Multispecific polypeptide constructs with constrained CD3 binding and related methods and uses

Abstract

To provide antibodies and drugs targeting CD3 / TCR pathway.SOLUTION: Provided is a multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, where the CD3 binding region is an anti-CD3 antibody or an antigen binding fragment being an Fv antibody fragment comprising a heavy chain variable region and a light chain variable region; the Fc is a heterodimeric Fc comprising a first Fc polypeptide and second Fc polypeptide, (the VH and VL of) the CD3 antibody or antigen binding fragment are linked to opposite polypeptides of the heterodimer; the first and second components are coupled by a non cleavable linker, the Fc region is positioned N terminally relative to the CD3 binding region; and 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 binds to a tumor associated antigen (TAA).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 656,331, filed April 11, 2018, entitled "MULTISPECIFIC POLYPEPTIDE CONSTRUCTS HAVING CONSTRAINED CD3 BINDING AND RELATED METHODS AND USES," the contents of which are incorporated by reference in their entirety.

[0002] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING This application is submitted with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 744952000240SeqList.TXT, created on April 10, 2019, and having a size of 212 kilobytes. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present invention generally relates to multispecific polypeptides with constrained CD3 binding. In some embodiments, the components of the multispecific polypeptide are connected by a non-cleavable linker. Methods of making and using these multispecific polypeptides in a variety of therapeutic, diagnostic, and prophylactic applications are also provided. [Background technology]

[0004] background Therapeutic antibodies that cause target cell depletion generally rely on effector function mediated through interactions with Fcγ receptors (FcγR) and complement proteins. FcγR-expressing effector cells are primarily those of the innate immune system. T cells are not the 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 distinct polypeptide chains called the epsilon, gamma, delta, and zeta chains. The CD3 complex serves as the signaling module of the T-cell receptor (TCR), noncovalently associating with the antigen-binding a / b chains of the TCR.

[0006] Direct engagement of CD3 leads to T cell activation and is therefore a desirable target for a variety of therapeutic and / or diagnostic applications. Thus, there is a need for antibodies and therapeutic agents that target the CD3 / TCR pathway. Summary of the Invention

[0007] overview The present disclosure provides multispecific polypeptide constructs that exhibit constrained CD3 binding. In some embodiments, the multispecific polypeptide constructs are composed of 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, such as a non-cleavable linker, and the Fc region is positioned N-terminal to the CD3-binding region; and one or both of the first and second components comprises an antigen-binding domain that binds to a tumor-associated antigen (TAA). In some embodiments, the CD3-binding region binds to CD3 (CD3ε). In some embodiments, the 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. In some embodiments, the first component comprises a first antigen-binding domain and the second component comprises a second antigen-binding domain, wherein each of the antigen-binding domains binds to a tumor-associated antigen (TAA). In some cases, the first antigen-binding domain is positioned at the amino terminus of the multispecific construct and the second antigen-binding domain is positioned at the carboxy terminus of the multispecific construct. In some embodiments, the first 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. In specific embodiments of the provided multispecific polypeptide constructs, at least one antigen-binding domain is positioned carboxy terminal to the CD3-binding region of the multispecific polypeptide construct.

[0008] Provided herein is a multispecific polypeptide construct comprising, from N-terminus to C-terminus, a first antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker, such as a non-cleavable linker; a CD3-binding region that binds to CD3 (CD3ε); and a second antigen-binding domain that binds to a tumor-associated antigen (TAA). Also provided is a multispecific polypeptide construct comprising, from N-terminus to C-terminus, an immunoglobulin Fc region; a linker, such as a non-cleavable linker; a CD3-binding region that binds to CD3 (CD3ε); and an antigen-binding domain that binds to a tumor-associated antigen (TAA). Provided is a multispecific polypeptide construct comprising, from N-terminus to C-terminus, an antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker, such as a non-cleavable linker; and a CD3-binding region that binds to CD3 (CD3ε).

