific polypeptide constructs of the present invention Nos. 1079 to 1081. [The present invention 1083] The non-cleavable linker includes (GGGGS)n (SEQ ID NO: 173), where n is 1 to 10, and any of the multispecific polypeptide constructs of the present invention Nos. 1079 to 1082. [The present invention 1084] The non-cleavable linker includes (GGGGGS)n (SEQ ID NO: 172), where n is 1 to 4, and any of the multispecific polypeptide constructs of the present invention Nos. 1079 to 1083. [The present invention 1085] A non-cleavable linker-containing multispecific polypeptide construct according to any one of aspects 1079 to 1084 of the present invention, which contains GGS. [Aspect 1086] A non-cleavable linker-containing multispecific polypeptide construct according to any one of aspects 1079 to 1084 of the present invention, which contains GGGGS (SEQ ID NO: 149). [Aspect 1087] A non-cleavable linker-containing multispecific polypeptide construct according to any one of aspects 1079 to 1084 of the present invention, which contains GGGGGG (SEQ ID NO: 135). [Aspect 1088] A non-cleavable linker-containing multispecific polypeptide construct according to any one of aspects 1079 to 1084 of the present invention, which contains (GGS)2 (SEQ ID NO: 10). [Aspect 1089] A non-cleavable linker-containing multispecific polypeptide construct according to any one of aspects 1079 to 1084 of the present invention, which contains GGSGGSGGS (SEQ ID NO: 11). [Aspect 1090] A non-cleavable linker-containing multispecific polypeptide construct according to any one of aspects 1079 to 1084 of the present invention, which contains GGSGGSGGSGGS (SEQ ID NO: 12). [Aspect 1091] The non-cleavable linker is A non-cleavable linker-containing multispecific polypeptide construct according to any one of aspects 1079 to 1084 of the present invention, which contains TIFF2025098077000006.tif4128. [Aspect 1092] The non-cleavable linker is A non-cleavable linker-containing multispecific polypeptide construct according to any one of aspects 1079 to 1084 of the present invention, which contains TIFF2025098077000007.tif4128. [Aspect 1093] The non-cleavable linker is A non-cleavable linker-containing multispecific polypeptide construct according to any one of aspects 1079 to 1084 of the present invention, which contains TIFF2025098077000008.tif4128. [Aspect 1094] The non-cleavable linker is A multispecific polypeptide construct according to any one of inventions 1079 to 1084, comprising TIFF2025098077000009.tif4128. [Invention 1095] A multispecific polypeptide construct according to any one of inventions 1001 to 1078, wherein the linker is a cleavable linker. [Invention 1096] A multispecific polypeptide construct comprising a first component comprising a heterodimeric Fc region and 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 the opposing polypeptides of the heterodimeric Fc; wherein the first and second components are coupled by a cleavable linker, and the heterodimeric Fc region is positioned N-terminal to the anti-CD3 antibody; wherein one or both of the first and second components comprise at least one antigen-binding domain that binds to a tumor-associated antigen (TAA); wherein one or both of the first and second components comprise at least one costimulatory receptor-binding region (CRBR) that binds to a costimulatory receptor, A multispecific polypeptide construct. [Invention 1097] The multispecific polypeptide construct of invention 1096, wherein one or both of the first and second components further comprise at least one inhibitory receptor-binding region (IRBR) that binds to an inhibitory receptor. [Invention 1098] A multispecific polypeptide construct comprising a first component comprising a heterodimeric Fc region and 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 the opposing polypeptides of the heterodimeric Fc; The first and second components are coupled by a cleavable linker, and the heterodimeric Fc region is positioned N-terminal to the anti-CD3 antibody; One or both of the first and second components comprise at least one antigen-binding domain that binds to a tumor-associated antigen (TAA); One or both of the first and second components comprise at least one inhibitory receptor-binding region (IRBR) that binds to an inhibitory receptor, Multispecific polypeptide construct. [Inventive concept 1099] The multispecific polypeptide construct of inventive concept 1098, wherein one or both of the first and second components further comprise at least one costimulatory receptor-binding region (CRBR) that binds to a costimulatory receptor. [Inventive concept 1100] The multispecific polypeptide construct according to any one of inventive concepts 1096 to 1099, wherein when the multispecific polypeptide construct is in the uncleaved state, the binding of the CD3-binding region to CD3 is substantially reduced compared to the cleaved state. [Inventive concept 1101] The multispecific polypeptide construct according to any one of inventive concepts 1096 to 1100, wherein in the cleaved state, the first and second components are not linked. [Inventive concept 1102] The multispecific polypeptide construct according to any one of inventive concepts 1096 to 1101, wherein the cleavable linker is a polypeptide that functions as a substrate for a protease. [Inventive concept 1103] The multispecific polypeptide construct of inventive concept 1102, wherein the protease is produced by immune effector cells, by a tumor, or by cells present in the tumor microenvironment. [Inventive concept 1104] The multispecific polypeptide construct of inventive concept 1103, wherein the protease is produced by immune effector cells, and the immune effector cells are activated T cells, natural killer (NK) cells, or NK T cells. [Inventive concept 1105] A multispecific polypeptide construct according to any of aspects 1102-1104 of the present invention, wherein the protease is selected from matriptase, matrix metalloprotease (MMP), granzyme B, and combinations thereof. [Aspect 1106] A multispecific polypeptide construct according to aspect 1105 of the present invention, wherein the protease is granzyme B. [Aspect 1107] A multispecific polypeptide construct according to any of aspects 1096-1106 of the present invention, wherein the cleavable linker comprises an amino acid sequence of the general formula P4 P3 P2 P1↓P1' (SEQ ID NO:150), where P4 is amino acid I, L, Y, M, F, V, or A; P3 is amino acid A, G, S, V, E, D, Q, N, or Y; P2 is amino acid H, P, A, V, G, S, or T; P1 is amino acid D or E; and P1' is amino acid I, L, Y, M, F, V, T, S, G, or A. [Aspect 1108] A multispecific polypeptide construct according to any of aspects 1096-1107 of the present invention, wherein the cleavable linker comprises an amino acid sequence of the general formula P4 P3 P2 P1↓P1' (SEQ ID NO:151), where P4 is amino acid I or L; P3 is amino acid E; P2 is amino acid P or A; P1 is amino acid D; and P1' is amino acid I, V, T, S, or G. [Aspect 1109] A multispecific polypeptide construct according to any of aspects 1096-1108 of the present invention, wherein the cleavable linker comprises the amino acid sequence IEPDI (SEQ ID NO:136), LEPDG (SEQ ID NO:152), LEADT (SEQ ID NO:137), IEPDG (SEQ ID NO:138), IEPDV (SEQ ID NO:139), IEPDS (SEQ ID NO:140), IEPDT (SEQ ID NO:141), or LEADG (SEQ ID NO:153). [Aspect 1110] The multispecific polypeptide construct of any one of aspects 1096 - 1109 of the present invention, wherein the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:22, 105 - 112, 136 - 141, 148, 150 - 153. [Aspect 1111] The multispecific polypeptide construct of any one of aspects 1096 - 1110 of the present invention, wherein the cleavable linker comprises the amino acid sequence shown in SEQ ID NO:105. [Aspect 1112] The multispecific polypeptide construct of aspect 1111 of the present invention, wherein the protease is matriptase. [Aspect 1113] The cleavable linker comprises the sequence P1QAR↓(A / V)(SEQ ID NO:154) (where P1 is any amino acid); or The cleavable linker comprises the sequence RQAR(A / V)(SEQ ID NO:155), The multispecific polypeptide construct of any one of aspects 1096 - 1112 of the present invention. [Aspect 1114] The multispecific polypeptide construct of any one of aspects 1096 - 1113 of the present invention, wherein the cleavable linker comprises the sequence RQARV(SEQ ID NO:156). [Aspect 1115] The multispecific polypeptide construct of any one of aspects 1096 - 1114 of the present invention, wherein the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:23, 154 - 156. [Aspect 1116] The multispecific polypeptide construct of aspect 1105 of the present invention, wherein the protease is MMP. [Aspect 1117] The multispecific polypeptide construct of aspect 1116 of the present invention, wherein MMP is MMP - 2. [Aspect 1118] A cleavable linker contains the general formula P3 P2 P1↓P1' (SEQ ID NO:157), where P3 is P, V, or A; P2 is Q or D; P1 is A or N; P1' is L, I, or M, and is any of the multispecific polypeptide constructs of the present invention from 1096 to 1117. [The present invention 1119] A cleavable linker contains the general formula P3 P2 P1↓P1' (SEQ ID NO:158), where P3 is P; P2 is Q or D; P1 is A or N; P1' is L or I, and is any of the multispecific polypeptide constructs of the present invention from 1096 to 1118. [The present invention 1120] A cleavable linker contains the sequence PAGL (SEQ ID NO:24), and is any of the multispecific polypeptide constructs of the present invention from 1096 to 1119. [The present invention 1121] A cleavable linker contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 - 31, 104 - 114, 117 - 118, 136 - 144, 148, 150 - 158, and is any of the multispecific polypeptide constructs of the present invention from 1096 to 1120. [The present invention 1122] The multispecific polypeptide construct is (i) a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and a VH domain or a 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 domain or VL domain of an anti - CD3 antibody or antigen - binding fragment and comprises at least One or both of the first and second polypeptides contain at least one antigen - binding domain that binds to a tumor - associated antigen (TAA), and one or both of the first and second components contain at least one co - stimulatory receptor - binding region (CRBR) that binds to a co - stimulatory receptor. The multispecific polypeptide construct comprises at least one CRBR and at least one antigen-binding domain. The multispecific polypeptide construct according to any one of claims 1069 to 1121 of the present invention. [Claim 1123 of the present invention] The multispecific polypeptide construct according to any one of claims 1001 to 1122 of the present invention, wherein one or more antigen-binding domains that bind to the TAA result in monovalent, divalent, trivalent, or tetravalent binding to the TAA. [Claim 1124 of the present invention] The multispecific polypeptide construct according to claim 1122 or claim 1123 of the present invention, wherein only one of the first or second polypeptides comprises at least one antigen-binding domain that binds to the TAA. [Claim 1125 of the present invention] The multispecific polypeptide construct according to any one of claims 1122 to 1124 of the present invention, wherein at least one antigen-binding domain is positioned on the amino-terminal side with respect to the Fc region of one of the first or second polypeptides of the multispecific polypeptide construct and / or on the carboxy-terminal side with respect to the CD3-binding region. [Claim 1126 of the present invention] The multispecific polypeptide construct according to any one of claims 1122 to 1124 of the present invention, wherein at least one antigen-binding domain is positioned on the amino-terminal side with respect to the Fc region of the multispecific construct and the second antigen-binding domain is positioned on the carboxy-terminal side with respect to the CD3-binding region of the multispecific construct. [Claim 1127 of the present invention] The multispecific polypeptide construct according to any one of claims 1122 to 1126 of the present invention, wherein only one of the first or second polypeptides comprises at least one co-stimulatory receptor-binding region (CRBR) that binds to a co-stimulatory receptor. [Claim 1128 of the present invention] A costimulatory receptor binding region (CRBR) is positioned on the amino-terminal side with respect to the Fc region of one of the first or second polypeptides of the multispecific polypeptide construct, or on the carboxy-terminal side with respect to the CD3 binding region, of any of the multispecific polypeptide constructs of the present invention 1122-1127. [The present invention 1129] The first polypeptide comprises, in order from the N-terminus to the C-terminus, a first antigen-binding domain that binds to a tumor-associated antigen (TAA), a first Fc polypeptide of a heterodimeric Fc region, a linker, the VL or VH of an anti-CD3 antibody or antigen-binding fragment, and a second antigen-binding domain that binds to a tumor-associated antigen (TAA); The second polypeptide comprises, in order from the N-terminus to the C-terminus, a second Fc polypeptide of a heterodimeric Fc region, a linker, optionally the same linker as present in the first polypeptide, the other of the VL or VH of an anti-CD3 antibody or antigen-binding fragment, and a costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor. Any of the multispecific polypeptide constructs of the present invention 1122-1128. [The present invention 1130] A heterodimeric Fc region comprises an Fc hole polypeptide and an Fc knob polypeptide, the VL of the anti-CD3 antibody or antigen-binding fragment is positioned on the C-terminal side with respect to the Fc hole, and the VH of the anti-CD3 antibody or antigen-binding fragment is positioned on the C-terminal side with respect to the Fc knob, of any of the multispecific polypeptide constructs of the present invention 1122-1129. [The present invention 1131] The antigen-binding domain, or each of the antigen-binding domains, independently comprises the extracellular domain of the native cognate binding partner of the TAA or a binding fragment thereof, or variants thereof that exhibit binding activity to the TAA, of any of the multispecific polypeptide constructs of the present invention 1001-1130. [The present invention 1132] The antigen-binding domain, or each of the antigen-binding domains, is independently an antibody selected from the group consisting of Fab fragments, F(ab')2 fragments, Fv fragments, scFv, scAb, dAb, single-domain heavy-chain antibodies, and single-domain light-chain antibodies, or an antigen-binding fragment thereof, of any of the multispecific polypeptide constructs of the present invention 1001 to 1131. [The present invention 1133] At least one costimulatory receptor-binding region (CRBR) is an extracellular domain of a native cognate binding partner of a costimulatory receptor or a binding fragment thereof, or a variant thereof that exhibits binding activity to the costimulatory receptor, or contains the same, of any of the multispecific polypeptide constructs of the present invention 1001 to 1132. [The present invention 1134] At least one costimulatory receptor-binding region (CRBR) is an antibody selected from the group consisting of Fab fragments, F(ab')2 fragments, Fv fragments, scFv, scAb, dAb, single-domain heavy-chain antibodies, and single-domain light-chain antibodies, or an antigen-binding fragment thereof, of any of the multispecific polypeptide constructs of the present invention 1001 to 1132. [The present invention 1135] The antibody or an antigen-binding fragment thereof is an Fv, scFv, Fab, or single-domain antibody (sdAb) of the multispecific polypeptide construct of the present invention 1132 or the present invention 1134. [The present invention 1136] The antibody or antigen-binding fragment is an sdAb of the multispecific polypeptide construct of the present invention 1132, the present invention 1134, or the present invention 1122. [The present invention 1137] One or both of the first and second components contain an antigen-binding domain that binds to a tumor-associated antigen (TAA), and the antigen-binding domain is a single-chain antibody fragment, of any of the multispecific polypeptide constructs of the present invention 1001 to 1136. [The present invention 1138] The single-chain antibody fragment is a single-domain antibody or a single-chain variable fragment (scFv) of the multispecific polypeptide construct of the present invention 1137. [The present invention 1139] The multispecific polypeptide construct of any one of inventions 1136 to 1138, wherein the sdAb is a human sdAb or a humanized sdAb. [Invention 1140] The multispecific polypeptide construct of any one of inventions 1136 to 1139, wherein the sdAb is a VHH, VNAR, modified VH domain, or modified VK domain. [Invention 1141] The multispecific polypeptide construct of invention 1132, invention 1134, or invention 1135, wherein the antibody or antigen-binding fragment thereof is a scFv. [Invention 1142] The multispecific polypeptide construct of invention 1132, invention 1134, or invention 1135, wherein the antibody or antigen-binding fragment thereof is a Fab. [Invention 1143] The multispecific polypeptide construct comprises (i) a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and the VH domain of an anti-CD3 antibody or antigen-binding fragment; (ii) a second polypeptide comprising a second Fc polypeptide of a heterodimeric Fc region, a linker, and the VL domain of an anti-CD3 antibody or antigen-binding fragment; (iii) a third polypeptide comprising the VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen; (iv) a fourth polypeptide comprising the VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a costimulatory receptor and the first and / or second polypeptide further comprises (1) the other of the VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen, and (2) the other of the VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a costimulatory receptor The multispecific polypeptide construct of any one of inventions 1001 to 1132 and 1134 to 1142. [Invention 1144] The multispecific polypeptide construct comprises (i) a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and a VH domain of an anti-CD3 antibody or antigen-binding fragment; (ii) a second polypeptide comprising a second Fc polypeptide of a heterodimeric Fc region, a linker, and a VL domain of an anti-CD3 antibody or antigen-binding fragment; and (iii) a third polypeptide comprising a VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a costimulatory receptor and the first and / or second polypeptide further comprises the other of VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a costimulatory receptor, the first and / or second polypeptide further comprises at least one antigen-binding domain that binds to a tumor-associated antigen (TAA), the multispecific polypeptide construct according to any one of 1001-1132 and 1134-1142 of the present invention. [Invention 1145] The multispecific polypeptide construct comprises (i) a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and a VH domain of an anti-CD3 antibody or antigen-binding fragment; (ii) a second polypeptide comprising a second Fc polypeptide of a heterodimeric Fc region, a linker, and a VL domain of an anti-CD3 antibody or antigen-binding fragment; and (iii) a third polypeptide comprising a VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen and the first and / or second polypeptide further comprises the other of VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen, the first and / or second polypeptide further comprises at least one costimulatory receptor-binding region (CRBR) that binds to a costimulatory receptor, The multispecific polypeptide construct of any one of the present inventions 1001 to 1132 and 1134 to 1142. [The present invention 1146] The multispecific polypeptide construct of the present invention 1144 or the present invention 1145, wherein only one of the first or second polypeptides contains the other of VH-CH1 (Fd) or VL-CL of the Fab antibody fragment. [The present invention 1147] The multispecific polypeptide construct of the present invention 1144 or the present invention 1145, wherein both of the first or second polypeptides contain the other of VH-CH1 (Fd) or VL-CL of the Fab antibody fragment. [The present invention 1148] The multispecific polypeptide construct of the present invention 1146 or the present invention 1147, wherein the other of VH-CH1 (Fd) or VL-CL of the Fab antibody fragment is positioned on the amino-terminal side with respect to the Fc region of one of the first or second polypeptides of the multispecific polypeptide construct and / or on the carboxy-terminal side with respect to the CD3 binding region. [The present invention 1149] The multispecific polypeptide construct of any one of the present inventions 1146 to 1148, wherein the other of VH-CH1 (Fd) or VL-CL of the Fab antibody fragment is positioned on the amino-terminal side with respect to the Fc region of the first polypeptide or the second polypeptide and on the carboxy-terminal side with respect to the CD3 binding region of the other of the first or second polypeptides. [The present invention 1150] The antigen-binding domain, or each of the antigen-binding domains, independently, is 1-92-LFA-3, 5T4, α4 integrin, αV integrin, α4β1 integrin, α4β7 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, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, 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, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor α (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPA multispecific polypeptide construct according to any one of 1001 to 1149 of the present invention that binds to a tumor antigen selected from IIb / 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, IL23, IL23R, IL27 / IL27R(wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged1, Jagged2, 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, TIGIT, 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. [1151 of the present invention] The antigen-binding domain comprises at least a first antigen-binding domain and a second antigen-binding domain, and the first antigen-binding domain and the second antigen-binding domain bind to the same TAA. Any multispecific polypeptide construct of the present invention from 1001 to 1150. [Inventive item 1152] The multispecific polypeptide construct of inventive item 1151, wherein the first antigen-binding domain and the second antigen-binding domain bind to different epitopes of the same TAA. [Inventive item 1153] The multispecific polypeptide construct of inventive item 1151, wherein the first antigen-binding domain and the second antigen-binding domain bind to the same epitope of the same TAA. [Inventive item 1154] The antigen-binding domain comprises at least a first antigen-binding domain and a second antigen-binding domain, and the first antigen-binding domain and the second antigen-binding domain bind to different TAAs. Any multispecific polypeptide construct of the present invention from 1001 to 1153. [Inventive item 1155] The co-stimulatory receptor binding region (CRBR) comprises at least a first CRBR and a second CRBR, and the first CRBR and the second CRBR bind to the same co-stimulatory receptor. Any multispecific polypeptide construct of the present invention from 1001, 1003 to 1017, 1020 to 1025, 1028 to 1097, and 1099 to 1154. [Inventive item 1156] The multispecific polypeptide construct of inventive item 1155, wherein the first co-stimulatory receptor binding region (CRBR) and the second CRBR bind to different epitopes of the same co-stimulatory receptor. [Inventive item 1157] The multispecific polypeptide construct of inventive item 1155, wherein the first co-stimulatory receptor binding region (CRBR) and the second CRBR bind to the same epitope of the same co-stimulatory receptor. [Inventive item 1158] The costimulatory receptor binding region (CRBR) of any of the multispecific polypeptide constructs of the present invention 1001, 1003-1017, 1020-1025, 1028-1097, and 1099-1157 comprises at least a first CRBR and a second CRBR. [The present invention 1159] The multispecific polypeptide construct of any of the present invention 1155-1158, wherein the first CRBR and the second CRBR bind to different costimulatory receptors. [The present invention 1160] The multispecific polypeptide construct of any of the present invention 1001, 1003-1017, 1020-1025, 1028-1097, and 1099-1159, wherein at least one costimulatory receptor binding region (CRBR) binds to a costimulatory receptor selected from 41BB (CD137), OX40 (CD134), CD27, glucocorticoid-induced TNFR-related protein (GITR), CD28, ICOS, CD40, B cell activation factor receptor (BAFF-R), B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI), and NKG2D. [The present invention 1161] The multispecific polypeptide construct of any of the present invention 1001, 1003-1017, 1020-1025, 1028-1097, and 1099-1160, wherein at least one costimulatory receptor binding region (CRBR) binds to a costimulatory receptor selected from 41BB (CD137), OX40 (CD134), and glucocorticoid-induced TNFR-related protein (GITR). [The present invention 1162] The multispecific polypeptide construct of any of the present invention 1001, 1003-1017, 1020-1025, 1028-1097, and 1099-1161, wherein at least one costimulatory receptor binding region (CRBR) binds to 41BB (CD137). [The present invention 1163] At least one costimulatory receptor comprises the amino acid sequence shown in SEQ ID NO:215, or an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:215, of any of the multispecific polypeptide constructs of the present invention 1001, 1003-1017, 1020-1025, 1028-1097, and 1099-1162. [Inventive item 1164] At least one costimulatory receptor comprises the amino acid sequence shown in SEQ ID NO:321, of any of the multispecific polypeptide constructs of the present invention 1001, 1003-1017, 1020-1025, 1028-1097, and 1099-1163. [Inventive item 1165] At least one inhibitory receptor binding region (IRBR) is the extracellular domain or a binding fragment thereof of the native cognate binding partner of the costimulatory receptor, or a variant thereof that exhibits binding activity with the costimulatory receptor or comprises the same, of any of the multispecific polypeptide constructs of the present invention 1002-1015, 1018-1023, 1026-1095, 1097-1121, 1123-1164. [Inventive item 1166] At least one inhibitory receptor binding region (IRBR) is an antibody or an antigen-binding fragment thereof selected from the group consisting of Fab fragments, F(ab')2 fragments, Fv fragments, scFv, scAb, dAb, single-domain heavy-chain antibodies, and single-domain light-chain antibodies, of any of the multispecific polypeptide constructs of the present invention 1002-1015, 1018-1023, 1026-1095, 1097-1121, 1123-1164. [Inventive item 1167] The antibody or antigen-binding fragment thereof is an Fv, scFv, Fab, or single-domain antibody (sdAb), of the multispecific polypeptide construct of the present invention 1166. [Inventive item 1168] The antibody or antigen-binding fragment thereof is an sdAb, of the multispecific polypeptide construct of the present invention 1166 or the present invention 1122. [Inventive item 1169] The multispecific polypeptide construct of the present invention 1168, wherein the sdAb is a human sdAb or a humanized sdAb. [The present invention 1170] The multispecific polypeptide construct of the present invention 1168 or the present invention 1169, wherein the sdAb is a VHH, VNAR, modified VH domain, or modified VK domain. [The present invention 1171] The multispecific polypeptide construct of the present invention 1166 or the present invention 1167, wherein the antibody or antigen-binding fragment thereof is a scFv. [The present invention 1172] The multispecific polypeptide construct of the present invention 1166 or the present invention 1167, wherein the antibody or antigen-binding fragment thereof is a Fab. [The present invention 1173] The multispecific polypeptide construct of any one of the present inventions 1002 - 1015, 1018 - 1023, 1026 - 1095, 1097 - 1121, 1123 - 1172, wherein the inhibitory receptor binding region (IRBR) comprises at least a first IRBR and a second IRBR, and the first IRBR and the second IRBR bind to the same inhibitory receptor. [The present invention 1174] The multispecific polypeptide construct of the present invention 1173, wherein the first inhibitory receptor binding region (IRBR) and the second IRBR bind to different epitopes of the same inhibitory receptor. [The present invention 1175] The multispecific polypeptide construct of the present invention 1173, wherein the first inhibitory receptor binding region (IRBR) and the second IRBR bind to the same epitope of the same inhibitory receptor. [The present invention 1176] The multispecific polypeptide construct of any one of the present inventions 1002 - 1015, 1018 - 1023, 1026 - 1095, 1097 - 1121, 1123 - 1175, wherein the inhibitory receptor binding region (IRBR) comprises at least a first IRBR and a second IRBR. [The present invention 1177] A multispecific polypeptide construct according to any one of aspects 1173 to 1176 of the present invention, wherein the first IRBR and the second IRBR bind to different inhibitory receptors. [Aspect 1178 of the present invention] A multispecific polypeptide construct according to any one of aspects 1002 to 1015, 1018 to 1023, 1026 to 1095, 1097 to 1121, 1123 to 1177 of the present invention, wherein at least one inhibitory receptor binding region (IRBR) binds to an inhibitory receptor selected from PD-1, CTLA-4, TIGIT, VISTA, or TIM3. [Aspect 1179 of the present invention] A multispecific polypeptide construct according to any one of aspects 1002 to 1015, 1018 to 1023, 1026 to 1095, 1097 to 1121, 1123 to 1178 of the present invention, wherein at least one inhibitory receptor binding region (IRBR) binds to PD-1. [Aspect 1180 of the present invention] A multispecific polypeptide construct according to any one of aspects 1001 to 1179 of the present invention, comprising a first linker peptide (LP1) between the first antigen-binding domain and the Fc region. [Aspect 1181 of the present invention] A multispecific polypeptide construct according to any one of aspects 1001 to 1180 of the present invention, comprising a second linker peptide (LP2) between the CD3-binding region and the second antigen-binding domain. [Aspect 1182 of the present invention] A multispecific polypeptide construct according to any one of aspects 1001 to 1180 of the present invention, comprising a first linker peptide (LP1) between the co-stimulatory receptor binding region (CRBR) and the Fc region. [Aspect 1183 of the present invention] A multispecific polypeptide construct according to any one of aspects 1001 to 1182 of the present invention, comprising a second linker peptide (LP2) between the CD3-binding region and the second co-stimulatory receptor binding region (CRBR). [Aspect 1184 of the present invention] Comprising a first linker peptide (LP1) between the antigen-binding domain or co-stimulatory receptor binding region and the Fc region and a second linker peptide (LP2) between the CD3-binding region and the antigen-binding domain or CRBR, Having a structural arrangement from the N-terminus to the C-terminus of the first antigen-binding domain or CRBR-LP1-Fc region-linker-CD3 binding region-LP2-the second antigen-binding domain or CRBR Any of the multispecific polypeptide constructs of the present invention 1001 to 1183. [The present invention 1185] The multispecific polypeptide construct of the present invention 1184, wherein the linker is a cleavable linker. [The present invention 1186] The multispecific polypeptide construct of the present invention 1184, wherein the linker is a non-cleavable linker. [The present invention 1187] Any of the multispecific polypeptide constructs of the present invention 1184 to 1186, wherein the two linking peptides are not identical to each other. [The present invention 1188] Any of the multispecific polypeptide constructs of the present invention 1180 to 1187, wherein LP1 or LP2 is independently a peptide about 1 to 20 amino acids in length. [The present invention 1189] The multispecific polypeptide construct of the present invention 1188, wherein LP1 or LP2 independently comprises a Gly-Ser linker or GGS shown in SEQ ID NOs: 10 to 13, 119, 135, 147, 149 or a peptide containing the same. [The present invention 1190] Any of the multispecific polypeptide constructs of the present invention 1001 to 1189, wherein the anti-CD3 antibody or antigen-binding fragment is an Fv antibody fragment. [The present invention 1191] The multispecific polypeptide construct of the present invention 1190, wherein the Fv antibody fragment is a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv). [The present invention 1192] The anti-CD3 antibody or antigen-binding fragment is VH CDR1 containing the amino acid sequence TYAMN (SEQ ID NO: 16); the amino acid sequence VH CD2 containing TIFF2025098077000010.tif4128; VH CDR3 containing the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:18); VL CDR1 containing the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:19); VL CDR2 containing the amino acid sequence GTNKRAP (SEQ ID NO:20); and VL CDR3 containing the amino acid sequence ALWYSNLWV (SEQ ID NO:21) Any of the multispecific polypeptide constructs of the present invention from 1001 to 1191, inclusive. [The present invention 1193] The anti-CD3 dsFv is VH having an amino acid sequence of any one of SEQ ID NO:14, 32 - 62, 287, 290, and 311, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO:14, 32 - 62, 287, 290, and 311; and VL having an amino acid sequence of any one of SEQ ID NO:15, 63 - 81, 241, 288, and 289, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO:15, 63 - 81, 241, 288, and 289 Any of the multispecific polypeptide constructs of the present invention 1160 or the present invention 1161, inclusive. [The present invention 1194] Any of the multispecific polypeptide constructs of the present invention from 1190 to 1193, inclusive, wherein the anti-CD3 dsFv contains the amino acid sequences of SEQ ID NO:14 and SEQ ID NO:15. [The present invention 1195] Does the anti-CD3 dsFv contain the amino acid sequence of SEQ ID NO:44 and the amino acid sequence of SEQ ID NO:72; does the anti-CD3 dsFv contain the amino acid sequence of SEQ ID NO:44 and the amino acid sequence of SEQ ID NO:241; does the anti-CD3 dsFv contain the amino acid sequence of SEQ ID NO:287 and the amino acid sequence of SEQ ID NO:288; or does the anti-CD3 dsFv contain the amino acid sequence of SEQ ID NO:311 and the amino acid sequence of SEQ ID NO:289, any of the multispecific polypeptide constructs of the present invention from 1190 to 1194. [The present invention 1196] Any of the multispecific polypeptide constructs of the present invention from 1001 to 1195, conjugated to an agent. [The present invention 1197] The agent is a therapeutic agent, an antitumor agent, a toxin or a fragment thereof, a detectable moiety, or a diagnostic agent, the multispecific polypeptide construct of the present invention 1196. [The present invention 1198] The agent is conjugated to the multispecific polypeptide construct via a linker, the multispecific polypeptide construct of the present invention 1197. [The present invention 1199] A polynucleotide encoding any of the multispecific polypeptide constructs of the present invention from 1001 to 1198. [The present invention 1200] A polynucleotide encoding any of the polypeptide chains of any of the multispecific polypeptide constructs of the present invention from 1001 to 1198. [The present invention 1201] A polynucleotide comprising a first nucleic acid sequence encoding a first polypeptide of any of the multispecific constructs of the present invention from 1001 to 1198 and a second nucleic acid sequence encoding a second polypeptide of the multispecific construct, wherein the first and second nucleic acid sequences are separated by an internal ribosome entry site (IRES) within the sequence or by a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, polynucleotide. [Invention 1202] The polynucleotide of Invention 1201, wherein the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter. [Invention 1203] The polynucleotide of Invention 1201 or Invention 1202, wherein the multispecific polypeptide construct comprises a third polypeptide chain, and the polynucleotide further comprises a third nucleic acid encoding the third polypeptide of the multispecific construct. [Invention 1204] The third nucleic acid is separated from the first and / or second polypeptides by an internal ribosome entry site (IRES) within the sequence, or by a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, and / or The third nucleic acid sequence is operably linked to the same promoter as the first and / or second nucleic acid sequences. The polynucleotide of Invention 1203. [Invention 1205] The polynucleotide of any one of Inventions 1201 to 1204, wherein the nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping is selected from T2A, P2A, E2A, or F2A. [Invention 1206] A vector comprising the polynucleotide of any one of Inventions 1199 to 1205. [Invention 1207] The vector of Invention 1206, which is an expression vector. [Invention 1208] The vector of Invention 1206 or 1207, which is a viral vector or a eukaryotic vector, optionally, the eukaryotic vector is a mammalian vector. [Invention 1209] A cell comprising one or more polynucleotides of any one of Inventions 1199 to 1205 or one or more vectors of any one of Inventions 1206 to 1208. [Invention 1210] The cell of the present invention 1209, which is recombinant or isolated. [The present invention 1211] The cell of the present invention 1210, which is a mammalian cell. [The present invention 1212] The cell of the present invention 1211, which is a HEK293 cell or a CHO cell. [The present invention 1213] A step of introducing one or more polynucleotides of any one of the present inventions 1199 to 1205 or one or more vectors of any one of the present inventions 1206 to 1208 into a cell, and A step of culturing the cell under conditions in which a multispecific polypeptide construct is produced A method for producing a multispecific polypeptide construct, comprising the above steps. [The present invention 1214] A method for producing a multispecific polypeptide construct, comprising a step of culturing any one of the cells of the present inventions 1209 to 1213 under conditions in which a multispecific polypeptide is produced by the cell. [The present invention 1215] The cell of the present invention 1213 or the present invention 1214, which is a mammalian cell. [The present invention 1216] The cell of the present invention 1215, which is a HEK293 cell or a CHO cell. [The present invention 1217] The method of the present invention 1213 or the present invention 1214, further comprising a step of isolating or purifying the multispecific polypeptide construct from the cell. [The present invention 1218] The method of any one of the present inventions 1213 to 1217, wherein the multispecific polypeptide construct is a heterodimer. [The present invention 1219] A multispecific polypeptide construct produced by the method of any one of the present inventions 1213 to 1218. [The present invention 1220] A pharmaceutical composition comprising any one of the multispecific polypeptide constructs of the present inventions 1001 to 1198 or the present invention 1219 and a pharmaceutically acceptable carrier. [The present invention 1221] The pharmaceutical composition of the present invention 1220 that is sterile. [The present invention 1222] A method of stimulating or inducing an immune response, comprising the step of contacting a target cell and a T cell with any one of the multispecific polypeptide constructs of the present invention 1001 to 1198 or the present invention 1219 or the pharmaceutical composition of the present invention 1220 or the present invention 1221, wherein the target cell expresses a tumor-associated antigen recognized by the multispecific polypeptide construct. [The present invention 1223] The method of the present invention 1222, wherein the target cell is a tumor cell expressing a tumor-associated antigen (TAA). [The present invention 1224] The method of the present invention 1222 or the present invention 1223, wherein the multispecific polypeptide construct comprises a cleavable linker that functions as a substrate for a protease, and the induction or stimulation of the immune response is increased in the presence of the protease. [The present invention 1225] The method of the present invention 1224, wherein the protease is produced by immune effector cells, by a tumor, or by cells present in the tumor microenvironment. [The present invention 1226] The method of the present invention 1224 or the present invention 1225, wherein the protease is produced by immune effector cells, and the immune effector cells are activated T cells, natural killer (NK) cells, or NK T cells. [The present invention 1227] The method of the present invention 1226, wherein the immune effector cells are in the vicinity of cells expressing an antigen. [The present invention 1228] The protease is produced by a tumor in the vicinity of cells expressing TAA in the tissue and / or by a tumor co-localized with TAA in the tissue, when the multispecific polypeptide construct is exposed to the protease, the protease cleaves the cleavable linker within the multispecific polypeptide construct, The method according to any one of the present inventions 1222 to 1227. [The present invention 1229] Any of the methods of the present invention from 1222 to 1228, wherein the protease is selected from matriptase, matrix metalloprotease (MMP), granzyme B, and combinations thereof. [The present invention 1230] The method of the present invention 1229, wherein the protease is granzyme B. [The present invention 1231] Any of the methods of the present invention from 1222 to 1230, wherein the contacting step is carried out ex vivo or in vitro. [The present invention 1232] Any of the methods of the present invention from 1222 to 1231, wherein the contacting step is carried out in vivo in a subject. [The present invention 1233] A method of stimulating or inducing an immune response in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of any of the multispecific conjugates of the present invention from 1001 to 1198 or 1219 or the pharmaceutical composition of the present invention 1220 or 1221. [The present invention 1234] Any of the methods of the present invention from 1222 to 1233, which increases cellular immunity. [The present invention 1235] Any of the methods of the present invention from 1222 to 1234, which increases T cell activity. [The present invention 1236] Any of the methods of the present invention from 1222 to 1235, which increases cytotoxic T lymphocyte (CTL) activity. [The present invention 1237] Any of the methods of the present invention from 1222 to 1236, in which the immune response against a tumor or cancer is increased. [The present invention 1238] Any of the methods of the present invention from 1222 to 1236 for treating a disease or condition in a subject. [The present invention 1239] A method of treating a disease or condition in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of any of the multispecific polypeptide constructs of the present invention from 1001 to 1198 or the pharmaceutical composition of the present invention 1220 or 1221. [The present invention 1240] The method of the present invention 1238 or the present invention 1239, wherein the disease or condition is a tumor or cancer. [The present invention 1241] The method of any one of the present inventions 1238 to 1240, wherein the disease or condition expresses a tumor-associated antigen (TAA). [The present invention 1242] The method of any one of the present inventions 1222 to 1241, wherein the subject is human. [The present invention 1243] Any multispecific polypeptide construct of the present inventions 1001 to 1198 or the pharmaceutical composition of the present invention 1220 or the present invention 1221 for use in the treatment of a disease or condition in a subject. [The present invention 1244] Use of any multispecific polypeptide construct of the present inventions 1001 to 1198 or the pharmaceutical composition of the present invention 1220 or the present invention 1221 for the manufacture of a medicament for use in the treatment of a disease or condition in a subject. [The present invention 1245] The multispecific polypeptide construct or pharmaceutical composition for use in the use of the present invention 1243, wherein the disease or condition is a tumor or cancer, or the use of the present invention 1244. [The present invention 1246] The multispecific polypeptide construct or pharmaceutical composition for use in the use of the present invention 1243 or the present invention 1245, wherein the disease or condition expresses a tumor-associated antigen (TAA), or the use of the present invention 1244 or the present invention 1245. [The present invention 1247] The multispecific polypeptide construct or pharmaceutical composition for use in the use of the present invention 1243, the present invention 1245, or the present invention 1246, wherein the subject is human, or the use of the present invention 1244, the present invention 1245, or the present invention 1246. [Brief Description of the Drawings]