[0009] In some of the provided embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a linker that does not contain a substrate recognition site specific for cleavage by a protease.

[0010] In some of the provided embodiments, positioning an Fc region N-terminal to the CD3 binding region reduces or prevents the ability of the CD3 binding region to bind to CD3. In some embodiments, the first component (component #1) and the second component (component #2) of the multispecific polypeptide construct are linked, disallowing binding to CD3 unless the antigen binding domain binds its cognate antigen. In some embodiments, component #1 contains at least one antigen binding domain and an Fc region. In some embodiments, component #2 contains at least a CD3 binding region and an antigen binding domain, the former being capable of binding to CD3 (when the multispecific construct binds to the antigen recognized by the antigen binding domain of component #1 or component #2). Thus, linking the CD3 binding region to an Fc region as described ensures that the multispecific polypeptide construct will not bind or otherwise engage CD3 unless the antigen binding domain binds its cognate antigen. This is advantageous because it prevents systemic binding of the CD3 binding region to T cells and concentrates the binding capacity of the CD3 binding region at the site of antigen expression, which is beneficial because it may reduce or eliminate the primary binding sink of peripheral T cells, allowing for more favorable distribution and localization to the site of antigen expression, e.g., tumor cells or the tumor microenvironment.

[0011] When the antigen-binding domain binds to its cognate antigen, the multispecific polypeptide construct can form an immune synapse between the antigen-expressing cell and the T cell via component #2. This simultaneous engagement mediates antigen-dependent T cell activation, cytotoxicity, cytokine release, degranulation, and proliferation. In some embodiments, the multispecific polypeptide construct can interact with FcγR and mediate innate immune effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the multispecific polypeptide construct can interact with complement proteins, i.e., C1q, and mediate complement-dependent cytotoxicity.

[0012] In some embodiments, the cognate antigen recognized by the antigen binding domain of the provided multispecific polypeptide constructs is a tumor-associated antigen (TAA).

[0013] Thus, in provided embodiments, the multispecific polypeptide construct is composed of 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, such as a non-cleavable linker, and the Fc region is positioned N-terminal to the CD3-binding region; and one or both of the first and second components comprises an antigen-binding domain that binds to a tumor-associated antigen (TAA). In some embodiments, the CD3-binding region binds to CD3 (CD3ε). In some embodiments, the 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. In some embodiments, the first component comprises a first antigen-binding domain and the second component comprises a second antigen-binding domain, wherein each of the antigen-binding domains binds to a tumor-associated antigen (TAA). In some cases, the first antigen-binding domain is positioned at the amino-terminus of the multispecific construct and the second antigen-binding domain is positioned at the carboxy-terminus of the multispecific construct. In some embodiments, the first 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. In specific embodiments of the provided multispecific polypeptide constructs, at least one antigen-binding domain is positioned carboxy terminal to the CD3 binding region of the multispecific polypeptide construct.

[0014] In some embodiments, the CD3-binding region is an antibody or antigen-binding fragment. In specific embodiments, the antibody or antigen-binding fragment is a two-chain polypeptide containing a variable heavy chain (VH) and a variable light chain (VL). In some embodiments, the antibody or antigen-binding fragment is an Fv. In specific embodiments, the Fv is a disulfide-stabilized Fv (dsFv) containing an interchain disulfide bond between the VH chain and the VL chain.