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[0064] **Detailed Description** The present disclosure provides fusion proteins that engage restricted T cells in the form of a multispecific polypeptide construct that binds at least to CD3, a second antigen, and a costimulatory receptor. Fusion proteins that engage T cells in the form of a multispecific polypeptide construct that binds at least to CD3, a second antigen, and an inhibitory receptor are also provided in the present disclosure. Fusion proteins that engage T cells in the form of a multispecific polypeptide construct that binds at least to CD3, a second antigen, a costimulatory receptor, and an inhibitory receptor are also provided in the present disclosure.

[0065] In some embodiments, the multispecific polypeptide constructs provided herein include 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 interchangeably as an anti-CD3 binding domain or a CD3 binding region), and at least a linker such as a polypeptide linker that joins the first component and the second component. In the provided multispecific polypeptide constructs, one or both of the first and second components contain at least one antigen binding domain that enables the restricted CD3 binding region to bind substantially to CD3 when engaged by binding to an antigen. In some embodiments, the antigen is a tumor-associated antigen (TAA). Further, in some aspects, one or both of the first and second components of the multispecific polypeptide construct also include at least one co-stimulatory receptor binding region (CRBR) that binds to a co-stimulatory receptor. In other aspects, one or both of the first and second components of the multispecific polypeptide construct also include at least one inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor. In a specific aspect of the present disclosure provided, one of the first and second components of the multispecific polypeptide construct contains a CRBR and the other of the first and second components contains an IRBR. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a cleavable linker.

[0066] In some embodiments, the provided multispecific polypeptide construct comprises an arrangement in which a first component containing an Fc region is on the N-terminal side relative to a second component containing a CD3 binding region. In such embodiments, the first and second components are joined via a linker on the C-terminal side relative to the end of the Fc region. In some embodiments, the antigen-binding domain and / or CRBR is positioned in the amino-terminal (N-terminal) region of the multispecific polypeptide construct. In some embodiments, the antigen-binding domain and / or CRBR is positioned in the carboxy-terminal (C-terminal) region of the multispecific polypeptide construct. In some embodiments, the antigen-binding domain and / or CRBR is positioned in both the N-terminal and C-terminal regions of the multispecific polypeptide construct. The various arrangements of the multispecific polypeptide constructs provided herein are shown in Figure 1.

[0067] In some embodiments, the provided multispecific polypeptide construct comprises an arrangement in which a first component containing an Fc region is on the N-terminal side relative to a second component containing a CD3 binding region. In such embodiments, the first and second components are joined via a linker on the C-terminal side relative to the end of the Fc region. In some embodiments, the antigen-binding domain and / or IRBR is positioned in the amino-terminal (N-terminal) region of the multispecific polypeptide construct. In some embodiments, the antigen-binding domain and / or IRBR is positioned in the carboxy-terminal (C-terminal) region of the multispecific polypeptide construct. In some embodiments, the antigen-binding domain and / or IRBR is positioned in both the N-terminal and C-terminal regions of the multispecific polypeptide construct. The various arrangements of the multispecific polypeptide constructs provided herein are shown in Figure 18A.

[0068] In some embodiments, the provided multispecific polypeptide construct comprises an arrangement in which a first component containing an Fc region is N-terminal to a second component containing a CD3 binding region. In such embodiments, the first and second components are joined via a linker that is C-terminal to the end of the Fc region. In some embodiments, the antigen binding domain and / or CRBR is positioned in the amino-terminal (N-terminal) region of the multispecific polypeptide construct, and the antigen binding domain and / or IRBR is positioned in the carboxy-terminal (C-terminal) region of the multispecific polypeptide construct. In some embodiments, the antigen binding domain and / or IRBR is positioned in the amino-terminal (N-terminal) region of the multispecific polypeptide construct, and the antigen binding domain and / or CRBR is positioned in the carboxy-terminal (C-terminal) region of the multispecific polypeptide construct. The various arrangements of the multispecific polypeptide constructs provided herein are shown in FIG. 18B.

[0069] The provided multispecific polypeptide construct exhibits restricted T cell engagement activity because it binds substantially to CD3 only after the antigen has bound via the antigen-binding domain. This is illustrated in the examples and drawings provided herein, which demonstrate that a protein that engages restricted CD3 can bind efficiently to TAA-positive cells and binds little or not at all to T cells. This unique property allows the protein that engages restricted CD3 to distribute to sites where TAA is present without binding to peripheral T cells. This format differs from other multispecific constructs that engage CD3 in which constitutive CD3 binding is prohibited or excluded, avoiding peripheral T cell binding and allowing preferential distribution to sites where the antigen recognized by the antigen-binding domain is present, providing a significant benefit. For example, as shown in the examples, the format that engages restricted CD3 allows for similar potency as the DART-Fc format (e.g., published PCT application number WO2017 / 030926), but binding to peripheral T cells is significantly attenuated. Further, while other CD3-engaging constructs mediate antigen-dependent T cell activation, the multispecific polypeptide constructs provided herein mediate both antigen-dependent T cell binding and activation.

[0070] The activity of the provided multispecific polypeptide construct that engages restricted T cells is, in several aspects, due to the positioning of the Fc region on the N-terminal side of the CD3-binding region. In several embodiments, such positioning reduces, attenuates, interferes with, and / or prevents CD3 binding by the CD3-binding region. The multispecific polypeptide constructs provided herein exhibit a reduced or eliminated ability to bind CD3 and activate T cells in the absence of antigen binding by the antigen-binding domain. In several embodiments, in the presence of an antigen-binding event mediated by the antigen-binding domain of the multispecific polypeptide construct, the ability to bind CD3 by the CD3-binding region is greatly enhanced. In several embodiments, in the presence of an antigen-binding event mediated by the antigen-binding domain of the multispecific polypeptide construct, the ability to activate T cells is greatly enhanced. Engagement of its cognate antigen by the antigen-binding domain within the multispecific polypeptide construct results in subsequent T cell engagement and mediates antigen-dependent T cell activation such as cytotoxicity, cytokine release, degranulation, and proliferation. In several embodiments, the provided multispecific polypeptide constructs can be used to increase the immune response, for example, to enhance T cell activity including cytolytic (or cytotoxic) T cell activity. Modulation of the immune response can, in several aspects, treat a disease or condition in a subject.

[0071] In some embodiments, the multispecific polypeptide constructs of the disclosure exhibit specificity for CD3, one or more other antigens, and one or more costimulatory receptors. In some embodiments, the multispecific polypeptide constructs of the disclosure exhibit specificity for CD3, one or more other antigens, and one or more inhibitory receptors. In some embodiments, the multispecific polypeptide constructs of the disclosure exhibit specificity for CD3, one or more other antigens, one or more costimulatory receptors, and one or more inhibitory receptors. In some embodiments, the multispecific polypeptide construct may contain one or more antigen-binding domains capable of binding to one or more TAAs, for example, 1, 2, or 3 antigen-binding domains, for example, 2 or 3 antigen-binding domains. In some embodiments, the multispecific polypeptide construct may contain one or more costimulatory receptor-binding regions (CRBRs) capable of binding to one or more costimulatory receptors, for example, 2 or 3 costimulatory receptors, for example, 1, 2, or 3 CRBRs. In some embodiments, the multispecific polypeptide construct may contain one or more inhibitory receptor-binding regions (IRBRs) capable of binding to one or more inhibitory receptors, for example, 2 or 3 inhibitory receptors, for example, 1, 2, or 3 IRBRs. Exemplary construct formats are shown in FIGS. 1-3 and FIGS. 18A-B.