[0015] Provided herein is a multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3-binding region, wherein the CD3-binding region is an anti-CD3 antibody or an antigen-binding fragment that is an Fv antibody fragment comprising a variable heavy chain region (VH) and a variable light chain region (VL); the Fc is a heterodimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide, wherein the VH and VL of the anti-CD3 antibody or antigen-binding fragment are linked to opposing polypeptides of the heterodimeric Fc; the first and second components are coupled by a non-cleavable linker, and the Fc region is located N-terminal to the CD3-binding region; the first component comprises a first antigen-binding domain, and the second component comprises a second antigen-binding domain, each of which binds to a tumor-associated antigen (TAA). In some embodiments, the CD3-binding region binds to CD3 (CD3ε). 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 multispecific construct comprises, from N- to C-terminus, a first antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a non-cleavable linker; a CD3-binding region that binds to CD3 (CD3ε); and a second antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0016] Provided herein is a multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3-binding region, wherein the CD3-binding region is an anti-CD3 antibody or an antigen-binding fragment that is a disulfide-stabilized Fv antibody fragment (dsFv) comprising a variable heavy chain (VH) and a variable light chain (VL); the Fc is a heterodimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide, wherein the VH and VL of the anti-CD3 antibody or antigen-binding fragment are linked to opposing polypeptides of the heterodimeric Fc; the first and second components are coupled by a non-cleavable linker, and the Fc region is located N-terminal to the CD3-binding region; and one or both of the first and second components comprises an antigen-binding domain that binds to a tumor-associated antigen (TAA). In a specific embodiment of the provided multispecific polypeptide constructs, at least one antigen-binding domain is located carboxy-terminal to the CD3-binding region of the multispecific polypeptide construct. In some embodiments, the CD3 binding region binds to CD3 (CD3ε).

[0017] Provided herein is a multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3-binding region, wherein the CD3-binding region is an anti-CD3 antibody or an antigen-binding fragment that is an Fv antibody fragment comprising a variable heavy chain (VH) and a variable light chain (VL); the Fc is a heterodimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide, wherein the VH and VL of the anti-CD3 antibody or antigen-binding fragment are linked to opposing polypeptides of the heterodimeric Fc; the first and second components are coupled by a non-cleavable linker, and the Fc region is positioned N-terminal to the CD3-binding region; and one or both of the first and second components comprise an antigen-binding domain that binds to a tumor-associated antigen (TAA), wherein the antigen-binding domain is a single-chain antibody fragment such as an sdAb or scFv. In specific embodiments of the provided multispecific polypeptide constructs, at least one antigen-binding domain is positioned carboxy-terminal to a CD3-binding region of the multispecific polypeptide construct. In some embodiments, the CD3-binding region binds to CD3 (CD3ε).

[0018] In the embodiments provided herein, the multispecific construct comprises, in order from N-terminus to C-terminus, a first antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a non-cleavable linker; a CD3-binding region that binds to CD3 (CD3ε); and a second antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0019] In the embodiments provided herein, the multispecific construct comprises, from N-terminus to C-terminus, an immunoglobulin Fc region; a non-cleavable linker; a CD3 binding region that binds to CD3 (CD3ε); and an antigen binding domain that binds to a tumor-associated antigen (TAA).

[0020] In the embodiments provided herein, the multispecific construct comprises, in order from N-terminus to C-terminus, an antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a non-cleavable linker; and a CD3-binding region that binds to CD3 (CD3ε).

[0021] Provided herein is a multispecific polypeptide construct comprising, from N-terminus to C-terminus, a first antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker, such as a non-cleavable linker; a CD3-binding region that binds to CD3 (CD3ε); and a second antigen-binding domain that binds to a tumor-associated antigen (TAA). Also provided is a multispecific polypeptide construct comprising, from N-terminus to C-terminus, an immunoglobulin Fc region; a linker, such as a non-cleavable linker; a CD3-binding region that binds to CD3 (CD3ε); and an antigen-binding domain that binds to a tumor-associated antigen (TAA). Provided is a multispecific polypeptide construct comprising, from N-terminus to C-terminus, an antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker, such as a non-cleavable linker; and a CD3-binding region that binds to CD3 (CD3ε).

[0022] In some aspects, the antigen-binding domain, or each of the antigen-binding domains, independently, is selected from an antibody or antigen-binding fragment, a natural cognate binding partner, anticalin (a modified lipocalin), darpin, fynomer, centyrin (a modified fibronectin III domain), a cystine-knot domain, affilin, affibody, or a modified CH3 domain. In some embodiments, the natural cognate binding partner includes the extracellular domain of the native cognate binding partner of the TAA or a binding fragment thereof, or a variant thereof that exhibits binding activity with the TAA.

[0023] In some embodiments, the antigen-binding domain comprises one or more copies of an antibody or antigen-binding fragment thereof. In some embodiments, the antigen-binding domain comprises one or more copies of an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a Fv fragment, a scFv, a scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. In some embodiments, the antigen-binding domain comprises one or more single-domain antibody (sdAb) fragments, e.g., a V H H, V NAR , modified V H domain, or modified V K Contains one or more copies of the domain. V H H can be generated from camelid heavy chain-only antibodies. NAR can be generated from cartilaginous fish heavy chain-only antibodies. Various methods, including interface engineering and specific germline family selection, have been used to generate conventional heterodimeric V H Domains and V K This has been done to generate monomeric sdAbs from the domains.

[0024] In some embodiments, one or more antigen-binding domains independently bind to an antigen that is a tumor-associated antigen (TAA). In some examples, the antigen-binding domain, or each of the antigen-binding domains independently, binds to an antigen selected from the group consisting of 1-92-LFA-3, 5T4, alpha4 integrin, alphaV integrin, alpha4beta1 integrin, alpha4beta7 integrin, AGR2, anti-Lewis Y, apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9 (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD 51, 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, 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.

[0025] In some embodiments, the Fc region is a homodimeric Fc region. In some embodiments, the immunoglobulin Fc region of the first component is an IgG isotype selected from the group consisting of an IgG1 isotype, an IgG2 isotype, an IgG3 isotype, and an IgG4 subclass. In some examples, the Fc region is a human IgG1, human IgG2, human IgG3, or human IgG4 Fc region, or an immunologically active fragment thereof. In some embodiments, the Fc region comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, or a sequence of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1. In some cases, the Fc region comprises a polypeptide comprising the 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. In some such embodiments, 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. In some examples, the Fc region comprises a polypeptide comprising the 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. In some examples, the Fc region comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:6, or a sequence of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:6.

[0026] In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence derived from an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6.

[0027] 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 is a polypeptide comprising an amino acid 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6.

[0028] In some embodiments, the Fc region is a heterodimeric Fc region.

[0029] In some embodiments, the Fc region is a heterodimer comprising a first Fc polypeptide and a second Fc polypeptide, wherein one or both of the first and second Fc polypeptides of the heterodimeric Fc region are variant Fc polypeptides comprising at least one modification to induce heterodimerization compared to the Fc region of human IgG1, human IgG2, or human IgG4. In some embodiments, the at least one modification is in or compared to the Fc region of human IgG1. In some embodiments, the at least one modification is in or compared to the Fc polypeptide set forth in SEQ ID NO:1 or an immunologically active fragment thereof. In some cases, 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, optionally compared to the Fc polypeptide set forth in SEQ ID NO:1 or an immunologically active fragment thereof. In some embodiments, each of the Fc polypeptides of the heterodimeric Fc independently comprises at least one amino acid modification. In some cases, the at least one modification is selected from a steric modification, a knobs-into-holes modification, a charge mutation to increase electrostatic complementarity of the polypeptide, a modification to alter the isoelectric point (pI variant), or a combination thereof.

[0030] In some examples, the amino acid modification is a charge mutation to increase the electrostatic complementarity of the polypeptides. In some embodiments, the first and / or second Fc polypeptide comprises a modification at a complementary position that is a substitution of an amino acid with an opposite charge to the complementary amino acid of the other polypeptide. In some embodiments, the first or second polypeptide comprises a modification at a complementary position that is a substitution of an amino acid with an opposite charge to the complementary amino acid of the other polypeptide. In some embodiments, at least the first or second Fc polypeptide each comprises a modification at a complementary position that is a substitution of an amino acid with an opposite charge to the complementary amino acid of the other polypeptide. In some embodiments, the first and second Fc polypeptide each comprise a modification at a complementary position that is a substitution of an amino acid with an opposite charge to the complementary amino acid of the other polypeptide.