[0072] In some embodiments, one or more CRBRs bind to co-stimulatory receptors expressed on the surface of T cells. Non-limiting examples of co-stimulatory receptors that can be targeted by CRBRs include, for example, 41BB, OX40, CD27, GITR, CD28, ICOS, CD40L, BAFFR, BCMA, TACI, or NKG2d. In some embodiments, one or more CRBRs are or include a binding domain (e.g., sdAb, scFv, or Fab) that binds to a co-stimulatory receptor. As shown herein, the presence of CRBRs in the provided multispecific polypeptide constructs increases or enhances T cell activity upon co-engagement of CD3 by the CD3 binding region.

[0073] In some cases, one or more CRBRs bind to co-stimulatory receptors that are not constitutively expressed in T cells. In some embodiments, one or more CRBRs bind to co-stimulatory receptors that are upregulated, induced, or expressed upon T cell activation. In some embodiments, the CRBR binds to a co-stimulatory receptor that is a member of the tumor necrosis factor (TNF) receptor family, such as a TNFRSF member that is upregulated, induced, or expressed upon T cell activation. In some embodiments, the CRBR binds to a co-stimulatory receptor that is a member of the B7 family, such as a B7 family member that is upregulated, induced, or expressed upon T cell activation. Such co-stimulatory receptors whose expression is upregulated or surface expression is induced in activated T cells include, for example, 41BB, OX40, and GITR. In some aspects, the CD3 binding region of the provided multispecific polypeptide construct induces or enhances T cell activation when the CD3 binding region binds to CD3, which, in turn, results in an increase or upregulation in the expression of such co-stimulatory receptors. In such examples, binding of the CRBR to a co-stimulatory receptor, such as 41BB, OX40, or GITR, increases or becomes possible only after engagement of CD3 by the CD3 binding region of the multispecific polypeptide construct, thereby controlling the co-stimulatory activity of the provided multispecific construct against target cells. In some cases, this can solve problems of other T cell engagers by avoiding an antigen "sink" effect due to binding to CD3 and / or co-stimulatory receptors expressed in peripheral T cells, such as resting T cells or unwanted cells, which can accelerate systemic clearance. In some embodiments, the provided multispecific polypeptide construct exhibits increased half-life and / or improved pharmacokinetic or pharmacodynamic properties, such as better tumor exposure, compared to other T cell engagers that can bind to peripheral cells, such as a bispecific T cell engager, for example, DART-Fc.

[0074] In some embodiments, one or more IRBRs bind to inhibitory receptors expressed on the surface of T cells. In some aspects, the inhibitory receptors are those whose expression is upregulated or increased in activated T cells. Non-limiting examples of inhibitory receptors that can be targeted by IRBRs include, for example, PD-1, CTLA-4, TIGIT, VISTA, TIM3, or LAG3. In some embodiments, one or more IRBRs are or include a binding domain (e.g., sdAb, scFv, or Fab) that binds to the inhibitory receptor.

[0075] In some embodiments, one or more IRBRs bind to inhibitory receptors and prevent or inhibit their interaction with their ligands, thereby blocking the inhibitory activity of T cells. In some embodiments, the IRBR is a PD-1 binding polypeptide that binds to PD-1. In some cases, the provided PD-1 binding polypeptide directly blocks or inhibits the interaction between PD-L1 / L2 and PD-1. In some aspects, the inclusion of an IRBR in a multispecific polypeptide construct suppresses or reduces the interaction between an inhibitory receptor and its ligand, e.g., between PD-Ll and / or PD-L2 and PD-1, thereby modulating the immune response. Transmission of inhibitory signals results in downmodulation of immune cell responses (and consequently downmodulation of the overall immune response), and blocking of inhibitory signals in immune cells results in upmodulation of immune cell responses (and consequently upmodulation of the immune response). In some cases, modulation by enhancement of the immune response can be used to treat certain diseases or conditions in which the immune response is suppressed, such as cancer. Generally, the ability to block inhibitory interactions occurs in the immune synapse and / or the tumor microenvironment when the CD3 binding region binds to CD3 on T cells and / or the antigen binding domain binds to a TAA. In specific embodiments, the provided multispecific polypeptide construct reduces, suppresses, or inhibits inhibitory signals mediated by inhibitory receptors in cells such as T cells.

[0076] In some embodiments, one or more antigen binding domains bind to antigens on tumor cells or cells of the tumor microenvironment. In some aspects, the provided multispecific polypeptide construct can be used to increase immune responses against tumors or cancers, such as cytotoxic activity, in T cells. In some embodiments, the provided multispecific polypeptide construct can be used to treat tumors or cancers in a subject.

[0077] In some embodiments, one or more antigen-binding domains bind to the same antigen. In some embodiments, the multispecific polypeptide construct comprises multiple antigen-binding domains that bind to different epitopes of the same antigen. In some embodiments, the multispecific polypeptide construct comprises multiple antigen-binding domains that bind to one or more different antigens. In some embodiments, the multispecific polypeptide construct comprises multiple antigen-binding domains that bind to different epitopes of the same antigen and also comprises additional antigen-binding domains that bind to one or more different antigens. In some examples, the provided multispecific polypeptide construct provides multivalent engagement of one or more TAAs, for example, through at least a first antigen-binding domain and a second antigen-binding domain. For example, in some embodiments, the polypeptide construct comprises at least a first antigen-binding single-domain antibody (sdAb) and a second antigen-binding sdAb. In some aspects, the first and second antigen-binding domains bind to the same antigen.

[0078] Furthermore, in some aspects, the CD3-binding region of the multispecific polypeptide constructs of the present disclosure is restricted by the presence of the Fc region or otherwise blocked and / or suppressed such that these constructs ensure that binding of T cells via CD3 does not occur or is reduced in peripheral blood. Accordingly, the multispecific polypeptide constructs of the present disclosure provide a number of advantages. In some aspects, these constructs limit the sink effect caused by binding to all T cells. In some aspects, these constructs reduce systemic toxicity.

[0079] In some embodiments, the provided multispecific polypeptide constructs of the present disclosure enable regulated in vivo distribution to a desired site in a subject, such as, for example, the site of tumor-associated antigen (TAA) expression. Sites of TAA expression include, for example, tumors and the surrounding tumor microenvironment.

[0080] In some embodiments, the 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 which CD3 binding is restricted and T cell interaction is eliminated or reduced when any or all of the antigen-binding domains are absent, and (2) an “active” state occurs in which the CD3-binding region can bind to CD3 and T cell interaction is enabled when any or all of the antigen-binding domains bind to an antigen.

[0081] In some embodiments, the Fc region is linked to the CD3-binding domain via a linker. In some embodiments, the Fc region is linked to the CD3-binding region via a non-cleavable linker. In some embodiments, the Fc region is linked to the CD3-binding region via a cleavable linker or other labile linker.

[0082] In some aspects, the multispecific polypeptide constructs of the present disclosure enable therapeutic efficacy in the absence of cleavage, e.g., in the absence of proteolysis. In some embodiments, the linker is a non-cleavable linker.

[0083] In some embodiments, the Fc region and the CD3-binding region are linked by a cleavable linker, such as 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 several mechanisms, e.g., through cleavage of the linker that joins 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 in which the Fc region and the CD3-binding region are linked by a cleavable linker, enhanced CD3 binding can occur after cleavage within the linker.

[0084] In some embodiments, the Fc region is a homodimeric Fc region. In some embodiments, the Fc region is a heterodimeric Fc region. In some embodiments, the Fc region is a monomeric Fc region. In some embodiments, the Fc region of the multispecific polypeptide construct can interact with FcγR and mediate innate immune effector functions such as antibody-dependent cell cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP). In some embodiments, the Fc region of the multispecific polypeptide construct can interact with a complement protein, i.e., C1q, and mediate complement-dependent cytotoxicity. Thus, in some aspects, the multispecific polypeptide constructs of the present disclosure enable multiple immune effector mechanisms including innate immune effectors and T cells.

[0085] In some embodiments, the linker is a cleavable linker. In some embodiments, the Fc region and the CD3 binding region are functionally linked by a cleavable linker. Cleavage of the linker between the Fc region and the CD3 binding region can separate the multispecific polypeptide construct into a first component and a second component. Depending on the composition of the multispecific polypeptide construct, the first and second components may have different functionalities. In some embodiments, the Fc region is a region that exhibits one or more effector functions such as an ADCC function, a CDC function, or an ADCP function. In such examples, the multispecific polypeptide constructs of the present disclosure can be used to create a self-amplifying system. For example, the multispecific construct can be used as follows: ADCC mediated by NK cells after TAA targeting and CD16 binding of the Fc region results in the release of granzyme B, which is capable of extracellular proteolysis, and cleavage of the linker between the first and second components of the multispecific polypeptide construct.

[0086] In some embodiments, the multispecific polypeptide construct provides a two-in-one therapeutic agent with a dual effector function where proteolytic activation of the multispecific polypeptide construct produces two components each with biological activity. The multispecific polypeptide constructs of the present disclosure can provide Fc-mediated effector functions such as, for example, ADCC (e.g., release of granzyme B by NK cells), ADCP, and / or CDC.

[0087] The restricted CD3 engagement construct can be used with any TAA binding domain and is intended to avoid interaction with peripheral T cells and enable better therapeutic exposure in tumors or the tumor microenvironment by mediating potent TAA-dependent T cell cytotoxicity. Incorporation of a protease-cleavable linker between the Fc and the CD3 binding domain component enables amplification of the T cell activation ability by allowing complete exposure of the CD3 binding domain. Depending on the specific linker included, the amplification step may be mediated by tumor-associated proteases or by granzyme released after antigen-dependent T cell activation. When a linker cleavable by tumor proteases is included, amplification is mediated by the tumor or the tumor microenvironment. On the other hand, when a linker cleavable by granzyme B is included, amplification can be self-mediated by T cells after antigen-dependent activation. Further, in cases where an effector-enabled Fc is included in the construct, amplification occurring through the ADCC mechanism can be mediated by granzyme released from NK cells.

[0088] In some embodiments, the cleavable linker can be cleaved by proteases produced in the tumor microenvironment and / or proteases produced or secreted by T cells upon T cell activation such that the cleavage is induced by the initial binding of the CD3 binding region in the tumor microenvironment to CD3 via binding to the TAA of the antigen binding domain. In some embodiments, the protease is granzyme B. In some aspects, the multispecific polypeptide construct of the present disclosure cleaves the linker within the multispecific polypeptide construct below the Fc immunoglobulin polypeptide, thereby, in some cases, harnessing the ability of proteases and / or granzyme B in the tumor microenvironment to generate two therapeutically active proteins with different effector cell engagements. In some aspects, upon cleavage of the cleavable linker, the cleaved first portion or component retains the Fc effector function and bivalent targeting via the first antigen binding domain of a first antigen such as, for example, a TAA, and the second portion or component retains the ability to engage T cells since separation of the CD3 binding region from the Fc region allows for CD3 binding. The cleaved second portion or component also retains, in some cases, the ability to bind the TAA, which can be bivalent binding via the second antigen binding domain.

[0089] The multispecific polypeptide constructs of the present disclosure are designed to ensure that the protease that cleaves the cleavable linker need not be tumor-biased (e.g., need not be differentially expressed only in the tumor site and / or tumor environment). Rather, these multispecific polypeptide constructs only require that the protease be present at the same location as the TAA. The valency of these constructs will drive the biodistribution and retention in the tumor and / or tumor microenvironment.

[0090] In some embodiments, the second part or component contains a CD3-binding region that is monovalent with respect to CD3 such that T cell activation does not occur unless TAA is present. In some aspects, if the multivalent polypeptide construct contains a cleavable linker, the cleaved second part or component enables TAA-dependent T cell-mediated cytotoxicity. In some cases, the cleaved second part or component ensures that no FcRn interaction occurs. Further, the cleaved second part or component is of a sufficiently small size, e.g., only approximately 50 kDa, which would ensure rapid clearance if, for any reason, the cleaved second part or component were distributed outside the tumor site and / or were aberrantly cleaved outside the tumor site.

[0091] In some embodiments, the multispecific polypeptide constructs of the disclosure enable simultaneous occurrence of cytotoxicity mediated by T cells and NK cells. In some cases, such activity can occur in a multispecific polypeptide construct containing a first antigen-binding domain, e.g., a first anti-TAA antigen-binding domain, and a second antigen-binding domain, e.g., a second anti-TAA antigen-binding domain, that can target different and / or non-competing epitopes of a given TAA.

[0092] In some aspects, the multispecific polypeptide constructs of the present disclosure offer a number of advantages compared to current bispecific therapeutics. The multispecific polypeptide constructs of the present disclosure are smaller than conventional therapeutic antibodies, e.g., 125 kDa versus 150 kDa, which should allow for better penetration into targets, e.g., tumors. First, the size of the overall multispecific polypeptide construct provides a long half-life for the uncleaved construct, and upon cleavage of the construct, the cleaved second portion or component will be small enough to ensure a short half-life. In some aspects, because CD3 binding by the CD3 binding region depends on TAA engagement prior to CD3 engagement occurring, the multispecific polypeptide constructs of the present disclosure exhibit reduced systemic toxicity or toxicity in regions outside of the tumor and / or tumor microenvironment. In some cases, the inclusion of a cleavable linker specific for proteases in the tumor environment reduces CD3 binding by the multispecific construct until proteolytic activation and TAA engagement amplify or enhance CD3 engagement. Additionally, the additional presence of a CRBR in the provided multispecific polypeptide construct provides antigen-dependent specific co-stimulatory signaling to further increase or enhance T cell activity at desired sites or locations such as tumors and / or the tumor microenvironment. Similarly, the inclusion of an IRBR in the provided multispecific polypeptide construct can enhance T cell activity by removing or blocking checkpoint inhibitory signals.

[0093] All publications and patent documents cited herein are hereby incorporated by reference as if each such publication or document was specifically and individually indicated to be incorporated by reference herein. The citation of publications and patent documents is not intended as an admission that anything is relevant prior art, nor does it constitute an admission as to the content or date thereof. Although the invention has been described in the specification, those skilled in the art will recognize that the invention can be practiced in various ways, and that the above description and the following examples are for purposes of illustration of the claims to follow and not for purposes of limitation.

[0094] I. Definitions Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. The terms “a” entity or “an” entity refer to one or more of that entity. For example, “a compound” refers to one or more compounds. Accordingly, the terms “(a)”, “(an)”, “one or more”, and “at least one” may be used interchangeably. Further, unless the context requires otherwise, the singular forms shall include the plural, and the plural forms shall include the singular. In general, the cell and tissue culture, molecular biology, protein and oligonucleotide or polynucleotide chemistry, and the nomenclature and techniques utilized in connection with hybridization described herein are well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzyme reactions and purification techniques are performed according to the manufacturer's specifications, or as commonly achieved in the art, or as described herein. The above techniques and procedures are generally performed according to conventional methods well known in the art, as described in various general and more specific references cited and described herein. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclature and the experimental procedures and techniques utilized in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and for the treatment of patients.

[0095] As used in accordance with the present disclosure, the following terms are to be understood to have the following meanings unless otherwise indicated.

[0096] As used herein, the term "antibody" refers to an immunoglobulin molecule and the antigen-binding portion or fragment of an immunoglobulin (Ig) molecule, i.e., a molecule containing an antigen-binding site that specifically binds (immunoreacts) with an antigen. The term antibody includes not only complete polyclonal or monoclonal antibodies, but also dAb, Fab, Fab', F(ab')2, Fv, single-chain (scFv), or single-domain antibodies (sdAb). Typically, an "antigen-binding fragment" contains at least one CDR of the heavy and / or light chains of an immunoglobulin that binds to at least one epitope of the antigen of interest. In this regard, an antigen-binding fragment may contain 1, 2, 3, 4, 5, or all 6 CDRs of the variable heavy (VH) and variable light (VL) sequences derived from an antibody that binds to the antigen, and generally, for an antibody containing VH and VL, 6 CDRs ("CDR1", "CDR2", and "CDR3" for each of the heavy and light chains), or for an antibody containing a single variable domain, 3 CDRs may be included. Antigen-binding fragments include single-domain antibodies, e.g., those containing only VH or only VL, e.g., V H H, V NAR , modified V H domains, or modified V K domains.

[0097] The basic antibody structural unit is known to contain a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, with each pair having one "light chain" (about 25 kDa) and one "heavy chain" (about 50 - 70 kDa). The amino-terminal portion of each chain contains a variable region of about 100 - 110 amino acids or more, which is mainly responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region that is mainly responsible for effector functions. Generally, antibody molecules obtained from humans are related to any of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other by the nature of the heavy chains present within the molecule. Certain classes have subclasses such as IgG1, IgG2, IgG3, IgG4, and others. Further, in humans, the light chain can be a κ chain or a λ chain.

[0098] As used herein, the term "monoclonal antibody" (mAb) or "monoclonal antibody composition" refers to a population of antibody molecules that contains only one molecular species of an antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. Specifically, the complementarity-determining regions (CDRs) of the monoclonal antibody are identical in all molecules of the population. MAbs contain an antigen-binding site that can immunoreact with a specific epitope of an antigen, characterized by a unique binding affinity.

[0099] As used herein, the term "epitope" includes a specific portion of an antigen that is targeted by an antibody, antibody fragment, or other binding domain. The term "epitope" includes any protein region to which specific binding is directed. The term "epitope" includes any protein determinant involved in specific binding to a binding molecule such as an antibody or antigen-binding fragment. Epitope determinants generally consist of surface groups having chemical activity of molecules such as amino acids or sugar side chains and generally have specific three-dimensional structural features and specific charge features. For example, an antibody can be produced against the N-terminal, central, or C-terminal peptide of a polypeptide. Further, an antibody can be produced against a linear or discontinuous epitope of a polypeptide.

[0100] As used herein, the terms "specific binding" and "specifically binds" refer to the ability of a binding molecule, such as an antibody or antigen-binding fragment, to preferentially bind to an antigen in a complex mixture of proteins and / or macromolecules. A binding molecule is said to "specifically bind" or "preferentially bind" if it reacts or associates with a particular cell or target antigen more frequently, rapidly, for a longer period of time, and / or with greater affinity than to another cell or target antigen. A binding molecule specifically binds or preferentially binds to its target if it binds to the target with greater affinity, avidity, rapidly, and / or for a longer period of time than to other substances. In some aspects, specific binding can refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. It is understood that specific binding or preferential binding does not necessarily require (although it can include) exclusive binding. Various known methods can be used to quantify or evaluate binding. The strength or affinity of a binding interaction can be represented by the dissociation constant (K d ) of the interaction, with a smaller K d representing greater affinity. One such method involves measuring the rates of formation and dissociation of the antigen-binding site / antigen complex, which rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally affect the rates in both directions. Thus, both the "association rate constant" (K on ) and the "dissociation rate constant" (K off ) can be determined by calculation of the concentrations as well as the actual rates of association and dissociation (see Nature 361:186-87 (1993)). The ratio of K off / K on allows cancellation of all parameters unrelated to affinity, and the dissociation constant K dequal to (see generally Davies et al. (1990) Annual Rev Biochem 59:439-473). A binding molecule, such as an antibody or antigen-binding fragment, specifically binds when the binding constant (K d ) is ≦ 1 μM, for example, in some embodiments, ≦ 100 nM, in some embodiments, ≦ 10 nM, in some embodiments, ≦ 100 pM to about 1 pM, as measured by an assay such as a radioligand binding assay or a similar assay known to those of skill in the art.

[0101] The terms “polypeptide” and “protein” are used interchangeably herein and refer to a molecular chain of two or more amino acids linked by peptide bonds. The term includes post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc. The term also includes molecules that contain one or more amino acid analogs or non-standard or non-natural amino acids that can be synthesized or recombinantly expressed using known protein engineering techniques. Further, a protein can be derivatized. A protein can be a single polypeptide chain or, for example, a multimer (dimer) of at least two polypeptide chains covalently linked to each other, such as by interchain disulfide bonds. Thus, monomers, dimers, and higher order multimeric polypeptides are included within the scope of the defined term. A multimeric polypeptide can be a homomultimer (of the same polypeptide chain) or a heteromultimer (of different polypeptide chains).

[0102] As used herein, the term "isolated protein" means that the protein of interest is (1) free of at least some other proteins found naturally in nature, (2) substantially free of other proteins from the same origin, e.g., from the same species, (3) expressed by cells from different species, (4) separated from at least about 50% of polynucleotides, lipids, carbohydrates, or other materials that associate in nature, (5) not associated (by covalent or non-covalent interactions) with a part of a protein that associates in nature, (6) functionally associated (by covalent or non-covalent interactions) with a polypeptide that does not associate in nature, or (7) not present in nature. Such isolated proteins may be encoded by genomic DNA, cDNA, mRNA, or other RNA, may be of synthetic origin, or may be a combination thereof. In certain embodiments, an isolated protein is substantially free of proteins, polypeptides, or other contaminating substances found in its natural environment that would interfere with its use (for therapy, diagnosis, prevention, research, or otherwise).

[0103] As used herein, the term "polynucleotide" means a nucleotide, ribonucleotide, or deoxynucleotide that is at least 10 bases in length, or a modified form of any type of nucleotide. The term includes single-stranded and double-stranded forms of DNA. Polynucleotides according to the present disclosure include nucleic acid molecules encoding the polypeptides of the multispecific polypeptide constructs shown herein.

[0104] As used herein, the term "isolated polynucleotide" means a polynucleotide that is (1) not associated with all or a portion of a polynucleotide found in nature due to its origin, (2) functionally linked to a polynucleotide not linked in nature, or (3) not present in nature as part of a larger sequence, and is derived from genomic, cDNA, synthetic, or combinations thereof.

[0105] As used herein, the term "functionally linked" refers to a relationship in which the components so described are in a position that permits them to function in the intended manner. A regulatory sequence "functionally linked" to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.

[0106] The term "substantial identity" as applied to polypeptides means that two peptide sequences share at least 80 percent sequence identity when optimally aligned by a program such as GAP or BESTFIT using a default gap weight, for example, in some embodiments, at least 90 percent sequence identity, in some embodiments, at least 95 percent sequence identity, and in some embodiments, at least 99 percent sequence identity. Specifically, conservative amino acid substitutions are contemplated. A conservative substitution is one that occurs within a family of amino acids that have related side chains. Genetically encoded amino acids are generally classified into the following families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include (i) serine and threonine, which are aliphatic hydroxy families; (ii) asparagine and glutamine, which are amide-containing families; (iii) alanine, valine, leucine, and isoleucine, which are aliphatic families; and (iv) phenylalanine, tryptophan, and tyrosine, which are aromatic families. For example, in particular, when the substitution does not involve amino acids within the framework site, it is reasonable to expect that isolated substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitutions of structurally related amino acids will not have a major effect on the binding or properties of the resulting molecule.Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein.

[0107] The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from a biological material.