[0031] In some instances, the amino acid modification is a knob-into-hole modification.

[0032] In some embodiments, the first Fc polypeptide of the heterodimeric Fc comprises a modification selected from Thr366Ser, Leu368Ala, Tyr407Val, and a combination thereof, and the second Fc polypeptide of the heterodimeric Fc comprises the modification T366W. In some cases, the first and second Fc polypeptides further comprise a modification of a non-cysteine ​​residue to a cysteine ​​residue, wherein the modification of the first polypeptide is at one of positions Ser354 and Y349, and the modification of the second Fc polypeptide is at the other of positions Ser354 and Y349. In some embodiments, the first Fc polypeptide comprises the modification T366W / S354C, and the second Fc polypeptide comprises the modifications T366S / L368A / Y407V / Y349C. In some embodiments, the first Fc polypeptide comprises the modifications L368D / K370S and the second Fc polypeptide comprises the modifications S364K / E357Q.

[0033] In some embodiments, the first Fc polypeptide comprises the modifications L368D / K370S and the second Fc polypeptide comprises the modifications S364K / E357Q.

[0034] In some embodiments, at least one of the first and second polypeptides comprises the modifications Q295E / N384D / Q418E / N421D.

[0035] 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 embodiments, one of the first or second Fc polypeptides of the heterodimeric Fc further comprises a modification at residue His435. In some cases, the modification is His435Arg.

[0036] In some embodiments, the Fc region, such as the first and / or second Fc polypeptide, comprises a polypeptide lacking Lys447.

[0037] In some of the provided embodiments, the first polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs: 82, 86, or 201, and the second polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs: 83, 87, 90, 92, 202, or 205. In some embodiments, the first Fc polypeptide and the second Fc polypeptide comprise a sequence selected from the group consisting of SEQ ID NOs: 82 and 83, respectively; SEQ ID NOs: 86 and 87, respectively; SEQ ID NOs: 201 and 202, respectively; SEQ ID NOs: 82 and 90, respectively; SEQ ID NOs: 86 and 92, respectively; and SEQ ID NOs: 201 and 205, respectively.

[0038] In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence derived from an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, comprising one or more modifications to prevent glycosylation, alter Fc receptor interaction, reduce Fc receptor binding, enhance interaction with CD32A, reduce complement protein C1q binding, increase half-life, enhance FcRn binding, alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), induce heterodimerization, prevent dimerization, stabilize homodimerization at the CH3:CH3 interface, and combinations thereof.

[0039] In some embodiments, modifications in the Fc region reduce binding to Fcγ receptors but minimally affect binding to neonatal Fc receptors (FcRn). In some embodiments, the variant or modified Fc polypeptide comprises the following mutations, using the Kabat numbering system: Met252Tyr and Met428Leu or Met252Tyr and Met428Val (M252Y, M428L, or M252Y, M428V).

[0040] In some embodiments, the Fc region comprises a polypeptide comprising at least one modification to enhance FcRn binding. In some instances, the modification is at a position selected from the group consisting of Met252, Ser254, Thr256, Met428, Asn434, and combinations thereof. In some cases, the modification is at a position selected from the group consisting of Met252Y, Ser254T, Thr256E, Met428L, Met428V, Asn434S, and combinations thereof. In some specific embodiments, the modification is at position Met252 and position Met428. In some cases, the modification is Met252Y and Met428L. In some cases, the modification is Met252Y and Met428V.

[0041] In some embodiments, the first polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs:94, 96, or 207, and the second polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs:98, 100, or 209. In some embodiments, the first Fc polypeptide and the second Fc polypeptide comprise a sequence selected from the group consisting of SEQ ID NOs:94 and 98, respectively; SEQ ID NOs:96 and 100, respectively; and SEQ ID NOs:207 and 209, respectively.

[0042] In some embodiments, the Fc region comprises a polypeptide comprising at least one modification to enhance FcγR binding. In some cases, the modification is at Ser239 or Ile332. In some embodiments, the glycosylation of the Fc region is modified to enhance FcγR binding compared to an unmodified Fc region. In some examples, the Fc region lacks fucose or has a reduced fucose content.

[0043] 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 an Fcγ receptor or C1q, hi some embodiments, the one or more amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235.

[0044] In some embodiments, the first polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs: 82, 86, 94, or 96, and the second polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs: 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 an Fcγ receptor or C1q. In some examples, the one or more amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235. In some aspects, the first polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs:84, 88, 95, or 97, and the second polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs:85, 89, 91, 93, 99, or 101.

[0045] In some embodiments, the first polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs: 84, 88, 95, 97, 203, or 208, and the second polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs: 85, 89, 91, 93, 99, 101, 204, 206, or 210. In some embodiments, the first Fc polypeptide and the second Fc polypeptide comprise a sequence selected from the group consisting of SEQ ID NOs: 84 and 85, respectively; SEQ ID NOs: 88 and 89, respectively; SEQ ID NOs: 203 and 204, respectively; SEQ ID NOs: 95 and 99, respectively; SEQ ID NOs: 97 and 101, respectively; SEQ ID NOs: 208 and 210, respectively; SEQ ID NOs: 84 and 91, respectively; SEQ ID NOs: 88 and 93, respectively; and SEQ ID NOs: 203 and 206, respectively.

[0046] In some 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 such embodiments, the CD3 binding region is monovalent.

[0047] In some embodiments, the anti-CD3 antibody or antigen-binding fragment is not a single-chain antibody, and optionally is not a single-chain variable fragment (scFv). In some embodiments, the Fc is a heterodimeric Fc, wherein the VH and VL comprising the anti-CD3 antibody or antigen-binding fragment are linked to opposing polypeptides of the heterodimeric Fc.

[0048] In some embodiments, the CD3 binding region is incapable or substantially incapable of binding or engaging CD3 unless at least one of the antigen binding domains binds to its TAA, hi some aspects, the CD3 binding region is incapable or substantially incapable of binding or engaging CD3 unless at least two of the antigen binding domains bind to its TAA.

[0049] In some embodiments, the multispecific polypeptide construct contains a linker that is a polypeptide linker. In some embodiments, the linker is a polypeptide up to 25 amino acids in length. In some cases, the linker is 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 ...8 to 10 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, 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, 8 to 24 amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14 amino acids, 8 to 24 amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14 amino acids, 8 to 24 amino acids, 8 to 24 amino acids, 8 to 12 amino acids, 8 to 18 amino acids A polypeptide of about 2 to 24 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. acids, about 2 to 20 amino acids, about 2 to 18 amino acids, about 2 to 14 amino acids, about 2 to 12 amino acids, about 2 to 10 amino acids, about 2 to 8 amino acids, about 2 to 6 amino acids, about 6 to 24 amino acids, about 6 to 20 amino acids, about 6 to 18 amino acids, about 6 to 14 amino acids, about 6 to 12 amino acids, about 6 to 10 amino acids, about 6 to 8 amino acids, about 8 to 24 amino acids, about 8 to 20 amino acids, about 8 to 18 amino acids, about 8 to 14 amino acids, about 8 to 12 amino acids, The polypeptide is a polypeptide of about 8 to 10 amino acids, about 10 to 24 amino acids, about 10 to 20 amino acids, about 10 to 18 amino acids, about 10 to 14 amino acids, about 10 to 12 amino acids, about 12 to 24 amino acids, about 12 to 20 amino acids, about 12 to 18 amino acids, about 12 to 14 amino acids, about 14 to 24 amino acids, about 14 to 20 amino acids, about 14 to 18 amino acids, about 18 to 24 amino acids, about 18 to 20 amino acids, or about 20 to 24 amino acids.In some embodiments, 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.