[0108] As used herein, the terms "label" or "labeled" refer to the incorporation of a detectable marker, for example, by incorporation of a radiolabeled amino acid into a polypeptide, or attachment of a biotinyl group detectable by a labeled avidin (e.g., streptavidin containing a fluorescent marker or enzyme activity detectable by optical or calorimetric means). In certain situations, the label or marker may also be therapeutic. A variety of methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., fluorophores, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pairing sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), but are not limited thereto. In some embodiments, the label is attached by spacer arms of various lengths to reduce possible steric hindrance.

[0109] As used herein, the term "tumor-associated antigen" or "TAA" primarily refers to a counter-structure that is present on tumor cells of a mammalian subject and is generally not found on normal cells of the mammalian subject. Tumor-specific antigens need not be exclusive to tumor cells and can be targeted by anti-tumor therapeutics such as the provided multispecific polypeptide constructs, provided that the proportion of cells having a particular mammalian tumor-associated antigen is sufficiently high or the level of tumor-associated antigen on the surface of the tumor is sufficiently high to provide prevention or treatment against the effects of mammalian tumors. In some embodiments, in a random statistical sample of cells from a mammalian subject having a tumor, at least 50% of the cells exhibiting TAA are cancer cells. In other embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the cells exhibiting TAA are cancer cells.

[0110] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, such as cells. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0111] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a mammalian subject, often a human. A pharmaceutical composition typically comprises an effective amount of an active agent (e.g., a multispecific polypeptide construct) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient, or diluent, respectively.

[0112] As used herein, the terms "treating," "treatment," or "therapy" of a disease or disorder, alone or in combination with another compound described herein, mean slowing, halting, or reversing the progression of a disease or disorder as evidenced by a decrease, cessation, or elimination of clinical or diagnostic symptoms by administration of a pharmaceutical composition of the disclosure. "Treating," "treatment," or "therapy" also mean a reduction in the severity of symptoms or a reduction in the recurrence rate in acute or chronic diseases or disorders. As used herein, with respect to cancer, the terms "treatment" of cancer, or "inhibiting," "inhibition," or "inhibit" are not limited, but are a statistically significant decrease in the rate of tumor growth, measured by standard criteria such as Response Evaluation Criteria for Solid Tumors (RECIST), cessation of tumor growth, or a decrease in the size, amount, metabolic activity, or volume of the tumor, or a statistically significant increase in progression-free survival (PFS) or overall survival (OS), among others. "Preventing," "prevention," or "prevention" of a disease or disorder means administration of a pharmaceutical composition, alone or in combination with another compound, to prevent the occurrence or onset of a disease or disorder, or some or all of the symptoms of a disease or disorder, or to reduce the likelihood of onset of a disease or disorder.

[0113] The term "effective amount" or "therapeutically effective amount" refers to the amount and / or concentration of a composition that, when administered to a patient, alone (i.e., as monotherapy) or in combination with an additional therapeutic agent, gives a statistically significant decrease in disease progression, for example, by alleviating or eliminating the symptoms and / or cause of the disease. An effective amount can be an amount that reduces, lessens, 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 physical function of a patient.

[0114] As used herein, "substantially pure" means that a particular species of substance is the predominant species present (i.e., present in greater abundance on a molar basis than other individual species in the composition), and a substantially purified fraction is a composition in which a particular species of substance constitutes at least about 50 percent (on a molar basis) of all the polymeric species present. Generally, a substantially pure composition will constitute more than about 80 percent of all the polymeric species present in the composition, for example, in some embodiments, more than about 85%, 90%, 95%, and 99%. In some embodiments, the species of substance is purified to substantial homogeneity such that the composition consists essentially of a single polymeric species (no contaminating species can be detected in the composition by conventional detection methods).

[0115] As used herein, "subject" is a mammal such as a human or other animal, typically a human. A subject, e.g., a patient, includes those in need of treatment for a disease or disorder. The term "patient" includes human and animal subjects. The subject may be male or female and may be of any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.

[0116] Other chemical terms herein are used according to their conventional usage in the art, as exemplified by McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

[0117] The term "about" as used herein refers to the ordinary error range for each respective value readily known to those of ordinary skill in the art. References to values or parameters with "about" herein include aspects (descriptions) relating to the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0118] II. Multispecific Polypeptide Constructs A multispecific polypeptide construct containing a first component containing an immunoglobulin Fc region and a second component containing a CD3 binding region, wherein the first and second components are coupled by a linker and the Fc region is positioned N-terminal to the CD3 binding region; and wherein one or both of the first and second components contain (1) an antigen-binding domain that binds to a tumor-associated antigen (TAA) and (2) a co-stimulatory receptor-binding region (CRBR) that binds to a co-stimulatory receptor, is provided herein. Also provided herein is a multispecific polypeptide construct containing a first component containing an immunoglobulin Fc region and a second component containing a CD3 binding region, wherein the first and second components are coupled by a linker and the Fc region is positioned N-terminal to the CD3 binding region; and wherein one or both of the first and second components contain (1) an antigen-binding domain that binds to a tumor-associated antigen (TAA) and (2) an inhibitory receptor-binding region (IRBR) that binds to an inhibitory receptor. In some embodiments, the multispecific polypeptide construct contains at least one antigen-binding domain that binds to a TAA. In some embodiments, the multispecific polypeptide construct contains at least a first antigen-binding domain that binds to a TAA and a second antigen-binding domain that binds to a TAA. In some embodiments, the multispecific polypeptide construct contains at least one CRBR that binds to a co-stimulatory receptor and / or at least one IRBR that binds to an inhibitory receptor. Exemplary formats of the multispecific constructs provided herein are shown in FIGS. 1-3 and FIGS. 18A and 18B.

[0119] In some embodiments, the multispecific polypeptide construct contains, in order from N-terminus to C-terminus, an immunoglobulin Fc region; a linker; a CD3 binding region that binds to CD3 (CD3ε); and at least two binding domains, one of which is an antigen-binding domain that binds to a TAA and the other of which is a CRBR that binds to a co-stimulatory receptor.

[0120] In some embodiments, the multispecific polypeptide construct comprises, in order from the N-terminus to the C-terminus, at least two binding domains, one being an antigen-binding domain that binds to a tumor-associated antigen (TAA) and the other being a CRBR that binds to a costimulatory receptor; an immunoglobulin Fc region; a linker; and a CD3-binding region that binds to CD3 (CD3ε).

[0121] In some embodiments, the multispecific polypeptide construct comprises, in order from the N-terminus to the C-terminus, an immunoglobulin Fc region; a linker; a CD3-binding region that binds to CD3 (CD3ε); and at least two binding domains, one being an antigen-binding domain that binds to a TAA and the other being an IRBR that binds to an inhibitory receptor.

[0122] In some embodiments, the multispecific polypeptide construct comprises, in order from the N-terminus to the C-terminus, an immunoglobulin Fc region; a linker; a CD3-binding region that binds to CD3 (CD3ε); and at least two binding domains, one being an antigen-binding domain that binds to a TAA and the other being an IRBR that binds to an inhibitory receptor.

[0123] In some embodiments, the multispecific polypeptide construct comprises, in order from the N-terminus to the C-terminus, at least two binding domains, one being an antigen-binding domain that binds to a tumor-associated antigen (TAA) and the other being an IRBR that binds to an inhibitory receptor; an immunoglobulin Fc region; a linker; and a CD3-binding region that binds to CD3 (CD3ε).

[0124] In some embodiments, the multispecific polypeptide construct contains, in order from the N-terminus to the C-terminus, at least one antigen-binding domain that binds to a tumor-associated antigen (TAA) or a CRBR that binds to a costimulatory receptor; an immunoglobulin Fc region; a linker; a CD3-binding region that binds to CD3 (CD3ε); and the other of at least one antigen-binding domain that binds to a TAA or a CRBR that binds to a costimulatory receptor. In some embodiments, the N-terminal portion or the C-terminal portion of the multispecific polypeptide construct contains one antigen-binding domain that binds to a TAA. In some embodiments, the N-terminal portion or the C-terminal portion of the multispecific polypeptide construct contains two antigen-binding domains that bind to a TAA. In some embodiments, each of the antigen-binding domains that bind to a TAA is the same. In some embodiments, at least the first antigen-binding domain and the second antigen-binding domain bind to different TAAs or different epitopes of the same TAA.

[0125] In some embodiments, the multispecific polypeptide construct contains, in order from the N-terminus to the C-terminus, at least one antigen-binding domain that binds to a tumor-associated antigen (TAA) or an IRBR that binds to an inhibitory receptor; an immunoglobulin Fc region; a linker; a CD3-binding region that binds to CD3 (CD3ε); and the other of at least one antigen-binding domain that binds to a TAA or an IRBR that binds to an inhibitory receptor. In some embodiments, the N-terminal portion or the C-terminal portion of the multispecific polypeptide construct contains one antigen-binding domain that binds to a TAA. In some embodiments, the N-terminal portion or the C-terminal portion of the multispecific polypeptide construct contains two antigen-binding domains that bind to a TAA. In some embodiments, each of the antigen-binding domains that bind to a TAA is the same. In some embodiments, at least the first antigen-binding domain and the second antigen-binding domain bind to different TAAs or different epitopes of the same TAA.

[0126] In some embodiments, the multispecific polypeptide construct contains, in order from the N-terminus to the C-terminus, a CRBR that binds to a costimulatory receptor; an immunoglobulin Fc region; a linker; a CD3 binding region that binds to CD3 (CD3ε); and at least one antigen-binding domain that binds to a TAA. In some embodiments, the C-terminal portion of the multispecific polypeptide construct contains one antigen-binding domain that binds to a TAA. In some embodiments, the C-terminal portion of the multispecific polypeptide construct contains two antigen-binding domains that bind to a TAA. In some embodiments, each of the antigen-binding domains that bind to a TAA is identical. In some embodiments, at least the first antigen-binding domain and the second antigen-binding domain bind to different TAAs, or different epitopes of the same TAA.

[0127] In some embodiments, the multispecific polypeptide construct contains, in order from the N-terminus to the C-terminus, an IRBR that binds to an inhibitory receptor; an immunoglobulin Fc region; a linker; a CD3 binding region that binds to CD3 (CD3ε); and at least one antigen-binding domain that binds to a TAA. In some embodiments, the C-terminal portion of the multispecific polypeptide construct contains one antigen-binding domain that binds to a TAA. In some embodiments, the C-terminal portion of the multispecific polypeptide construct contains two antigen-binding domains that bind to a TAA. In some embodiments, each of the antigen-binding domains that bind to a TAA is identical. In some embodiments, at least the first antigen-binding domain and the second antigen-binding domain bind to different TAAs, or different epitopes of the same TAA.

[0128] In some embodiments, the multispecific polypeptide construct contains, in order from the N-terminus to the C-terminus, at least one antigen-binding domain that binds to a TAA; an immunoglobulin Fc region; a linker; a CD3-binding region that binds to CD3 (CD3ε); and a CRBR that binds to a co-stimulatory receptor. In some embodiments, the N-terminal portion of the multispecific polypeptide construct contains one antigen-binding domain that binds to a TAA. In some embodiments, the N-terminal portion of the multispecific polypeptide construct contains two antigen-binding domains that bind to a TAA. In some embodiments, each of the antigen-binding domains that bind to a TAA is identical. In some embodiments, at least the first antigen-binding domain and the second antigen-binding domain bind to different TAAs, or different epitopes of the same TAA.

[0129] In some embodiments, the multispecific polypeptide construct contains, in order from the N-terminus to the C-terminus, at least one antigen-binding domain that binds to a TAA; an immunoglobulin Fc region; a linker; a CD3-binding region that binds to CD3 (CD3ε); and an IRBR that binds to an inhibitory receptor. In some embodiments, the N-terminal portion of the multispecific polypeptide construct contains one antigen-binding domain that binds to a TAA. In some embodiments, the N-terminal portion of the multispecific polypeptide construct contains two antigen-binding domains that bind to a TAA. In some embodiments, each of the antigen-binding domains that bind to a TAA is identical. In some embodiments, at least the first antigen-binding domain and the second antigen-binding domain bind to different TAAs, or different epitopes of the same TAA.

[0130] In some embodiments, the multispecific polypeptide construct contains, in order from the N-terminus to the C-terminus, at least one antigen-binding domain that binds to a TAA; an immunoglobulin Fc region; a linker; a CD3-binding region that binds to CD3 (CD3ε); and at least two binding domains, one of which is an antigen-binding domain that binds to a TAA and the other of which is a CRBR that binds to a costimulatory receptor. In some embodiments, the C-terminal portion of the multispecific polypeptide construct contains two antigen-binding domains that bind to a TAA. In some embodiments, each of the antigen-binding domains that bind to a TAA is identical. In some embodiments, at least the first antigen-binding domain and the second antigen-binding domain bind to different TAAs or different epitopes of the same TAA.

[0131] In some embodiments, the multispecific polypeptide construct contains, in order from the N-terminus to the C-terminus, at least one antigen-binding domain that binds to a TAA; an immunoglobulin Fc region; a linker; a CD3-binding region that binds to CD3 (CD3ε); and at least two binding domains, one of which is an antigen-binding domain that binds to a TAA and the other of which is an IRBR that binds to an inhibitory receptor. In some embodiments, the C-terminal portion of the multispecific polypeptide construct contains two antigen-binding domains that bind to a TAA. In some embodiments, each of the antigen-binding domains that bind to a TAA is identical. In some embodiments, at least the first antigen-binding domain and the second antigen-binding domain bind to different TAAs or different epitopes of the same TAA.

[0132] In some embodiments, the multispecific polypeptide construct contains, in order from the N-terminus to the C-terminus, at least two binding domains, one being an antigen-binding domain that binds to a tumor-associated antigen (TAA) and the other being a CRBR that binds to a costimulatory receptor; an immunoglobulin Fc region; a linker; a CD3-binding region that binds to CD3 (CD3ε); and at least one antigen-binding domain that binds to a TAA. In some embodiments, the N-terminal portion of the multispecific polypeptide construct contains two antigen-binding domains that bind to a TAA. In some embodiments, each of the antigen-binding domains that bind to a TAA is identical. In some embodiments, at least the first antigen-binding domain and the second antigen-binding domain bind to different TAAs, or different epitopes of the same TAA.

[0133] In some embodiments, the multispecific polypeptide construct contains, in order from the N-terminus to the C-terminus, at least two binding domains, one being an antigen-binding domain that binds to a tumor-associated antigen (TAA) and the other being an IRBR that binds to an inhibitory receptor; an immunoglobulin Fc region; a linker; a CD3-binding region that binds to CD3 (CD3ε); and at least one antigen-binding domain that binds to a TAA. In some embodiments, the N-terminal portion of the multispecific polypeptide construct contains two antigen-binding domains that bind to a TAA. In some embodiments, each of the antigen-binding domains that bind to a TAA is identical. In some embodiments, at least the first antigen-binding domain and the second antigen-binding domain bind to different TAAs, or different epitopes of the same TAA.

[0134] In some embodiments, the multispecific polypeptide construct contains, in order from the N-terminus to the C-terminus: at least two binding domains, one of which is an antigen-binding domain that binds to a tumor-associated antigen (TAA) and the other of which is a CRBR that binds to a costimulatory receptor; an immunoglobulin Fc region; a linker; a CD3-binding region that binds to CD3 (CD3ε); and at least two binding domains, one of which is an antigen-binding domain that binds to a TAA and the other of which is an IRBR that binds to an inhibitory receptor. In some embodiments, each of the antigen-binding domains that bind to a TAA is identical. In some embodiments, at least the first antigen-binding domain and the second antigen-binding domain bind to different TAAs, or different epitopes of the same TAA.

[0135] In some embodiments, the multispecific polypeptide construct contains, in order from the N-terminus to the C-terminus: at least two binding domains, one of which is an antigen-binding domain that binds to a tumor-associated antigen (TAA) and the other of which is an IRBR that binds to an inhibitory receptor; an immunoglobulin Fc region; a linker; a CD3-binding region that binds to CD3 (CD3ε); and at least two binding domains, one of which is an antigen-binding domain that binds to a TAA and the other of which is a CRBR that binds to a costimulatory receptor. In some embodiments, each of the antigen-binding domains that bind to a TAA is identical. In some embodiments, at least the first antigen-binding domain and the second antigen-binding domain bind to different TAAs, or different epitopes of the same TAA.

[0136] In some embodiments, the multispecific polypeptide construct is a dimer, and dimerization is formed by covalent or non-covalent interactions between 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 some embodiments, the multispecific polypeptide construct contains a homodimeric Fc region, and in some cases, both polypeptide chains of the multispecific polypeptide construct are identical (homodimer). In some embodiments, the multispecific polypeptide construct contains a heterodimeric Fc region, and in some cases, the polypeptide chains of the multispecific polypeptide construct are different (heterodimer). In a specific example of a heterodimeric multispecific polypeptide construct, the CD3 binding region is a two-chain polypeptide containing a VH chain and a VL chain, for example, an Fv antibody fragment containing VH and VL. In some embodiments, the Fv antibody fragment includes a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv).

[0137] In some embodiments, the multispecific polypeptide construct is formed from or comprises two polypeptides: a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a 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, and a VL domain of an anti-CD3 antibody or antigen-binding fragment (e.g., Fv). In some embodiments, the first polypeptide contains one or two antigen-binding domains, or chains thereof, that bind to a TAA. In some embodiments, the second polypeptide contains one or two antigen-binding domains, or chains thereof, that bind to a TAA. In some embodiments, each antigen-binding domain that binds to a TAA is positioned N-terminal to the Fc polypeptide and / or C-terminal to the chain of the CD3-binding region. In some embodiments, the first polypeptide, the second polypeptide, or both the first and second polypeptides further comprise a CRBR such that the multispecific polypeptide construct contains at least one CRBR. In some embodiments, the multispecific polypeptide construct contains two CRBRs. In some embodiments, the CRBR of the first and / or second polypeptide may be positioned N-terminal to the Fc polypeptide and / or C-terminal to the chain of the CD3-binding region. In some embodiments, the first polypeptide, the second polypeptide, or both the first and second polypeptides further comprise an IRBR such that the multispecific polypeptide construct contains at least one IRBR. In some embodiments, the multispecific polypeptide construct contains two IRBRs. In some embodiments, the IRBR of the first and / or second polypeptide may be positioned N-terminal to the Fc polypeptide and / or C-terminal to the chain of the CD3-binding region.In some embodiments, the first polypeptide or the second polypeptide or both the first and second polypeptides further comprise a CRBR and the first polypeptide or the second polypeptide or both the first and second polypeptides further comprise an IRBR such that the multispecific polypeptide construct contains at least one CRBR and at least one IRBR.

[0138] Various exemplary multispecific polypeptide constructs containing at least two polypeptide chains are described herein, for example, in FIGS. 1-3 and 18A-B. Any of such arrangements of the multispecific polypeptide constructs is contemplated.

[0139] In some embodiments, the multispecific polypeptide construct contains at least two antigen-binding domains that bind to tumor-associated antigens (TAAs) and at least one co-stimulatory receptor-binding region (CRBR) that binds to a co-stimulatory receptor. In some embodiments, the multispecific polypeptide construct contains: (1) a first polypeptide that, in order from the N-terminus to the C-terminus, contains a first antigen-binding domain that binds to a tumor-associated antigen (TAA), 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 antigen-binding domain that binds to a tumor-associated antigen (TAA); and (2) a second polypeptide that, in order from the N-terminus to the C-terminus, contains a second Fc polypeptide of a heterodimeric Fc region, the same linker (e.g., the same cleavable linker), the other chain (the other of VH or VL) of the anti-CD3 antibody or antigen-binding fragment, and a co-stimulatory receptor-binding region (CRBR) that binds to a co-stimulatory receptor. In some embodiments, the multispecific polypeptide construct contains: (1) a first polypeptide that, in order from the N-terminus to the C-terminus, includes a first antigen-binding domain that binds to a tumor-associated antigen (TAA), 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 antigen-binding domain that binds to a tumor-associated antigen (TAA); and (2) a second polypeptide that, in order from the N-terminus to the C-terminus, includes a co-stimulatory receptor-binding region (CRBR) that binds to a co-stimulatory receptor, a second Fc polypeptide of a heterodimeric Fc region, the same linker (e.g., the same cleavable linker), and the other chain (the other of VH or VL) of the anti-CD3 antibody or antigen-binding fragment.

[0140] In some embodiments, the multispecific polypeptide construct contains at least two antigen-binding domains that bind to tumor-associated antigens (TAAs) and at least one inhibitory receptor-binding region (IRBR) that binds to an inhibitory receptor. In some embodiments, the multispecific polypeptide construct contains: (1) a first polypeptide that, in order from the N-terminus to the C-terminus, includes a first antigen-binding domain that binds to a tumor-associated antigen (TAA), 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 antigen-binding domain that binds to a tumor-associated antigen (TAA); and (2) a second polypeptide that, in order from the N-terminus to the C-terminus, includes a second Fc polypeptide of a heterodimeric Fc region, the same linker (e.g., the same cleavable linker), the other chain (the other of VH or VL) of an anti-CD3 antibody or antigen-binding fragment, and an inhibitory receptor-binding region (IRBR) that binds to an inhibitory receptor. In some embodiments, the multispecific polypeptide construct contains: (1) a first polypeptide that, in order from the N-terminus to the C-terminus, includes a first antigen-binding domain that binds to a tumor-associated antigen (TAA), 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 antigen-binding domain that binds to a tumor-associated antigen (TAA); and (2) a second polypeptide that, in order from the N-terminus to the C-terminus, includes an inhibitory receptor-binding region (IRBR) that binds to an inhibitory receptor, a second Fc polypeptide of a heterodimeric Fc region, the same linker (e.g., the same cleavable linker), and the other chain (the other of VH or VL) of an anti-CD3 antibody or antigen-binding fragment.

[0141] In some embodiments, the multispecific polypeptide construct contains at least two antigen-binding domains that bind to tumor-associated antigens (TAAs), at least one co-stimulatory receptor-binding region (CRBR) that binds to a co-stimulatory receptor, and at least one inhibitory receptor-binding region (IRBR) that binds to an inhibitory receptor. In some embodiments, the multispecific polypeptide construct contains the following: (1) a first polypeptide that, in order from the N-terminus to the C-terminus, includes a first antigen-binding domain that binds to a tumor-associated antigen (TAA), 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 antigen-binding domain that binds to a tumor-associated antigen (TAA); and (2) a second polypeptide that, in order from the N-terminus to the C-terminus, includes one of CRBR or IRBR, a second Fc polypeptide of a heterodimeric Fc region, the same linker (e.g., the same cleavable linker), the other chain (the other of VH or VL) of an anti-CD3 antibody or antigen-binding fragment, and the other of CRBR or IRBR.

[0142] Each of the components of the multispecific polypeptide construct of the present disclosure is described in more detail below.

[0143] 1. Anti-CD3 binding domain The multispecific polypeptide constructs of the present disclosure include one or more copies of an anti-CD3 binding domain. The anti-CD3 binding domains of the present disclosure activate T cells through engagement of CD3ε in T cells. The anti-CD3 binding domains of the present disclosure operate, stimulate, activate, and / or otherwise increase CD3-mediated T cell activation. The biological activities of CD3 include, for example, T cell activation and other signaling through the interaction between CD3 and the antigen-binding subunits of the T cell receptor (TCR). For example, the anti-CD3 binding domains of the present disclosure activate T cells either fully or partially through engagement of CD3ε in T cells by, for example, partially or fully modulating CD3-mediated T cell activation, such as by operating, stimulating, activating, or otherwise increasing it.

[0144] In preferred embodiments, the anti-CD3 binding domains of the present disclosure specifically bind to the ε chain of CD3, also known as CD3ε. The anti-CD3ε binding domains of the present disclosure activate T cells through engagement of CD3ε in T cells. The anti-CD3ε binding domains of the present disclosure include, for example, monoclonal antibodies such as mammalian monoclonal antibodies, primate monoclonal antibodies, fully human monoclonal antibodies, and also include humanized monoclonal antibodies and chimeric antibodies, as well as antigen-binding fragments thereof. In some embodiments, the anti-CD3ε binding domain includes one or more copies of an antibody or an antigen-binding fragment thereof.

[0145] In some embodiments, the anti-CD3ε binding domain comprises one or more copies of an antibody or an antigen-binding fragment thereof selected from the group consisting of a Fab fragment, an 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. In some embodiments, the anti-CD3 binding domain comprises an Fv antibody fragment that binds to CD3ε (referred to herein 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 CD3 binding.

[0146] In some embodiments, the CD3 binding region is not a single-chain antibody. For example, in some aspects, the CD3 binding region is not a single-chain variable fragment (scFv).

[0147] In some embodiments, the CD3 binding region is an Fv antibody fragment containing a variable heavy chain (also referred to as Hv or VH) and a variable light chain (also referred to as Lv or VL), such as any of those described. In aspects of such embodiments, the immunoglobulin Fc region is a heterodimeric Fc region containing two different Fc polypeptides capable of heterodimeric association between both polypeptides of the Fc heterodimer, such as any of those described in Section II.2. In such embodiments, the variable heavy chain (Hv) and variable light chain (Lv) of the CD3 binding region are linked to opposing chains of the heterodimeric Fc.

[0148] In some embodiments, the anti-CD3ε binding domain comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, and at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence is a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO:16); an amino acid sequence Selected from a VH CD2 sequence comprising at least TIFF2025098077000011.tif4128; and a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:18).

[0149] In some embodiments, the anti-CD3ε binding domain comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, and at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence is selected from a VL CDR1 sequence comprising at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:19); a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO:20); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO:21).

[0150] In some embodiments, the anti-CD3ε binding domain comprises a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO:16); the amino acid sequence A VH CD2 sequence comprising at least TIFF2025098077000012.tif4128; a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:18), a VL CDR1 sequence comprising at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:19); a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO:20); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO:21).

[0151] In some embodiments, the anti-CD3ε binding domain comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, and at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence comprises a VH CDR1 sequence having a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence TYAMN (SEQ ID NO:16); a VH CD2 sequence having a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to TIFF2025098077000013.tif4128; and a VH CDR3 sequence having a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:18).

[0152] In some embodiments, the anti-CD3ε binding domain comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, and at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence comprises a VL CDR1 sequence having a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:19); a VL CDR2 sequence having a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence GTNKRAP (SEQ ID NO:20); and a VL CDR3 sequence having a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence ALWYSNLWV (SEQ ID NO:21).

[0153] In some embodiments, the anti-CD3ε binding domain comprises a VH CDR1 sequence comprising at least the amino acid sequence GFTFNTYAMN (SEQ ID NO:312); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATY (SEQ ID NO:318); a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:18), a VL CDR1 sequence comprising at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:19); a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO:20); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO:21).

[0154] In some embodiments, the anti-CD3ε binding domain comprises a VH CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence TYAMN (SEQ ID NO:16); an amino acid sequence TIFF2025098077000014.tif4128 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the VH CD2 sequence; a VH CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:18), a VL CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:19); a VL CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence GTNKRAP (SEQ ID NO:20); and a VL CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence ALWYSNLWV (SEQ ID NO:21).

[0155] In some embodiments, the anti-CD3ε binding domain comprises a VH CDR1 sequence comprising at least the amino acid sequence GFTFNTYAMN (SEQ ID NO:312); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATY (SEQ ID NO:318); a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:18), a VL CDR1 sequence comprising at least the amino acid sequence GSSTGAVTTSNYAN (SEQ ID NO:319); a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO:320); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNHWV (SEQ ID NO:315).

[0156] In some embodiments, the anti-CD3ε binding domain comprises a VH CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence GFTFNTYAMN (SEQ ID NO: 312); a VH CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence RIRSKYNNYATY (SEQ ID NO: 313); a VH CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence GSSTGAVTTSNYAN (SEQ ID NO: 319); a VL CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence GTNKRAP (SEQ ID NO: 320); and a VL CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence ALWYSNHWV (SEQ ID NO: 315).

[0157] 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 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81 and 241. 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 that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81 and 241. 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 consisting of SEQ ID NOs: 32-62 and a light chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81 and 241. In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62 and a light chain variable amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81 and 241.

[0158] 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 that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81 and 241. In some embodiments, the anti-CD3ε binding domain comprises a combination of a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62 and a light chain variable region amino acid sequence that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81 and 241.

[0159] In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 32-81, 241, 287-291, and 311. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 32-81, 241, 287-291, and 311. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of a variable heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 32-62, and 287, 290, and 311 and a variable light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 63-81, 241, 288, and 289. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of a variable heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 32-62, 287, 290, and 311 and a variable light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 63-81, 241, 288, and 289.

[0160] 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 Fv antibody fragment comprises a combination of a variable heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 32-43, 45-47, 48, and 287, and a variable light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 15, 63, 65-71, 73, 75, 77, and 288. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of a variable heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 14, 32-43, 45-47, 48, and 287 and a variable light chain amino acid sequence selected from the group consisting of SEQ ID NO: 15, 63, 65-71, 73, 75, 77, and 288.

[0161] In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (Lv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:15. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:14 and a variable light chain (Lv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:15. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO:14. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO:15. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO:14 and a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO:15.

[0162] In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:287. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:288. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:287 and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:288. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:287. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:288. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:287 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:288.

[0163] In a specific embodiment, the Fv is V H -V LIt is a disulfide-stabilized Fv (dsFv) in which the heterodimer is stabilized by an interchain disulfide bond. In some embodiments, the interchain disulfide bond is modified by mutations at positions in the framework positions of the VH chain and / or the VL chain. In some embodiments, by Kabat numbering, the VH chain contains a mutation to Cys at position 44 and the VL chain contains a mutation to Cys at position 100. For example, in some embodiments, by Kabat numbering, the VH chain contains the mutation G44C and the VL chain contains the mutation G100C. In some embodiments, the disulfide-stabilized anti-CD3 Fv comprises an anti-CD3 VH containing a mutation to Cys at position 105 and an anti-CD3 VL containing a mutation to Cys at position 43 by Kabat numbering.

[0164] In some embodiments, the anti-CD3ε Fv comprises a combination of a variable heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 44 and 49-62, 290, and 311, and a variable light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 64, 72, 74, 76, 78-81, 241, and 289. In some of such embodiments, the anti-CD3 Fv is a dsFv having a VH chain containing the mutation G44C and a VL chain containing the mutation G100C by Kabat numbering, respectively. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of a variable heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 44 and 49-62, 290, and 311, and a variable light chain amino acid sequence selected from the group consisting of SEQ ID NO: 64, 72, 74, 76, 78-81, 241, and 289.

[0165] In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:44. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (Lv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:72. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:44, and a variable light chain (Lv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:72. In some of such embodiments, the anti-CD3 Fv is a dsFv having a VH chain containing the mutation G44C and a VL chain containing the mutation G100C, respectively, by Kabat numbering. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO:44. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO:72. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO:44 and a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO:72.

[0166] In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (Lv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 241. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 44, and a variable light chain (Lv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 241. In some of such embodiments, the anti-CD3 Fv is a dsFv having a VH chain containing the mutation G44C and a VL chain containing the mutation G100C, each by Kabat numbering. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO: 241. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO: 44 and a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO: 241.

[0167] In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:290. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:289. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:290, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:289. In some of such embodiments, the anti-CD3 Fv is a dsFv having a VH chain containing the mutation G44C and a VL chain containing the mutation G100C, each by Kabat numbering. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:290. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:289. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:290 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:289.

[0168] In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 311. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 289. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 311, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 289. In some of such embodiments, the anti-CD3 Fv is a dsFv having a VH chain containing the mutation G44C and a VL chain containing the mutation G100C, each by Kabat numbering. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 311. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 289. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 311 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 289.

[0169] 2. Immunoglobulin Fc polypeptide The multispecific polypeptide constructs of the present disclosure include an immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is an IgG isotype selected from the group consisting of IgG1 isotype, IgG2 isotype, IgG3 isotype, and IgG4 subclass. In some embodiments, the Fc region is a human Fc. In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6. In some embodiments, the immunoglobulin Fc region contains an Fc chain that is an immunologically active fragment of any of SEQ ID NOs: 1-6. In some embodiments, the immunoglobulin Fc region contains an Fc polypeptide chain that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences of SEQ ID NOs: 1-6, or an immunologically active fragment thereof.

[0170] In some embodiments, the multispecific polypeptide construct is a dimer formed by polypeptides each containing an Fc. In some specific embodiments, the same or substantially the same polypeptide will dimerize to create a homodimer. In some embodiments, the dimer is a homodimer in which the two polypeptide chains of the multispecific polypeptide construct are identical. In other cases, the Fc region is formed by an Fc domain that has been mutated or modified to promote heterodimerization in which different polypeptides dimerize to give a heterodimer. Thus, in some embodiments, the dimer is a heterodimer in which the two polypeptide chains of the multispecific polypeptide construct are different. Exemplary modifications for promoting heterodimerization are known and include the following.

[0171] Generally, in addition to the antigen-binding ability, which is the main function of immunoglobulins, the Fc region is responsible for effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Furthermore, the FcRn sequence present in the Fc region plays a role in controlling the IgG level in serum by increasing the in vivo half-life through conjugation with the in vivo FcRn receptor. In some embodiments, in the Fc for use with the provided multispecific polypeptide construct, such functions may be altered, for example, they may be decreased or enhanced.

[0172] 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 provide Fc-mediated effector functions such as, for example, ADCC (e.g., release of granzyme B by NK cells), ADCP, and / or CDC. In some embodiments where the multispecific polypeptide construct contains a cleavable linker, cleavage of the linker produces two components each having biological activity: a CD3-binding region capable of binding to and engaging CD3 on T cells (which can also include, in some aspects, a CRBR for inducing a co-stimulatory signal on T cells), and an Fc region linked to a TAA antigen-binding domain capable of exhibiting a target-specific effector function. In certain embodiments provided herein, the multispecific polypeptide construct includes a non-cleavable linker and, in some aspects, may not exhibit independent Fc-mediated effector functions.

[0173] In some embodiments, the Fc region comprises an Fc polypeptide that has been mutated or modified to alter one or more effector functions. Various examples of mutations of the Fc polypeptide for altering, e.g., reducing, effector functions are known and include the following. In some embodiments, unless otherwise specified with respect to a particular SEQ ID NO, references to amino acid substitutions in the Fc region are by EU numbering according to Kabat (also referred to as Kabat numbering). EU numbering is known and is according to the EU index reported in the latest IMGT Scientific Chart (IMGT®, the international ImMunoGeneTics information system®, http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created: 17 May 2001, last updated: 10 Jan 2013)) and Kabat, E. A. et al. Sequences of Proteins of Immunological interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0174] In some embodiments, provided multispecific polypeptide constructs containing an Fc region that exhibits reduced effector function may be desirable candidates for applications where restricted CD3 binding is desired but certain effector functions (such as CDC and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays may be performed to confirm reduced / abrogated CDC activity and / or ADCC activity. For example, to ensure that a multispecific polypeptide construct and / or its cleaved components lack FcγR binding (and thus are likely to lack ADCC activity) but retain FcRn binding ability, an Fc receptor (FcR) binding assay may be performed. The main cells for mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Non-limiting examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent 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. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assay methods may be utilized (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.)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest can be evaluated in vivo 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 cleaved components are unable to bind to C1q and thus lack CDC activity. See, for example, the C1q binding ELISA and C3c binding ELISA of WO 2006 / 029879 and WO 2005 / 100402. A CDC assay can be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and determination of in vivo clearance / half-life can also be performed using methods known in the art (see, for example, Petkova, S. B. et al., Int'l. Immunol. 18 (12): 1759-1769 (2006)).

[0175] In some embodiments, the immunoglobulin Fc region or an 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. TIFF2025098077000015.tif31128

[0176] In some embodiments, the immunoglobulin Fc region or an 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:1.

[0177] In some embodiments, an IgG1 Fc polypeptide or variant thereof, such as any of the following, can be made in the G1m1 allotype or the G1m3 allotype. In some embodiments, the Fc region may contain amino acids of the human G1m1 allotype, such as residues containing Asp (D) and Leu (L) at positions 356 and 358, as shown, for example, in SEQ ID NO:1. In some cases, the Fc polypeptide may contain the amino acid substitutions E356D and M358L to reconstruct the residues of the allotype G1m1. In other embodiments, the Fc region may contain amino acids of the human G1m3 allotype, such as residues Glu (E) and Met (M) at positions 356 and 358, as shown, for example, in SEQ ID NOs:194 and 195, according to EU numbering. In some cases, the Fc polypeptide may contain the amino acid substitutions D356E and L358M to reconstruct the residues of the allotype G1m3.

[0178] In some embodiments, the human IgG1 Fc region is modified to alter antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). For example, amino acid modifications described in 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 reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11. The entire content of each of these is hereby incorporated by reference in its entirety.

[0179] In some embodiments, an Fc region, such as the human IgG1 Fc region, is modified to enhance ADCC activity or CDC activity. Examples of mutations that enhance ADCC include modifications at Ser239 and Ile332, such as 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 using the Kabat numbering system (e.g., Lys326Ala and / or Glu333Ala (K326A and E333A)).

[0180] In some embodiments, the Fc region is modified to provide reduced Fc-mediated effector function, for example, through binding to reduced Fc receptor binding, such as FcγR binding rather than normal FcRn binding. In some embodiments, the human IgG1 Fc region fusion protein of the present disclosure lacks fucose attached to the N-linked glycan chain at N297 or has reduced fucose. There are a number of 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. In some embodiments, the human IgG1 Fc region is modified at amino acid Asn297 (boxed, Kabat numbering) (e.g., Asn297Ala (N297A) or Asn297Asp (N297D)) to prevent glycosylation of the fusion protein.

[0181] In some embodiments, the Fc region of the fusion protein is modified 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), or Ala327 (A327). For example, Leu234Ala (L234A), Leu235Ala (L235A), Asp265Asn (D265N), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Asn325Glu (N325E), or Ala327Ser (A327S). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu235 (within the box of SEQ ID NO:1, Kabat numbering) to alter Fc receptor interaction (e.g., Leu235Glu (L235E) or Leu235Ala (L235A)). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (within the box of SEQ ID NO:1, Kabat numbering) to alter Fc receptor interaction (e.g., Leu234Ala (L234A)). In some embodiments, the Fc region of the fusion protein is modified at both amino acids 234 and 235 (e.g., Leu234Ala and Leu235Ala (L234A / L235A) or Leu234Val and Leu235Ala (L234V / L235A)). In a preferred embodiment, the modification within the Fc region reduces binding to the Fc receptor gamma receptor but has minimal effect on binding to the neonatal Fc receptor (FcRn).

[0182] 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, Thr256Glu (M252Y, S254T, T256E respectively) (Kabat numbering, Dall'Acqua et al 2006, J. Biol Chem Vol. 281(33)23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al 2010 Nature Biotech, Vol. 28(2)157-159) (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest). In some embodiments, the mutant or modified Fc polypeptide contains the following mutations using the Kabat numbering system: Met252Tyr and Met428Leu or Met252Tyr and Met428Val (M252Y, M428L or M252Y, M428V).

[0183] In some embodiments, the Fc region of the fusion protein lacks the amino acid at one or more of the following positions to reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (L235). In these embodiments, the Fc deletion of these three amino acids reduces complement protein C1q binding. TIFF2025098077000016.tif30128

[0184] 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 can include E233P, L234V, and / or L235A.

[0185] In some embodiments, the Fc region of the fusion protein is modified at Gly236 (within the box of SEQ ID NO:1) to reduce Fc receptor binding. For example, Gly236 is deleted from the fusion protein. In some embodiments, the human IgG1 Fc region is modified at amino acid Gly236 (e.g., Gly236Ala (G236A)) to enhance its interaction with CD32A.

[0186] In specific embodiments, for example, mutations in the Fc region to reduce Fc effector function via reduced binding of the Fc receptor to FcγR include any of the mutations G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G, or E233P / L234V / L235A / G236del.

[0187] In some embodiments, the human IgG1 Fc region lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).

[0188] In some embodiments, the fusion or its immunologically active fragment 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:2.

[0189] In some embodiments, the immunoglobulin Fc region of the fusion protein or its immunologically active fragment is of the human IgG2 isotype and has the following amino acid sequence. TIFF2025098077000017.tif30128

[0190] In some embodiments, the fusion or its immunologically active fragment 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:3.

[0191] In some embodiments, the human IgG2 Fc region is modified at amino acid Asn297 (boxed, for example, Asn297Ala (N297A) or Asn297Asp (N297D) to prevent glycosylation of the antibody). In some embodiments, the human IgG2 Fc region lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).

[0192] In some embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG3 isotype and has the following amino acid sequence. TIFF2025098077000018.tif31128

[0193] In some embodiments, the antibody or its immunologically active fragment 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:4.

[0194] In some embodiments, the human IgG3 Fc region is modified at amino acid Asn297 (boxed, Kabat numbering) (e.g., Asn297Ala (N297A) or Asn297Asp (N297D)) to prevent glycosylation of the antibody. In some embodiments, the human IgG3 Fc region is modified at amino acid 435 (e.g., Arg435His (R435H)) to extend the half-life. In some embodiments, the human IgG3 Fc region lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).

[0195] 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. TIFF2025098077000019.tif31128

[0196] In some embodiments, the antibody or its immunologically active fragment 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:5.

[0197] 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. TIFF2025098077000020.tif30128

[0198] In some embodiments, the antibody or its immunologically active fragment 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:6.

[0199] In other embodiments, the human IgG4 Fc region is modified at amino acid 235 (e.g., Leu235Glu (L235E)) to alter Fc receptor interactions. In some embodiments, the human IgG4 Fc region is modified at amino acid Asn297 (boxed, Kabat numbering) (e.g., Asn297Ala (N297A) or Asn297Asp (N297D)) to prevent glycosylation of the antibody. In some embodiments, the human IgG4 Fc region lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).

[0200] In some embodiments, the human IgG Fc region is modified to stabilize homodimerization at the CH3:CH3 interface by introducing two disulfide bonds by changing Ser354 to Cys (S354C) and changing Tyr349 to Cys (Y349C) (S354C / Y349C).

[0201] In certain embodiments of the multispecific polypeptide constructs provided herein, the human IgG Fc region is modified to induce heterodimerization. A variety of methods for promoting heterodimerization of complementary Fc polypeptides are known. For example, see 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, WO2009 / 089004, WO2011143545, WO2014 / 067011, WO2012 / 058768, WO2018027025; Published US Patent Application Nos. US20140363426, US20150307628, US20180016354, US20150239991; and US Patents Nos. 5731168, 7183076, 9701759, 9605084, and 9650446. Methods for promoting heterodimerization of the Fc chains include, for example, mutagenesis of the Fc region by including a set of "knob-into-hole" mutations or by including mutations to achieve electrostatic steering of the Fc that is favorable for attractive interactions between different polypeptide chains.For example, in some embodiments, the Fc polypeptide of the heterodimer has mutations to alter the charge polarity at the Fc dimer interface such that electrostatically matched co-expression of the Fc chains supports favorable attractive interactions, thereby promoting the formation of the desired Fc heterodimer, and unfavorable repulsive charge interactions suppress the formation of undesired Fc homodimers (Guneskaran et al., (2010) JBC, 285:19637-19646). When co-expressed in cells, the chains can associate with each other, but due to charge repulsion, the chains do not substantially self-associate. Other strategies for generating heterodimeric Fc include mixing of human IgG and IgA CH3 domain segments to create complementary CH3 heterodimers, called SEED Fc.

[0202] Methods and variants of heterodimerization also include those described in the published international PCT application WO2014 / 145806, including "knobs and holes" mutations (also called "skew" variants), mutations related to "electrostatic steering" or "charge pairs", and pI variants. Heterodimeric variants also include any of those described in the published US application numbers US2012 / 0149876 or US2018 / 011883.

[0203] In some embodiments, to promote heterodimerization, both polypeptides of the Fc heterodimer contain paired or complementary amino acid modifications. Exemplary pairs of amino acid modifications of the polypeptide of the Fc fusion are shown in Table 1.

[0204] (Table 1) Pairs of amino acids of heterodimeric Fc TIFF2025098077000021.tif47133

[0205] In some embodiments, the modification involves introducing a protrusion (knob) into the first Fc polypeptide and a cavity (hole) into the second Fc polypeptide such that the protrusion is positioned in the cavity to facilitate complexation of the first and second Fc-containing polypeptides. The amino acids targeted for substitution and / or modification to create the protrusion or cavity in the polypeptide are typically interfacial amino acids that interact with or contact one or more amino acids at the interface of the second polypeptide.

[0206] In some embodiments, the first Fc polypeptide modified to contain a protrusion (hole) amino acid protrudes from the interface of the first Fc polypeptide and thus has at least one side chain positioned in a compensatory cavity (hole) at the adjacent interface of the second polypeptide, including substitution of the native or first amino acid with an amino acid. The substituted amino acid most often has a larger side chain volume than the first amino acid residue. Methods for determining and / or evaluating the properties of amino acid residues are known to those of skill in the art to identify those that are ideal substituted amino acids for creating a protrusion. In some embodiments, the substituted residue for formation of the protrusion is a naturally occurring amino acid residue and includes, for example, arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). In some examples, the first residue identified for substitution is an amino acid residue with a small side chain, such as, for example, alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0207] In some embodiments, the second Fc polypeptide modified to contain a cavity (hole) is recessed from the interface of the second polypeptide and thus has at least one side chain of an amino acid that can receive a corresponding protrusion from the interface of the first polypeptide, including substitution of a native or first amino acid to the amino acid. The substituted amino acid most often has a smaller side chain volume than the first amino acid residue. Methods for determining and / or evaluating the properties of amino acid residues are known to those skilled in the art to identify those that are ideal substitution residues for cavity formation. Generally, substitution residues for cavity formation are naturally occurring amino acids, including, for example, alanine (A), serine (S), threonine (T), and valine (V). In some examples, the first amino acid identified for substitution is an amino acid with a large side chain, such as, for example, tyrosine, arginine, phenylalanine, or tryptophan.

[0208] The CH3 interface of human IgG1 contains, for example, 16 residues of each domain located in four antiparallel β-strands buried 1090 Å2 from each surface (see, for example, 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. Patent No. 5,731,168). Modification of the CH3 domain to create a protrusion or cavity is described, for example, in U.S. Patent No. 5,731,168; International Patent Applications WO98 / 50431 and WO2005 / 063816; and Ridgway et al., (1996) Prot. Engin. 617-621. In some examples, modification of the CH3 domain to create a protrusion or cavity typically targets residues located in two central antiparallel β-strands. The goal is to minimize the risk that the created protrusion is received by the compensatory cavity of the partner CH3 domain rather than being received by protruding into the surrounding solvent.

[0209] For example, in some embodiments, the heterodimeric Fc can preferentially pair with a second CH3 domain having amino acid modifications (T366S / L368A / Y407V) at positions Thr366, Leu368, and Tyr407 to smaller amino acids, such as Ser, Ala, Val, respectively, when exchanged with a bulkier amino acid, such as Try (T366W). Heterodimerization via CH3 modification can be further stabilized by the introduction of disulfide bonds, for example, by the change of Ser354 to Cys (S354C) and Tyr349 to Cys (Y349C) in the opposing CH3 domains (reviewed in Carter, 2001 Journal of Immunological Methods, 248:7-15).

[0210] In a specific embodiment, the multispecific polypeptide construct contains a first and a second Fc that can mediate Fc heterodimerization, the first Fc polypeptide contains the mutations T366W and S354C, and the second Fc polypeptide contains the mutations T366S, L368A, Y407V, and Y349C. In some embodiments, the first Fc polypeptide is selected from Fc polypeptides comprising the sequences shown in SEQ ID NO:291 or 297, and the second Fc polypeptide is selected from Fc polypeptides comprising the sequences shown in SEQ ID NO:292, 295, or 299. In some embodiments, the first Fc polypeptide has or comprises the amino acid sequence shown in any of SEQ ID NO:82, 86, 94, or 96, and the second Fc polypeptide has or comprises the amino acid sequence shown in any of SEQ ID NO:83, 87, 90, 92, 98, or 100.

[0211] In some embodiments, the Fc polypeptide exhibits characteristics that provide Fc-mediated effector functions. In specific examples, the first Fc polypeptide has the sequence shown in SEQ ID NO: 291 or includes it, and the second Fc polypeptide is or includes SEQ ID NO: 292 or 295. In some embodiments, the first Fc polypeptide has the sequence shown in SEQ ID NO: 82 or includes it, and the second Fc polypeptide is or includes the sequence shown in SEQ ID NO: 83 or 90. In some embodiments, the first Fc polypeptide has the sequence shown in SEQ ID NO: 86 or includes it, and the second Fc polypeptide is or includes the sequence shown in SEQ ID NO: 87 or 92. The first and second Fc polypeptides can be formatted in any polypeptide chain of the construct.

[0212] In some embodiments, one or both of the first and second Fc polypeptides may further comprise one or more amino acid mutations to further reduce one or more Fc effector functions such as reduced Fc receptor binding. Exemplary mutations for reducing Fc effector functions include any of those described. In some embodiments, the modification can be a deletion of one or more positions Glu233 (E233), Leu234 (L234), or Leu235 (L235), such as a deletion of Glu233 (E233), Leu234 (L234), and Leu235 (L235). In some embodiments, the first Fc polypeptide is selected from Fc polypeptides comprising the sequences shown in SEQ ID NO: 293 or 298, and the second Fc polypeptide is selected from Fc polypeptides comprising the sequences shown in SEQ ID NO: 294, 296, or 300. In some embodiments, the first Fc polypeptide is or comprises the amino acid sequence shown in any of SEQ ID NO: 84, 88, 95, or 97, and the second Fc polypeptide is or comprises the amino acid sequence shown in any of SEQ ID NO: 85, 89, 91, 93, 99, or 101.

[0213] In a specific example, the first Fc polypeptide is or comprises the sequence shown in SEQ ID NO: 293, and the second Fc polypeptide is or comprises SEQ ID NO: 294 or 296. In some embodiments, the first Fc polypeptide is or comprises the sequence shown in SEQ ID NO: 84, and the second Fc polypeptide is or comprises the sequence shown in SEQ ID NO: 85 or 91. In some embodiments, the first Fc polypeptide is or comprises the sequence shown in SEQ ID NO: 88, and the second Fc polypeptide is or comprises the sequence shown in SEQ ID NO: 89 or 93. The first and second Fc polypeptides can be formatted in either polypeptide chain of the construct.

[0214] In some embodiments, the first Fc polypeptide or the second Fc polypeptide further comprises the mutations M252Y and / or M428V. In a specific example, the first Fc polypeptide is or comprises the sequence shown in SEQ ID NO:297, and the second Fc polypeptide is or comprises the sequence shown in SEQ ID NO:299. In some embodiments, the first Fc polypeptide is or comprises the sequence shown in SEQ ID NO:94, and the second Fc polypeptide is or comprises the sequence shown in SEQ ID NO:98. In some embodiments, the first Fc polypeptide is or comprises the sequence shown in SEQ ID NO:96, and the second Fc polypeptide is or comprises the sequence shown in SEQ ID NO:100. In other examples, the first Fc polypeptide is or comprises the sequence shown in SEQ ID NO:298, and the second Fc polypeptide is or comprises the sequence shown in SEQ ID NO:300. In some embodiments, the first Fc polypeptide is or comprises the sequence shown in SEQ ID NO:95, and the second Fc polypeptide is or comprises the sequence shown in SEQ ID NO:99. In some embodiments, the first Fc polypeptide is or comprises the sequence shown in SEQ ID NO:97, and the second Fc polypeptide is or comprises the sequence shown in SEQ ID NO:101. The first and second Fc polypeptides can be formatted in any polypeptide chain of the construct.

[0215] Additional examples of variants that can facilitate the promotion of heterodimers are 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 T366S / L368A / Y407V and T366W, or 366S / L368A / Y407V / Y349C and T366W / S354C (each pair represents a mutation in the first Fc polypeptide and the second Fc polypeptide), any combination or pair of steric variants (e.g., sc variants) of the first Fc polypeptide and the second Fc polypeptide. In a specific embodiment, the provided construct contains first and second Fc polypeptides containing the mutation pairs L368D / K370S and S364K and E357Q.

[0216] An additional mechanism that can be used in the generation of heterodimers is what is sometimes referred to as "electrostatic steering" as described in Gunasekaran et al., J. Biol. Chem. 285 (25): 19637 (2010). This is sometimes referred to herein as a "charge pair". In this aspect, electrostatics is used to bias the formation towards heterodimerization. As will be understood by those skilled in the art, these also have an effect on the pI and thus on purification and can thus, in some cases, also be considered pI variants. However, since they are generated to enforce heterodimerization and not used as purification tools, they are classified as "steric variants". In one aspect, 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 aspect, the first Fc polypeptide may contain the mutations C220E / P228E / 368E, and the second Fc polypeptide may contain the mutations C220R / E224R / P228R / K409R.

[0217] In some embodiments, heterodimerization can be facilitated by pI variants. In some instances, pI variants can include those that increase the pI of the protein (basic changes). In other instances, pI variants can include those that decrease the pI of the protein (acidic changes). In some cases, all combinations of these variants can be made, including combinations where one Fc polypeptide can be wild-type or a variant that does not exhibit a significantly different pI from wild-type, and the other Fc polypeptide can be more basic or more acidic. Alternatively, each Fc polypeptide can be altered such that one is made more basic and one is made more acidic. In some embodiments, at least one Fc polypeptide is a negative pI variant Fc containing the mutations Q295E / N384D / Q418E / N421D.

[0218] In some embodiments, combinations of steric heterodimerization variants (e.g., knob and hole) with pI variants or charge pair variants can be used.

[0219] In a specific embodiment, the provided construct contains (a) a first Fc polypeptide comprising the skew variant S364K / E357Q; and (b) a second Fc polypeptide containing the skew variants L368D / K370S and the pI variant N208D / Q295E / N384D / Q418E / N421D. In some embodiments, one or both of the first and second polypeptides may contain additional mutations to reduce Fc effector activity, such as the exemplary mutations E233P / L234V / L235A / G236del / S267K. Examples of such first and second Fc polypeptides that can mediate Fc heterodimerization include the sequences shown in SEQ ID NO:285 and 286. The first and second Fc polypeptides can be formatted in either polypeptide chain of the construct.

[0220] The resulting multispecific polypeptide construct can be purified by suitable methods, such as affinity chromatography on a protein A column or a protein G column. When two nucleic acid molecules encoding different polypeptides are transformed into cells, homodimer and heterodimer formation will occur. The conditions for expression can be adjusted so that heterodimer formation is favored over homodimer formation.

[0221] Techniques for the recovery of heterodimers from homodimers based on differential affinity for heterodimeric affinity reagents are known. In some aspects, such techniques involve the design of heterodimers 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 to abolish or reduce the affinity for the protein A reagent in one of the polypeptides of the Fc heterodimer. See, for example, WO2017134440, WO2010151792, Jendeberg et al., (1997) J. Immunol. Methods, 201(1): 25-34. In some of these embodiments, the Fc region can be modified at the protein A binding site of one member of the heterodimer to prevent protein A binding and thereby enable more efficient purification of the heterodimeric fusion protein. Exemplary modifications at this binding site are Ile253, e.g., Ile253Arg (I253R). In some embodiments, the modification can be H435R or H435R / Y436F. In some embodiments, the Fc polypeptide of the Fc heterodimer may contain modifications (pA+ / pG-) such that it can bind to protein A but cannot bind to protein G. Exemplary pA+ / pG- amino acid modifications for human IgG1 contain serine at position 428, serine at position 434, and optionally histidine at position 436, or Fc containing these residues at the corresponding positions in human IgG 2, 3, or 4. In some aspects, such amino acid modifications at positions 428 and 434 of a given IgG Fc polypeptide, and optionally such amino acid modifications at position 436, reduce or prevent binding of protein G and enhance purification of the protein.

[0222] In some embodiments, any such modification that confers differential affinity for an affinity reagent 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 can form homodimers because there is no steric hindrance at the dimerization interface that exists in the case of the T336W modified Fc. Thus, in some embodiments, the I253R modification is combined with the T366S / L368A / Y407V modified Fc so that purification of the potentially formed homodimeric Fc is not permitted. Similar modifications can be utilized by combining T366S / L368A / Y407V and H453R.

[0223] In some embodiments, the Fc region of the heterodimeric molecule may further contain one or more other Fc mutations such as any of those described above. In some embodiments, the heterodimeric molecule contains an Fc region that includes mutations that reduce effector function.

[0224] In some embodiments, one Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in either SEQ ID NO:291 (e.g., SEQ ID NO:82 or 86) or 297 (e.g., SEQ ID NO:94 or 96), and the other Fc polypeptide of the heterodimeric Fc contains the amino acid sequence set forth in either SEQ ID NO:201 (e.g., SEQ ID NO:83 or 87), 295 (e.g., SEQ ID NO:90 or 92), or 299 (e.g., SEQ ID NO:98 or 100). In some embodiments, one Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in either SEQ ID NO:293 (e.g., SEQ ID NO:84 or 88) or 298 (e.g., SEQ ID NO:95 or 97), and the other Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in either SEQ ID NO:294 (e.g., SEQ ID NO:85 or 89), 296 (e.g., SEQ ID NO:91 or 93), or 300 (e.g., SEQ ID NO:99 or 101).

[0225] In some embodiments, the human IgG Fc region is modified to prevent dimerization. In these embodiments, the fusion proteins of the present disclosure are monomeric. For example, modification of the charged residue at residue Thr366, e.g., Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu (T366K, T366R, T366D, or T366E, respectively), prevents CH3-CH3 dimerization.

[0226] In some embodiments, the Fc region of the fusion protein is modified 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), or Ala327 (A327). For example, Leu234Ala (L234A), Leu235Ala (L235A), Asp265Asn (D265N), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Asn325Glu (N325E), or Ala327Ser (A327S). In a preferred embodiment, the modification within the Fc region reduces binding to the Fc receptor gamma receptor but has a minimal effect on binding to the neonatal Fc receptor (FcRn).

[0227] 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 may contain a modified IgG1 hinge having the sequence EPKSSDKTHTCPPC (SEQ ID NO:7), where Cys220, which forms a disulfide with the C-terminal cysteine of the light chain, is mutated to serine (e.g., Cys220Ser (C220S)). In other embodiments, the fusion protein contains a shortened hinge having the sequence DKTHTCPPC (SEQ ID NO:8).

[0228] In some embodiments, the fusion protein has a modified hinge derived from IgG4 that is modified (e.g., Ser228Pro (S228P)) to prevent or reduce chain exchange and has the sequence ESKYGPPCPPC (SEQ ID NO:9). In some embodiments, the fusion protein contains a linker polypeptide. In other embodiments, the fusion protein contains a linker and a hinge polypeptide.

[0229] 3. Linker The provided multispecific polypeptide construct contains a linker that joins or couples a first component containing an immunoglobulin Fc region and a second component containing a CD3-binding region. In some embodiments, the linker is positioned at the end of the C-terminal region of the Fc region such that the Fc region is on the N-terminal side of the CD3-binding region. Since the provided multispecific polypeptide construct is a multimer such as a dimer containing first and second polypeptides that together form the first and second components, the provided construct includes a linker that joins the Fc portion of the first polypeptide and the CD3-binding region, and a linker that joins the Fc portion of the second polypeptide and the CD3-binding region. In some embodiments, the first polypeptide includes a first Fc polypeptide of a heterodimeric Fc region, a linker, and a first domain (e.g., VH) of the CD3-binding region, and the second polypeptide includes a second Fc polypeptide of the heterodimeric Fc region, a linker, and a second domain (e.g., VL) of the CD3-binding region. Typically, the linkers present in the first and second polypeptides of the multispecific polypeptide construct are the same. Thus, in some embodiments, each domain of the CD3-binding domain is linked via a linker such as the same linker to the opposing polypeptide of Fc such as a heterodimeric Fc.

[0230] A variety of polypeptide linkers for use in fusion proteins are known (see, e.g., Chen et al. (2013) Adv. Drug. Deliv. 65:1357-1369; and International PCT Publication Nos. WO2014 / 099997, WO2000 / 24884; U.S. Patent No. 5,258,498; U.S. Patent No. 5,525,491; U.S. Patent No. 5,525,491, U.S. Patent No. 6,132,992).

[0231] In some embodiments, the linker is selected such that when the CD3 binding region is conjugated to the Fc region of the multispecific polypeptide conjugate, the CD3 binding region is constrained and cannot or substantially cannot bind or engage CD3 on the surface of a cell, such as a T cell, when the multispecific polypeptide construct contacts the cell. A variety of assays, including assays for evaluating T cell binding, NFAT activation using reporter systems, cytolytic T cell activity, cytokine production, and / or expression of T cell activation markers, can be utilized to evaluate the binding or engagement of CD3 by the multispecific polypeptide construct. Exemplary assays are shown in the provided examples. Typically, the linker ensures the correct folding of the polypeptide construct, does not exhibit charges that are incompatible with the activity or function of the linked polypeptides, and does not form bonds or other interactions with amino acid residues in one or more of the domains that would attenuate or alter the activity of the linked polypeptides. In some embodiments, the linker is a polypeptide linker. The polypeptide linker can be a flexible linker or a rigid linker, or a combination of both.

[0232] In some instances, the linker is short, medium, or long. In some embodiments, the linker is up to 40 amino acids in length. In some embodiments, up to 25 amino acids in length. In some embodiments, the linker is at least about 2 amino acids in length. In some instances, a suitable length is, for example, at least 1 amino acid residue, typically less than about 40 amino acid residues, such as 2-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, 8-12 amino acids in length. In some embodiments, the linker is about 2-24 amino acids, 2-20 amino acids, 2-18 amino acids, 2-14 amino acids, 2-12 amino acids, 2-10 amino acids, 2-8 amino acids, 2-6 amino acids, 6-24 amino acids, 6-20 amino acids, 6-18 amino acids, 6-14 amino acids, 6-12 amino acids, 6-10 amino acids, 6-8 amino acids, 8-24 amino acids, 8-20 amino acids, 8-18 amino acids, 8-14 amino acids, 8-12 amino acids, 8-10 amino acids, 10-24 amino acids, 10-20 amino acids, 10-18 amino acids, 10-14 amino acids, 10-12 amino acids, 12-24 amino acids, 12-20 amino acids, 12-18 amino acids, 12-14 amino acids, 14-24 amino acids, 14-20 amino acids, 14-18 amino acids, 18-24 amino acids, 18-20 amino acids, or 20-24 amino acids. In some embodiments, the linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0233] In certain aspects, the longer the linker length, the greater the CD3 binding when the multispecific polypeptide conjugate binds to its antigen, e.g., a TAA. Thus, in some aspects, the linker is longer than 12 amino acids, e.g., longer than 13, 14, 15, 16, 17, or 18 amino acids. In some embodiments, the linker is 12 - 40 amino acids in length, 12 - 30 amino acids, 12 - 24 amino acids, 12 - 18 amino acids, 12 - 15 amino acids, 15 - 40 amino acids, 15 - 30 amino acids, 15 - 24 amino acids, 15 - 18 amino acids, 18 - 40 amino acids, 18 - 30 amino acids, 18 - 24 amino acids, 24 - 40 amino acids, 24 - 30 amino acids, or 30 - 40 amino acids.

[0234] The linker may be naturally occurring, synthetic, or a combination of both. Particularly suitable linker polypeptides mainly contain amino acid residues selected from glycine (Gly), serine (Ser), alanine (Ala), and threonine (Thr). For example, the linker may contain at least 75% (calculated based on the total number of residues present in the peptide linker), e.g., at least 80%, at least 85%, or at least 90% of amino acid residues selected from Gly, Ser, Ala, and Thr. The linker may consist only of residues of Gly, Ser, Ala, and / or Thr. In some embodiments, the linker contains 1 - 25 glycine residues, 5 - 20 glycine residues, 5 - 15 glycine residues, or 8 - 12 glycine residues. In some aspects, suitable peptide linkers typically contain at least 50% glycine residues, e.g., at least 75% glycine residues. In some embodiments, the peptide linker contains only glycine residues. In some embodiments, the peptide linker contains only glycine and serine residues.

[0235] In some embodiments, these linkers are mainly composed of the amino acids glycine and serine and are referred to herein as GS linkers. In s...

Claims

1. 1. A multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, the first and second components are coupled by a linker, and the Fc region is positioned N-terminal to the CD3 binding region; one or both of the first and second components comprises at least one antigen-binding domain that binds to a tumor-associated antigen (TAA); One or both of the first and second components comprises at least one costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor; Multispecific polypeptide constructs.

2. 1. A multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, the first and second components are coupled by a linker, and the Fc region is positioned N-terminal to the CD3 binding region; one or both of the first and second components comprises at least one antigen-binding domain that binds to a tumor-associated antigen (TAA); One or both of the first and second components comprises at least one inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor; Multispecific polypeptide constructs.

3. 1. A multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, the first and second components are coupled by a linker, and the Fc region is positioned N-terminal to the CD3 binding region; one or both of the first and second components comprises at least one antigen-binding domain that binds to a tumor-associated antigen (TAA); one or both of the first and second components comprises at least one costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor; One or both of the first and second components comprises at least one inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor; Multispecific polypeptide constructs.

4. 4. The multispecific polypeptide construct of any one of claims 1 to 3, wherein the CD3 binding region binds to CD3 (CD3ε).

5. 5. The multispecific polypeptide construct according to any one of claims 1 to 4, wherein at least one antigen binding domain is positioned amino-terminal to the Fc region and / or carboxy-terminal to the CD3 binding region of the multispecific polypeptide construct.

6. 6. The multispecific polypeptide construct of any one of claims 1 to 5, wherein the first component comprises a first antigen-binding domain and the second component comprises a second antigen-binding domain, each of the antigen-binding domains binding to a tumor-associated antigen (TAA).

7. 7. The multispecific polypeptide construct of claim 6, wherein the first antigen-binding domain is positioned amino-terminal to the Fc region of the multispecific construct and the second antigen-binding domain is positioned carboxy-terminal to the CD3 binding region of the multispecific construct.

8. 8. The multispecific polypeptide construct of any one of claims 1 and 3 to 7, wherein at least one costimulatory receptor binding region (CRBR) is positioned amino-terminal to the Fc region and / or carboxy-terminal to the CD3 binding region of the multispecific polypeptide construct.

9. 9. The multispecific polypeptide construct of any one of claims 1 and 3 to 8, wherein at least one costimulatory receptor binding region (CRBR) is positioned carboxy-terminal to the CD3 binding region of the multispecific polypeptide construct.

10. 8. The multispecific polypeptide construct according to any one of claims 2 to 7, wherein at least one inhibitory receptor binding region (IRBR) is positioned amino-terminal to the Fc region and / or carboxy-terminal to the CD3 binding region of the multispecific polypeptide construct.

11. 11. The multispecific polypeptide construct according to any one of claims 2 to 7 and 10, wherein at least one inhibitory receptor binding region (IRBR) is positioned carboxy-terminal to the CD3 binding region of the multispecific polypeptide construct.

12. 12. The multispecific polypeptide construct of any one of claims 1 to 11, wherein the first component comprises a first antigen-binding domain and the second component comprises a second antigen-binding domain, each of the antigen-binding domains binding to a tumor-associated antigen (TAA).

13. 13. The multispecific polypeptide construct of claim 12, wherein the first antigen-binding domain is positioned amino-terminal to the Fc region of the multispecific construct and the second antigen-binding domain is positioned carboxy-terminal to the CD3 binding region of the multispecific construct.

14. 14. The multispecific polypeptide construct of claim 12 or claim 13, wherein the first or second component further comprises a costimulatory receptor binding region (CRBR).

15. 14. The multispecific polypeptide construct of claim 12 or claim 13, wherein the first or second component further comprises an inhibitory receptor binding region (IRBR).

16. From the N-terminus to the C-terminus, a costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor and / or an antigen-binding domain that binds to a tumor-associated antigen (TAA); Immunoglobulin Fc region; Linker; a CD3 binding region that binds to CD3 (CD3ε); and Costimulatory receptor binding domain (CRBR) that binds to a costimulatory receptor and / or an antigen-binding domain that binds to a tumor-associated antigen (TAA) A multispecific polypeptide construct comprising: A multispecific polypeptide construct comprising at least one CRBR and at least one antigen-binding domain.

17. 17. The multispecific polypeptide construct of claim 16, comprising only one costimulatory receptor binding region (CRBR).

18. From the N-terminus to the C-terminus, an inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor and / or an antigen-binding domain that binds to a tumor-associated antigen (TAA); Immunoglobulin Fc region; Linker; a CD3 binding region that binds to CD3 (CD3ε); and Inhibitory receptor binding regions (IRBRs) that bind to inhibitory receptors and / or antigen-binding domains that bind to tumor-associated antigens (TAAs) A multispecific polypeptide construct comprising: A multispecific polypeptide construct comprising at least one IRBR and at least one antigen-binding domain.

19. 20. The multispecific polypeptide construct of claim 18, comprising only one inhibitory receptor binding region (IRBR).

20. From the N-terminus to the C-terminus, an inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor or a costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor, and / or an antigen-binding domain that binds to a tumor-associated antigen (TAA); Immunoglobulin Fc region; Linker; a CD3 binding region that binds to CD3 (CD3ε); and An antigen-binding domain that binds to the other of IRBR or CRBR, and / or a tumor-associated antigen (TAA) A multispecific polypeptide construct comprising: A multispecific polypeptide construct comprising at least one IRBR, at least one CRBR, and at least one antigen-binding domain.

21. 21. The multispecific polypeptide construct of any one of claims 16 to 20, comprising two antigen binding domains that bind to the TAA.

22. 22. The multispecific polypeptide construct of claim 21 , wherein the antigen binding domains bind to the same tumor-associated antigen (TAA).

23. 23. The multispecific polypeptide construct of claim 21 or claim 22, wherein one antigen binding domain is positioned amino terminal to the Fc region and one antigen binding domain is positioned carboxy terminal to the CD3 binding region.

24. From the N-terminus to the C-terminus, Immunoglobulin Fc region; Linker; a CD3 binding region that binds to CD3 (CD3ε); and An antigen-binding domain that binds to tumor-associated antigens (TAA) and a costimulatory receptor-binding region (CRBR) that binds to costimulatory receptors A multispecific polypeptide construct comprising:

25. From the N-terminus to the C-terminus, an antigen-binding domain that binds to tumor-associated antigens (TAA) and a costimulatory receptor-binding region (CRBR) that binds to costimulatory receptors; Immunoglobulin Fc region; A linker; and CD3-binding domain that binds to CD3 (CD3ε) A multispecific polypeptide construct comprising:

26. From the N-terminus to the C-terminus, Immunoglobulin Fc region; Linker; a CD3 binding region that binds to CD3 (CD3ε); and An antigen-binding domain that binds to tumor-associated antigens (TAA) and an inhibitory receptor-binding region (IRBR) that binds to inhibitory receptors A multispecific polypeptide construct comprising:

27. From the N-terminus to the C-terminus, an antigen-binding domain that binds to tumor-associated antigens (TAA) and an inhibitory receptor-binding region (IRBR) that binds to inhibitory receptors; Immunoglobulin Fc region; A linker; and CD3-binding domain that binds to CD3 (CD3ε) A multispecific polypeptide construct comprising:

28. 28. The multispecific polypeptide construct according to any one of claims 1 to 27, wherein the Fc region is a homodimeric Fc region.

29. 29. The multispecific polypeptide construct according to any one of claims 1 to 28, wherein the Fc region is the Fc region of human IgG1, human IgG2, human IgG3 or human IgG4, or an immunologically active fragment thereof.

30. 30. The multispecific polypeptide construct of any one of claims 1 to 29, wherein the Fc region comprises a polypeptide comprising an amino acid sequence as shown in SEQ ID NO:1, or a sequence of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:

1.

31. the Fc region comprises a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:2, or a sequence of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:2; the Fc region comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4, or a sequence of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:4; or the Fc region comprises a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:5, or a sequence of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:5; 30. A multispecific polypeptide construct according to any one of claims 1 to 29.

32. 32. The multispecific polypeptide construct according to any one of claims 1 to 23, 29 to 31, wherein the Fc region is a heterodimeric Fc region.

33. 33. The multispecific polypeptide construct of claim 32, wherein one or both Fc polypeptides of the heterodimeric Fc region comprise at least one modification to induce heterodimerization compared to the polypeptide of the homodimeric Fc region, and optionally compared to the Fc polypeptide shown in SEQ ID NO:1 or an immunologically active fragment thereof.

34. 34. The multispecific polypeptide construct of claim 33, wherein each of the Fc polypeptides of the heterodimeric Fc independently comprises at least one amino acid modification.

35. 35. The multispecific polypeptide construct of claim 34, wherein each of the Fc polypeptides of the heterodimeric Fc comprises a knob-into-hole modification or a charge mutation to increase electrostatic complementarity of the polypeptides.

36. 36. The multispecific polypeptide construct of claim 35, wherein the amino acid modification is a knob-into-hole modification.

37. 37. The multispecific polypeptide construct of any one of claims 32 to 36, wherein the CD3 binding region comprises a variable heavy region (VH) and a variable light region (VL), wherein the VL is C-terminal to a first Fc polypeptide of the heterodimeric Fc region and the VH is C-terminal to a second Fc polypeptide of the heterodimeric Fc region, wherein the first Fc polypeptide comprises a hole mutation and the second Fc polypeptide comprises a knob mutation.

38. 38. The multispecific polypeptide construct of any one of claims 32 to 37, wherein the first Fc polypeptide of said heterodimeric Fc comprises a modification selected from Thr366Ser, Leu368Ala, Tyr407Val, and a combination thereof, and the second Fc polypeptide of said heterodimeric Fc comprises the modification Thr366Trp.

39. 39. The multispecific polypeptide construct of claim 38, wherein the first and second Fc polypeptides further comprise a modification of a non-cysteine ​​residue to a cysteine ​​residue, the modification of the first polypeptide being at one of positions Ser354 and Tyr349 and the modification of the second Fc polypeptide being at the other of positions Ser354 and Tyr349.

40. 36. The multispecific polypeptide construct of claim 35, wherein the amino acid modification is a charge mutation to increase electrostatic complementarity of the polypeptide.

41. 41. The multispecific polypeptide construct of any one of claims 32 to 35 and 40, wherein each of the first and / or second Fc polypeptide or the first and second Fc polypeptide comprises a modification at a complementary position, the modification being a substitution of an amino acid having an opposite charge to the complementary amino acid of the other polypeptide.

42. 42. The multispecific polypeptide construct of any one of claims 33 to 41, wherein one of the first or second Fc polypeptides of the heterodimeric Fc further comprises a modification at residue Ile253.

43. 43. The multispecific polypeptide construct of claim 42, wherein said modification is Ile253Arg.

44. 44. The multispecific polypeptide construct of any one of claims 33 to 43, wherein one of the first or second Fc polypeptides of the heterodimeric Fc further comprises a modification at residue His435.

45. The multispecific polypeptide construct of claim 44, wherein the modification is His435Arg.

46. 46. ​​The multispecific polypeptide construct of any one of claims 1 to 45, wherein the Fc region comprises a polypeptide lacking Lys447.

47. 47. The multispecific polypeptide construct of any one of claims 1 to 46, wherein the Fc region comprises a polypeptide comprising at least one modification for enhancing FcRn binding.

48. 48. The multispecific polypeptide construct of claim 47, wherein the modification is at a position selected from the group consisting of Met252, Ser254, Thr256, Met428, Asn434, and combinations thereof.

49. 49. The multispecific polypeptide construct of claim 48, wherein the modification is at a position selected from the group consisting of Met252Y, Ser254T, Thr256E, Met428L, Met428V, Asn434S, and combinations thereof.

50. The multispecific polypeptide construct of claim 48, wherein said modifications are at positions Met252 and Met428.

51. The multispecific polypeptide construct of claim 50, wherein the modifications are Met252Y and Met428L.

52. The multispecific polypeptide construct of claim 50, wherein said modifications are Met252Y and Met428V.

53. 53. The multispecific polypeptide construct of any one of claims 32 to 52, wherein the first polypeptide of the heterodimeric Fc comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 82, 86, 94, or 96, and the second polypeptide of the heterodimeric Fc comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 83, 87, 90, 92, 98, or 100.

54. 54. The multispecific polypeptide construct of any one of claims 32 to 53, wherein the first polypeptide of the heterodimeric Fc comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 291, 293, 297, or 298, and the second polypeptide of the heterodimeric Fc comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 292, 294, 295, 296, 299, or 300.

55. The Fc region Polypeptides containing at least one amino acid modification that reduces effector function and / or reduces binding to an effector molecule selected from Fcγ receptors or C1q 55. The multispecific polypeptide construct of any one of claims 1 to 54, comprising:

56. 56. The multispecific polypeptide construct of claim 55, wherein the one or more amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235.

57. 57. The multispecific polypeptide construct of any one of claims 32 to 56, wherein the first polypeptide of the heterodimeric Fc comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 84, 88, 95, or 97, and the second polypeptide of the heterodimeric Fc comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 85, 89, 91, 93, 99, or 101.

58. 57. The multispecific polypeptide construct of any one of claims 32 to 56, wherein the first polypeptide of the heterodimeric Fc comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 291, 293, 297, or 298, and the second polypeptide of the heterodimeric Fc comprises a sequence of amino acids as set forth in any of SEQ ID NOs: 292, 294, 295, 296, 299, or 300.

59. 55. The multispecific polypeptide construct of any one of claims 1 to 54, wherein the Fc region comprises a polypeptide comprising at least one modification for enhancing FcγR binding.

60. 60. The multispecific polypeptide construct of claim 59, wherein the modification is at Ser239 or Ile332.

61. 60. The multispecific polypeptide construct of any one of claims 1 to 54 and 59, wherein glycosylation of the Fc region is modified to enhance FcγR binding compared to an unmodified Fc region.

62. 62. The multispecific polypeptide construct of claim 61 , wherein the Fc region lacks fucose or has reduced fucose content.

63. 63. The multispecific polypeptide construct of any one of claims 1 to 62, wherein the CD3 binding region is an anti-CD3 antibody or antigen-binding fragment.

64. 64. The multispecific polypeptide construct of claim 63, wherein the anti-CD3 antibody or antigen-binding fragment comprises a variable heavy region (VH) and a variable light region (VL).

65. 65. The multispecific construct of claim 64, wherein the VL of the anti-CD3 antibody or antigen-binding fragment is linked to a first Fc polypeptide of a heterodimeric Fc comprising a modification selected from Thr366Ser, Leu368Ala, Tyr407Val, and a combination thereof, and the VH of the anti-CD3 antibody or antigen-binding fragment is linked to a second Fc polypeptide of a heterodimeric Fc comprising the modification Thr366Trp.

66. 66. The multispecific polypeptide construct of any one of claims 1 to 65, wherein the CD3 binding region is monovalent.

67. 67. The multispecific polypeptide construct of any one of claims 1 to 66, wherein the CD3 binding region is a variable fragment (Fv) comprising a variable heavy region (VH) and a variable light region (VL).

68. 67. The multispecific polypeptide construct of any one of claims 63 to 66, wherein the anti-CD3 antibody or antigen-binding fragment is not a single chain antibody, and optionally is not a single chain variable fragment (scFv).

69. The multispecific polypeptide construct of any one of claims 64 to 68, wherein the Fc is a heterodimeric Fc, and the VH and VL constituting the anti-CD3 antibody or antigen-binding fragment are linked to opposing polypeptides of the heterodimeric Fc.

70. 70. The multispecific polypeptide construct of any of claims 1 to 69, wherein the CD3 binding region is incapable or substantially incapable of binding or engaging CD3 unless at least one of the antigen binding domains is bound to its TAA.

71. 71. The multispecific polypeptide construct of any one of claims 1 to 70, wherein the CD3 binding region is incapable or substantially incapable of binding or engaging CD3 unless at least two of the antigen binding domains are bound to its TAA.

72. 72. The multispecific polypeptide construct of any one of claims 1 to 71, wherein the linker is a polypeptide linker.

73. 73. The multispecific polypeptide construct of claim 72, wherein the linker is a polypeptide up to 25 amino acids in length.

74. The linker may be 2-24 amino acids, 2-20 amino acids, 2-18 amino acids, 2-14 amino acids, 2-12 amino acids, 2-10 amino acids, 2-8 amino acids, 2-6 amino acids, 6-24 amino acids, 6-20 amino acids, 6-18 amino acids, 6-14 amino acids, 6-12 amino acids, 6-10 amino acids, 6-8 amino acids, 8-24 amino acids, 8-20 amino acids, 8-18 amino acids, 8-14 amino acids, 8-12 amino acids , 8-10 amino acids, 10-24 amino acids, 10-20 amino acids, 10-18 amino acids, 10-14 amino acids, 10-12 amino acids, 12-24 amino acids, 12-20 amino acids, 12-18 amino acids, 12-14 amino acids, 14-24 amino acids, 14-20 amino acids, 14-18 amino acids, 18-24 amino acids, 18-20 amino acids, or 20-24 amino acids, or about 2-24 amino acids, 2-20 amino acids.

74. The multispecific polypeptide construct of claim 72 or claim 73, which is a polypeptide of 2 to 18 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to 24 amino acids, 6 to 20 amino acids, 6 to 18 amino acids, 6 to 14 amino acids, 6 to 12 amino acids, 6 to 10 amino acids, 6 to 8 amino acids, 8 to 24 amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14 amino acids, 8 to 12 amino acids, 8 to 10 amino acids, 10 to 24 amino acids, 10 to 20 amino acids, 10 to 18 amino acids, 10 to 14 amino acids, 10 to 12 amino acids, 12 to 24 amino acids, 12 to 20 amino acids, 12 to 18 amino acids, 12 to 14 amino acids, 14 to 24 amino acids, 14 to 20 amino acids, 14 to 18 amino acids, 18 to 24 amino acids, 18 to 20 amino acids, or 20 to 24 amino acids.

75. 75. The multispecific polypeptide construct of any one of claims 72 to 74, wherein the linker is a polypeptide that is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

76. 76. The multispecific polypeptide construct of any one of claims 72 to 75, wherein the linker is a polypeptide that is 3 to 18 amino acids in length.

77. 76. The multispecific polypeptide construct of any one of claims 72 to 75, wherein the linker is a polypeptide that is 12 to 18 amino acids in length.

78. 76. The multispecific polypeptide construct of any one of claims 72 to 75, wherein the linker is a polypeptide that is 15 to 18 amino acids in length.

79. 79. The multispecific polypeptide construct of any one of claims 1 to 78, wherein the linker is a non-cleavable linker.

80. 80. The multispecific polypeptide construct of claim 79, wherein the non-cleavable linker does not contain a substrate recognition site that is specifically recognized for cleavage by a protease.

81. 81. The multispecific polypeptide construct of claim 79 or claim 80, wherein the non-cleavable linker comprises GS, GGS, GGGGS (SEQ ID NO:149), GGGGGS (SEQ ID NO:135), and combinations thereof.

82. 82. The multispecific polypeptide construct of any one of claims 79 to 81, wherein the non-cleavable linker comprises (GGS)n, wherein n is 1 to 10.

83. 83. The multispecific polypeptide construct of any one of claims 79 to 82, wherein the non-cleavable linker comprises (GGGGS)n (SEQ ID NO:173), wherein n is 1 to 10.

84. 84. The multispecific polypeptide construct of any one of claims 79 to 83, wherein the non-cleavable linker comprises (GGGGGS)n (SEQ ID NO:172), wherein n is 1 to 4.

85. 85. The multispecific polypeptide construct of any one of claims 79 to 84, wherein the non-cleavable linker comprises GGS.

86. 85. The multispecific polypeptide construct of any one of claims 79 to 84, wherein the non-cleavable linker comprises GGGGS (SEQ ID NO:149).

87. 85. The multispecific polypeptide construct of any one of claims 79 to 84, wherein the non-cleavable linker comprises GGGGGS (SEQ ID NO:135).

88. The non-cleavable linker is (GGS) 2 85. The multispecific polypeptide construct of any one of claims 79 to 84, comprising: (SEQ ID NO:10).

89. 85. The multispecific polypeptide construct of any one of claims 79 to 84, wherein the non-cleavable linker comprises GGSGGSGGS (SEQ ID NO:11).

90. 85. The multispecific polypeptide construct of any one of claims 79 to 84, wherein the non-cleavable linker comprises GGSGGSGGSGGS (SEQ ID NO:12).

91. The non-cleavable linker is 85. The multispecific polypeptide construct of any one of claims 79 to 84, comprising:

92. The non-cleavable linker is 85. The multispecific polypeptide construct of any one of claims 79 to 84, comprising:

93. The non-cleavable linker is 85. The multispecific polypeptide construct of any one of claims 79 to 84, comprising:

94. The non-cleavable linker is 85. The multispecific polypeptide construct of any one of claims 79 to 84, comprising:

95. 79. The multispecific polypeptide construct of any one of claims 1 to 78, wherein the linker is a cleavable linker.

96. 1. A multispecific polypeptide construct comprising a first component comprising a heterodimeric Fc region and 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 cleavable 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 binds to a tumor-associated antigen (TAA); One or both of the first and second components comprises at least one costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor; Multispecific polypeptide constructs.

97. 97. The multispecific polypeptide construct of claim 96, wherein one or both of the first and second components further comprises at least one inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor.

98. 1. A multispecific polypeptide construct comprising a first component comprising a heterodimeric Fc region and 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 cleavable 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 binds to a tumor-associated antigen (TAA); One or both of the first and second components comprises at least one inhibitory receptor binding region (IRBR) that binds to an inhibitory receptor; Multispecific polypeptide constructs.

99. 99. The multispecific polypeptide construct of claim 98, wherein one or both of the first and second components further comprises at least one costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor.

100. 100. The multispecific polypeptide construct of any one of claims 96 to 99, wherein binding of the CD3 binding region to CD3 is substantially reduced when the multispecific polypeptide construct is in an uncleaved state compared to a cleaved state.

101. 101. The multispecific polypeptide construct of any one of claims 96 to 100, wherein in the cleaved state the first and second components are unlinked.

102. The multispecific polypeptide construct of any one of claims 96 to 101, wherein the cleavable linker is a polypeptide that functions as a substrate for a protease.

103. The multispecific polypeptide construct of claim 102, wherein the protease is produced by an immune effector cell, by the tumor, or by a cell present in the tumor microenvironment.

104. The multispecific polypeptide construct of claim 103, wherein 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 a NK T cell.

105. 105. The multispecific polypeptide construct of any one of claims 102 to 104, wherein the protease is selected from matriptase, matrix metalloprotease (MMP), granzyme B, and combinations thereof.

106. The multispecific polypeptide construct of claim 105, wherein the protease is granzyme B.

107. 107. The multispecific polypeptide construct of any one of claims 96 to 106, wherein the cleavable linker comprises an amino acid sequence of the general formula P4 P3 P2 P1↓P1' (SEQ ID NO:150), wherein P4 is an amino acid I, L, Y, M, F, V, or A; P3 is an amino acid A, G, S, V, E, D, Q, N, or Y; P2 is an amino acid H, P, A, V, G, S, or T; P1 is an amino acid D or E; and P1' is an amino acid I, L, Y, M, F, V, T, S, G, or A.

108. 108. The multispecific polypeptide construct of any one of claims 96 to 107, wherein the cleavable linker comprises an amino acid sequence of the general formula P4 P3 P2 P1↓P1' (SEQ ID NO:151), wherein P4 is the amino acid I or L; P3 is the amino acid E; P2 is the amino acid P or A; P1 is the amino acid D; and P1' is the amino acid I, V, T, S, or G.

109. 109. The multispecific polypeptide construct of any one of claims 96 to 108, wherein the cleavable linker comprises the amino acid sequence IEPDI (SEQ ID NO:136), LEPDG (SEQ ID NO:152), LEADT (SEQ ID NO:137), IEPDG (SEQ ID NO:138), IEPDV (SEQ ID NO:139), IEPDS (SEQ ID NO:140), IEPDT (SEQ ID NO:141), or LEADG (SEQ ID NO:153).

110. 110. The multispecific polypeptide construct of any one of claims 96-109, wherein the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 105-112, 136-141, 148, 150-153.

111. 111. The multispecific polypeptide construct of any one of claims 96 to 110, wherein the cleavable linker comprises the amino acid sequence shown in SEQ ID NO:

105.

112. The multispecific polypeptide construct of claim 111, wherein the protease is matriptase.

113. the cleavable linker comprises the sequence P1QAR↓(A / V) (SEQ ID NO:154), where P1 is any amino acid; or The cleavable linker comprises the sequence RQAR(A / V) (SEQ ID NO:155), 113. The multispecific polypeptide construct of any one of claims 96 to 112.

114. 114. The multispecific polypeptide construct of any one of claims 96 to 113, wherein the cleavable linker comprises the sequence RQARV (SEQ ID NO:156).

115. 15. The multispecific polypeptide construct of any one of claims 96 to 114, wherein the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 154 to 156.

116. The multispecific polypeptide construct of claim 105, wherein the protease is an MMP.

117. The multispecific polypeptide construct of claim 116, wherein the MMP is MMP-2.

118. 118. The multispecific polypeptide construct of any one of claims 96 to 117, wherein the cleavable linker comprises the general formula P3 P2 P1↓P1' (SEQ ID NO:157), where P3 is P, V, or A; P2 is Q or D; P1 is A or N; and P1' is L, I, or M.

119. 119. The multispecific polypeptide construct of any one of claims 96 to 118, wherein the cleavable linker comprises the general formula P3 P2 P1↓P1' (SEQ ID NO:158), wherein P3 is P; P2 is Q or D; P1 is A or N; and P1' is L or I.

120. 120. The multispecific polypeptide construct of any one of claims 96 to 119, wherein the cleavable linker comprises the sequence PAGL (SEQ ID NO:24).

121. 121. The multispecific polypeptide construct of any one of claims 96-120, wherein the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-31, 104-114, 117-118, 136-144, 148, 150-158.

122. The multispecific polypeptide construct comprises: (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 the heterodimeric Fc region, a linker, optionally the same linker as is present in the first polypeptide, and the other of the VH domain or VL domain of the anti-CD3 antibody or antigen-binding fragment. At least one or both of the first and second polypeptides comprises at least one antigen-binding domain that binds to a tumor-associated antigen (TAA), and one or both of the first and second components comprises at least one costimulatory receptor-binding region (CRBR) that binds to a costimulatory receptor; The multispecific polypeptide construct comprises at least one CRBR and at least one antigen-binding domain; 122. A multispecific polypeptide construct according to any one of claims 69 to 121.

123. The multispecific polypeptide construct of any one of claims 1 to 122, wherein the one or more antigen binding domains that bind to the TAA provide monovalent, bivalent, trivalent or tetravalent binding to the TAA.

124. 124. The multispecific polypeptide construct of claim 122 or claim 123, wherein only one of the first or second polypeptide comprises at least one antigen binding domain that binds to a TAA.

125. 125. The multispecific polypeptide construct of any of claims 122 to 124, wherein at least one antigen-binding domain is positioned amino-terminal to an Fc region and / or carboxy-terminal to a CD3 binding region of one of the first or second polypeptides of said multispecific polypeptide construct.

126. 125. The multispecific polypeptide construct of any of claims 122 to 124, wherein at least one antigen binding domain is positioned amino terminal to an Fc region of the multispecific construct and a second antigen binding domain is positioned carboxy terminal to a CD3 binding region of the multispecific construct.

127. 127. The multispecific polypeptide construct of any one of claims 122-126, wherein only one of the first or second polypeptide comprises at least one costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor.

128. 128. The multispecific polypeptide construct of any of claims 122-127, wherein the costimulatory receptor binding region (CRBR) is positioned amino-terminal to an Fc region or carboxy-terminal to a CD3 binding region of one of the first or second polypeptides of said multispecific polypeptide construct.

129. the first polypeptide comprises, in order from N-terminus to C-terminus, a first antigen-binding domain that binds to a tumor-associated antigen (TAA), a first Fc polypeptide of a heterodimeric Fc region, a linker, a VL or VH of an anti-CD3 antibody or antigen-binding fragment, and a second antigen-binding domain that binds to the tumor-associated antigen (TAA); the second polypeptide comprises, in order from N-terminus to C-terminus, a second Fc polypeptide of a heterodimeric Fc region, a linker, optionally the same linker as is present in the first polypeptide, the other of the VL or VH of an anti-CD3 antibody or antigen-binding fragment, and a costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor; 129. A multispecific polypeptide construct according to any one of claims 122 to 128.

130. 130. The multispecific polypeptide construct of any one of claims 122-129, wherein the heterodimeric Fc region comprises an Fc hole polypeptide and an Fc knob polypeptide, wherein the VL of the anti-CD3 antibody or antigen binding fragment is positioned C-terminal to the Fc hole and the VH of the anti-CD3 antibody or antigen binding fragment is positioned C-terminal to the Fc knob.

131. 131. The multispecific polypeptide construct of any one of claims 1 to 130, wherein the antigen-binding domain, or each of said antigen-binding domains independently, comprises the extracellular domain of a native cognate binding partner of the TAA or a binding fragment thereof, or a variant thereof that exhibits binding activity with the TAA.

132. 132. The multispecific polypeptide construct of any of claims 1 to 131, wherein the antigen-binding domain, or each of said antigen-binding domains independently, 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.

133. 133. The multispecific polypeptide construct of any of claims 1-132, wherein at least one costimulatory receptor binding region (CRBR) is or comprises the extracellular domain of a native cognate binding partner of the costimulatory receptor or a binding fragment thereof, or a variant thereof that exhibits binding activity with the costimulatory receptor.

134. 133. The multispecific polypeptide construct of any of claims 1 to 132, wherein 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.

135. 135. The multispecific polypeptide construct of claim 132 or claim 134, wherein the antibody or antigen-binding fragment thereof is an Fv, scFv, Fab, or a single domain antibody (sdAb).

136. 132, 134, or 122, wherein the antibody or antigen-binding fragment is an sdAb.

137. 137. The multispecific polypeptide construct of any one of claims 1 to 136, wherein one or both of the first and second components comprises an antigen binding domain that binds to a tumor associated antigen (TAA), and wherein the antigen binding domain is a single chain antibody fragment.

138. 138. The multispecific polypeptide construct of claim 137, wherein the single chain antibody fragment is a single domain antibody or a single chain variable fragment (scFv).

139. 139. The multispecific polypeptide construct of any one of claims 136 to 138, wherein the sdAb is a human sdAb or a humanized sdAb.

140. 140. The multispecific polypeptide construct of any one of claims 136-139, wherein the sdAb is a VHH, a VNAR, a modified VH domain, or a modified VK domain.

141. 136. The multispecific polypeptide construct of claim 132, claim 134, or claim 135, wherein the antibody or antigen-binding fragment thereof is an scFv.

142. 136. The multispecific polypeptide construct of claim 132, claim 134, or claim 135, wherein the antibody or antigen-binding fragment thereof is a Fab.

143. The multispecific polypeptide construct comprises: (i) a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and a VH domain of an anti-CD3 antibody or antigen-binding fragment; (ii) a second polypeptide comprising a second Fc polypeptide of the heterodimeric Fc region, a linker, and a VL domain of an anti-CD3 antibody or antigen-binding fragment; (iii) a third polypeptide comprising the VH-CH1(Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen; (iv) a fourth polypeptide comprising the VH-CH1(Fd) or VL-CL of a Fab antibody fragment that binds to a costimulatory receptor. Including, The first and / or second polypeptide comprises (1) the other of VH-CH1(Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen, and (2) The other of VH-CH1(Fd) or VL-CL of the Fab antibody fragment that binds to the costimulatory receptor Further comprising:

143. A multispecific polypeptide construct according to any one of claims 1 to 132 and 134 to 142.

144. The multispecific polypeptide construct comprises: (i) a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and a VH domain of an anti-CD3 antibody or antigen-binding fragment; (ii) a second polypeptide comprising a second Fc polypeptide of the heterodimeric Fc region, a linker, and a VL domain of an anti-CD3 antibody or antigen-binding fragment; and (iii) a third polypeptide comprising the VH-CH1(Fd) or VL-CL of a Fab antibody fragment that binds to a costimulatory receptor; Including, the first and / or second polypeptide further comprises the other of the VH-CH1(Fd) or VL-CL of a Fab antibody fragment that binds to a costimulatory receptor; the first and / or second polypeptide further comprises at least one antigen-binding domain that binds to a tumor-associated antigen (TAA); 143. A multispecific polypeptide construct according to any one of claims 1 to 132 and 134 to 142.

145. The multispecific polypeptide construct comprises: (i) a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and a VH domain of an anti-CD3 antibody or antigen-binding fragment; (ii) a second polypeptide comprising a second Fc polypeptide of the heterodimeric Fc region, a linker, and a VL domain of an anti-CD3 antibody or antigen-binding fragment; and (iii) a third polypeptide comprising the VH-CH1(Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen; Including, the first and / or second polypeptide further comprises the other of the VH-CH1(Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen, the first and / or second polypeptide further comprises at least one costimulatory receptor binding region (CRBR) that binds to a costimulatory receptor; 143. A multispecific polypeptide construct according to any one of claims 1 to 132 and 134 to 142.

146. 146. The multispecific polypeptide construct of claim 144 or claim 145, wherein only one of the first or second polypeptide comprises the other of the VH-CH1(Fd) or VL-CL of the Fab antibody fragment.

147. 146. The multispecific polypeptide construct of claim 144 or claim 145, wherein both the first or second polypeptide comprise the other of the VH-CH1(Fd) or VL-CL of the Fab antibody fragment.

148. 148. The multispecific polypeptide construct of claim 146 or claim 147, wherein the other of the VH-CH1(Fd) or VL-CL of the Fab antibody fragment is positioned amino terminal to the Fc region and / or carboxy terminal to the CD3 binding region of one of the first or second polypeptides of the multispecific polypeptide construct.

149. 149. The multispecific polypeptide construct of any of claims 146-148, wherein the other of the VH-CH1(Fd) or VL-CL of the Fab antibody fragment is positioned amino terminal to the Fc region of the first polypeptide or the second polypeptide and carboxy terminal to the CD3 binding region of the other of the first or second polypeptide.

150. The antigen-binding domain, or each of the antigen-binding domains independently, is: 1-92-LFA-3, 5T4, α4 integrin, αV integrin, α4β1 integrin, α4β7 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, CD11 a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52 , CD56, 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, DLL3, DLL4, DPP-4, DSG1, EDA, EDB , EGFR, 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, I L23, IL23R, IL27 / IL27R (wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged1, Jagged2, 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, TIGIT, TIM-3, TLR2, TLR4, 150. The multispecific polypeptide construct of any one of claims 1 to 149, which binds to a tumor antigen selected from 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.

151. 151. The multispecific polypeptide construct of any one of claims 1 to 150, wherein the antigen binding domain comprises at least a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain and the second antigen binding domain bind to the same TAA.

152. The multispecific polypeptide construct of claim 151, wherein the first antigen-binding domain and the second antigen-binding domain bind to different epitopes of the same TAA.

153. The multispecific polypeptide construct of claim 151, wherein the first antigen-binding domain and the second antigen-binding domain bind to the same epitope of the same TAA.

154. 154. The multispecific polypeptide construct of any of claims 1 to 153, wherein the antigen binding domain comprises at least a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain and the second antigen binding domain bind to different TAAs.

155. The multispecific polypeptide construct of any one of claims 1, 3-17, 20-25, 28-97, and 99-154, wherein the costimulatory receptor binding region (CRBR) comprises at least a first CRBR and a second CRBR, and the first CRBR and the second CRBR bind to the same costimulatory receptor.

156. The multispecific polypeptide construct of claim 155, wherein the first costimulatory receptor binding region (CRBR) and the second CRBR bind to different epitopes of the same costimulatory receptor.

157. The multispecific polypeptide construct of claim 155, wherein the first costimulatory receptor binding region (CRBR) and the second CRBR bind to the same epitope of the same costimulatory receptor.

158. 158. The multispecific polypeptide construct of any one of claims 1, 3-17, 20-25, 28-97 and 99-157, wherein the costimulatory receptor binding region (CRBR) comprises at least a first CRBR and a second CRBR.

159. The multispecific polypeptide construct of any one of claims 155-158, wherein the first CRBR and the second CRBR bind to different costimulatory receptors.

160. 160. The multispecific polypeptide construct of any one of claims 1, 3-17, 20-25, 28-97 and 99-159, wherein at least one costimulatory receptor binding region (CRBR) binds to a costimulatory receptor selected from 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.

161. 161. The multispecific polypeptide construct of any one of claims 1, 3-17, 20-25, 28-97 and 99-160, wherein at least one costimulatory receptor binding region (CRBR) binds to a costimulatory receptor selected from 41BB (CD137), OX40 (CD134), and glucocorticoid-inducible TNFR-related protein (GITR).

162. 162. The multispecific polypeptide construct of any one of claims 1, 3-17, 20-25, 28-97 and 99-161, wherein at least one costimulatory receptor binding region (CRBR) binds to 41BB (CD137).

163. The multispecific polypeptide construct of any one of claims 1, 3-17, 20-25, 28-97 and 99-162, wherein at least one costimulatory receptor comprises an amino acid sequence set forth in SEQ ID NO:215 or an amino acid sequence having at least 99% sequence identity with the amino acid sequence set forth in SEQ ID NO:

215.

164. 23. The multispecific polypeptide construct of any one of claims 1, 3-17, 20-25, 28-97 and 99-163, wherein at least one costimulatory receptor comprises the amino acid sequence set forth in SEQ ID NO:

321.

165. The multispecific polypeptide construct of any one of claims 2-15, 18-23, 26-95, 97-121, 123-164, wherein at least one inhibitory receptor binding region (IRBR) is or comprises the extracellular domain of a native cognate binding partner of a costimulatory receptor or a binding fragment thereof, or a variant thereof that exhibits binding activity with a costimulatory receptor.

166. The multispecific polypeptide construct of any one of claims 2-15, 18-23, 26-95, 97-121, 123-164, wherein at least one inhibitory receptor binding region (IRBR) 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.

167. 167. The multispecific polypeptide construct of claim 166, wherein the antibody or antigen-binding fragment thereof is an Fv, scFv, Fab, or a single domain antibody (sdAb).

168. The multispecific polypeptide construct of claim 166 or claim 122, wherein the antibody or antigen-binding fragment thereof is an sdAb.

169. 169. The multispecific polypeptide construct of claim 168, wherein the sdAb is a human sdAb or a humanized sdAb.

170. 170. The multispecific polypeptide construct of claim 168 or claim 169, wherein the sdAb is a VHH, a VNAR, a modified VH domain, or a modified VK domain.

171. 168. The multispecific polypeptide construct of claim 166 or claim 167, wherein the antibody or antigen-binding fragment thereof is an scFv.

172. 168. The multispecific polypeptide construct of claim 166 or claim 167, wherein the antibody or antigen-binding fragment thereof is a Fab.

173. The multispecific polypeptide construct of any one of claims 2-15, 18-23, 26-95, 97-121, 123-172, wherein the inhibitory receptor binding region (IRBR) comprises at least a first IRBR and a second IRBR, and the first IRBR and the second IRBR bind to the same inhibitory receptor.

174. The multispecific polypeptide construct of claim 173, wherein the first inhibitory receptor binding region (IRBR) and the second IRBR bind to different epitopes of the same inhibitory receptor.

175. The multispecific polypeptide construct of claim 173, wherein the first inhibitory receptor binding region (IRBR) and the second IRBR bind to the same epitope of the same inhibitory receptor.

176. The multispecific polypeptide construct of any one of claims 2-15, 18-23, 26-95, 97-121, 123-175, wherein the inhibitory receptor binding region (IRBR) comprises at least a first IRBR and a second IRBR.

177. 177. The multispecific polypeptide construct of any one of claims 173-176, wherein the first IRBR and the second IRBR bind to different inhibitory receptors.

178. The multispecific polypeptide construct of any one of claims 2-15, 18-23, 26-95, 97-121, 123-177, wherein at least one receptor binding region (IRBR) binds to an inhibitory receptor selected from PD-1, CTLA-4, TIGIT, VISTA, or TIM3.

179. 178. The multispecific polypeptide construct of any one of claims 2-15, 18-23, 26-95, 97-121, 123-178, wherein at least one inhibitory receptor binding region (IRBR) binds to PD-1.

180. 180. The multispecific polypeptide construct of any one of claims 1 to 179, comprising a first connecting peptide (LP1) between the first antigen binding domain and the Fc region.

181. 181. The multispecific polypeptide construct of any of claims 1 to 180, comprising a second connecting peptide (LP2) between the CD3 binding region and the second antigen binding domain.

182. 181. The multispecific polypeptide construct of any one of claims 1 to 180, comprising a first connecting peptide (LP1) between the costimulatory receptor binding region (CRBR) and the Fc region.

183. 183. The multispecific polypeptide construct of any one of claims 1-182, comprising a second connecting peptide (LP2) between the CD3 binding region and the second costimulatory receptor binding region (CRBR).

184. a first connecting peptide (LP1) between the antigen-binding domain or the costimulatory receptor binding region and the Fc region, and a second connecting peptide (LP2) between the CD3 binding region and the antigen-binding domain or the CRBR; having the structural arrangement, from N-terminus to C-terminus, of a first antigen-binding domain or CRBR - LP1 - Fc region - linker - CD3 binding region - LP2 - a second antigen-binding domain or CRBR; 184. The multispecific polypeptide construct of any one of claims 1 to 183.

185. The multispecific polypeptide construct of claim 184, wherein the linker is a cleavable linker.

186. The multispecific polypeptide construct of claim 184, wherein the linker is a non-cleavable linker.

187. 187. The multispecific polypeptide construct of any one of claims 184-186, wherein the two connecting peptides are not identical to each other.

188. 188. The multispecific polypeptide construct of any one of claims 180-187, wherein LP1 or LP2 is independently a peptide of about 1 to 20 amino acids in length.

189. 189. The multispecific polypeptide construct of claim 188, wherein LP1 or LP2 independently comprise a peptide that is or comprises a Gly-Ser linker or GGS as shown in SEQ ID NOs: 10-13, 119, 135, 147, 149.

190. 190. The multispecific polypeptide construct of any of claims 1-189, wherein the anti-CD3 antibody or antigen-binding fragment is an Fv antibody fragment.

191. The multispecific polypeptide construct of claim 190, wherein the Fv antibody fragment is a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv).

192. The anti-CD3 antibody or antigen-binding fragment is VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO:16); a VH CDR2 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:18); a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:19); a VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO:20); and a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO:21).

192. The multispecific polypeptide construct of any one of claims 1 to 191, comprising:

193. Anti-CD3 dsFv, a VH having an amino acid sequence of any of SEQ ID NOs: 14, 32-62, 287, 290, and 311, 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: 14, 32-62, 287, 290, and 311; and VL having an amino acid sequence of any of SEQ ID NOs: 15, 63-81, 241, 288, and 289, 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: 15, 63-81, 241, 288, and 289.

162. The multispecific polypeptide construct of claim 160 or claim 161, comprising:

194. 194. The multispecific polypeptide construct of any one of claims 190 to 193, wherein the anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO:14 and the amino acid sequence of SEQ ID NO:

15.

195. The multispecific polypeptide construct of any one of claims 190 to 194, wherein the anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO:44 and the amino acid sequence of SEQ ID NO:72; the anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO:44 and the amino acid sequence of SEQ ID NO:241; the anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO:287 and the amino acid sequence of SEQ ID NO:288; or the anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO:311 and the amino acid sequence of SEQ ID NO:

289.

196. 196. The multispecific polypeptide construct of any one of claims 1 to 195, conjugated to an agent.

197. 197. The multispecific polypeptide construct of claim 196, wherein said agent is a therapeutic agent, an anti-tumor agent, a toxin or fragment thereof, a detectable moiety, or a diagnostic agent.

198. 198. The multispecific polypeptide construct of claim 197, wherein the agent is conjugated to the multispecific polypeptide construct via a linker.

199. A polynucleotide encoding the multispecific polypeptide construct of any one of claims 1 to 198.

200. A polynucleotide encoding any of the polypeptide chains of the multispecific polypeptide construct of any one of claims 1 to 198.

201. A polynucleotide comprising a first nucleic acid sequence encoding a first polypeptide of a multispecific construct according to any one of claims 1 to 198 and a second nucleic acid sequence encoding a second polypeptide of the multispecific construct, the 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; Polynucleotide.

202. 202. The polynucleotide of claim 201, wherein the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter.

203. The polynucleotide of claim 201 or claim 202, wherein the multispecific polypeptide construct comprises a third polypeptide chain, and wherein the polynucleotide further comprises a third nucleic acid encoding the third polypeptide of the multispecific construct.

204. the third nucleic acid is separated from the first and / or second polypeptide by an internal ribosome entry site (IRES) or by a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and / or the third nucleic acid sequence is operably linked to the same promoter as the first and / or second nucleic acid sequence; The polynucleotide of claim 203.

205. The polynucleotide of any one of claims 201-204, wherein the nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping is selected from T2A, P2A, E2A, or F2A.

206. A vector comprising the polynucleotide of any one of claims 199 to 205.

207. The vector of claim 206, which is an expression vector.

208. 208. The vector of claim 206 or 207, which is a viral vector or a eukaryotic vector, optionally wherein the eukaryotic vector is a mammalian vector.

209. 207. A cell comprising one or more polynucleotides of any one of claims 199-205 or one or more vectors of any one of claims 206-208.

210. The cell of claim 209, which is recombinant or isolated.

211. The cell of claim 210, which is a mammalian cell.

212. The cell of claim 211, which is a HEK293 cell or a CHO cell.

213. Introducing one or more polynucleotides according to any one of claims 199 to 205 or one or more vectors according to any one of claims 206 to 208 into a cell; and Culturing the cells under conditions whereby the multispecific polypeptide construct is produced. A method for making a multispecific polypeptide construct comprising:

214. 214. A method of making a multispecific polypeptide construct, comprising culturing a cell of any of claims 209-213 under conditions such that the multispecific polypeptide is produced by the cell.

215. 215. The cell of claim 213 or claim 214, which is a mammalian cell.

216. The cell of claim 215, which is a HEK293 cell or a CHO cell.

217. The method of claim 213 or claim 214, further comprising the step of isolating or purifying the multispecific polypeptide construct from the cell.

218. The method of any one of claims 213-217, wherein the multispecific polypeptide construct is a heterodimer.

219. 219. A multispecific polypeptide construct produced by the method of any one of claims 213-218.

220. 220. A pharmaceutical composition comprising the multispecific polypeptide construct of any one of claims 1 to 198 or claim 219 and a pharma- ceutically acceptable carrier.

221. 221. The pharmaceutical composition of claim 220, which is sterile.

222. 221. A method for stimulating or inducing an immune response comprising contacting a target cell and a T cell with a multispecific polypeptide construct of any one of claims 1 to 198 or claim 219 or with the pharmaceutical composition of claim 220 or claim 221, wherein the target cell expresses a tumor associated antigen that is recognized by the multispecific polypeptide construct.

223. The method of claim 222, wherein the target cell is a tumor cell expressing a tumor-associated antigen (TAA).

224. The method of claim 222 or claim 223, wherein the multispecific polypeptide construct comprises a cleavable linker that functions as a substrate for a protease, and wherein induction or stimulation of an immune response is increased in the presence of the protease.

225. The method of claim 224, wherein the protease is produced by an immune effector cell, by the tumor, or by a cell present in the tumor microenvironment.

226. The method of claim 224 or claim 225, wherein the protease is produced by an immune effector cell, the immune effector cell being an activated T cell, a natural killer (NK) cell, or a NK T cell.

227. The method of claim 226, wherein the immune effector cells are in proximity to the cells expressing the antigen.

228. a protease produced by the tumor in the vicinity of cells expressing the TAA in the tissue and / or co-localized with the TAA in the tissue; when the multispecific polypeptide construct is exposed to a protease, the protease cleaves the cleavable linker within the multispecific polypeptide construct; The method of any one of claims 222-227.

229. The method of any one of claims 222-228, wherein the protease is selected from matriptase, matrix metalloprotease (MMP), granzyme B, and combinations thereof.

230. The method of claim 229, wherein the protease is granzyme B.

231. The method of any one of claims 222-230, wherein the contacting step is carried out ex vivo or in vitro.

232. The method of any one of claims 222-231, wherein the contacting step is performed in vivo in a subject.

233. A method of stimulating or inducing an immune response in a subject comprising administering to a subject in need thereof a therapeutically effective amount of a multispecific conjugate of any one of claims 1 to 198 or claim 219 or a pharmaceutical composition of claim 220 or claim 221.

234. The method of any one of claims 222-233, which increases cellular immunity.

235. The method of any one of claims 222-234, wherein the method increases T cell activity.

236. The method of any one of claims 222-235, wherein the method increases cytolytic T cell (CTL) activity.

237. The method of any one of claims 222-236, wherein the immune response against a tumor or cancer is increased.

238. The method of any one of claims 222-236, for treating a disease or condition in a subject.

239. 221. 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 multispecific polypeptide construct according to any one of claims 1 to 198 or a pharmaceutical composition according to claim 220 or claim 221.

240. The method of claim 238 or claim 239, wherein the disease or condition is a tumor or cancer.

241. The method of any one of claims 238-240, wherein the disease or condition expresses a tumor associated antigen (TAA).

242. The method of any one of claims 222-241, wherein the subject is a human.

243. 222. A multispecific polypeptide construct according to any one of claims 1 to 198 or a pharmaceutical composition according to claim 220 or claim 221 for use in the treatment of a disease or condition in a subject.

244. 222. Use of a multispecific polypeptide construct according to any one of claims 1 to 198 or a pharmaceutical composition according to claim 220 or claim 221 for the manufacture of a medicament for use in the treatment of a disease or condition in a subject.

245. 245. A multispecific polypeptide construct or pharmaceutical composition for use according to claim 243, or the use according to claim 244, wherein the disease or condition is a tumor or cancer.

246. A multispecific polypeptide construct or pharmaceutical composition for use according to claim 243 or claim 245, or the use according to claim 244 or claim 245, wherein the disease or condition expresses a tumor associated antigen (TAA).

247. A multispecific polypeptide construct or a pharmaceutical composition for use according to claim 243, claim 245 or claim 246, or a use according to claim 244, claim 245 or claim 246, wherein the subject is a human.

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