[0050] In some embodiments, the linker is 3 to 18 amino acids in length. In some embodiments, the linker is 12 to 18 amino acids in length. In some embodiments, the linker is 15 to 18 amino acids in length. In some embodiments, the linker is 18 amino acids in length.

[0051] In some embodiments, the non-cleavable linker does not contain a substrate recognition site that is specifically recognized for cleavage by a protease. In some embodiments, the protease is produced by an immune effector cell, by a tumor, or by a cell present in the tumor microenvironment. In some embodiments, the protease is produced by an immune effector cell, and the immune effector cell is an activated T cell, a natural killer (NK) cell, or an NK T cell. In some embodiments, the protease is selected from matriptase, matrix metalloproteinase (MMP), granzyme B, and combinations thereof. In some embodiments, the protease is granzyme B.

[0052] In some embodiments, the linker comprises the amino acid sequence TIFF0007824455000001.tif4128 and combinations thereof. In some embodiments, the linker comprises the amino acid sequence (GGS)n, where n is 1 to 10. In some embodiments, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:173), where n is 1 to 10. In some embodiments, the linker comprises the amino acid sequence (GGGGGS)n (SEQ ID NO:172), where n is 1 to 4.

[0053] In some embodiments, the linker is or comprises GGS. In some embodiments, the linker is or comprises GGGGS (SEQ ID NO:149). In some embodiments, the linker is or comprises GGGGGS (SEQ ID NO:135). In some embodiments, the linker is or comprises GGSGGS ("(GGS)2") (SEQ ID NO:10). In some embodiments, the linker is or comprises GGSGGSGGS ("(GGS)3") (SEQ ID NO:11). In some embodiments, the linker is or comprises GGSGGSGGSGGS ("(GGS)4") (SEQ ID NO:12). In some embodiments, the linker is In some embodiments, the linker is or comprises: In some embodiments, the linker is or comprises: In some embodiments, the linker is or comprises: TIFF0007824455000005.tif4128. In some embodiments, the linker is or comprises GGGGG (SEQ ID NO:192).

[0054] In some embodiments, the antigen-binding domain of the first component (which in some cases is the first antigen-binding domain) and the immunoglobulin Fc region are operably linked via one or more additional amino acid linkers (referred to herein as intracomponent linkers). The intracomponent peptide linker of the first component (also referred to as LP1) can be a peptide linker such as any of those described in Section II.3. The intracomponent peptide linkers present in the first component, i.e., linking the Fc region and the antigen-binding domain, can be of various lengths, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length. In some embodiments, these intracomponent linkers are primarily composed of the amino acids glycine and serine, and are referred to herein as GS linkers. In some embodiments, the GS linker is TIFF0007824455000006.tif19150.

[0055] In some embodiments, the multispecific polypeptide construct comprises at least (i) a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and a VH domain of an anti-CD3 antibody or antigen-binding fragment thereof; and (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 thereof, wherein one or both of the first and second polypeptides comprises at least one antigen-binding domain that binds to a tumor-associated antigen (TAA). In some embodiments, the VH of the anti-CD3 antibody or antigen-binding fragment is on the same polypeptide as the at least one antigen-binding domain that binds to the tumor-associated antigen (TAA). In some embodiments, the polypeptide comprising the VL of the anti-CD3 antibody or antigen-binding fragment does not contain at least one antigen-binding domain that binds to the tumor-associated antigen (TAA). In specific embodiments of the provided multispecific polypeptide constructs, at least one antigen-binding domain is positioned carboxy-terminal to the CD3-binding region of the multispecific polypeptide construct.

[0056] In some embodiments, the second component comprises one or more copies of a CD3 binding domain.

[0057] In some embodiments, the anti-CD3 binding domain is an anti-CD3 antibody or antigen-binding fragment comprising one or more copies of an antibody or antigen-binding fragment capable of binding to or engaging CD3, such as CD3ε. In some embodiments, the anti-CD3 binding domain comprises one or more copies of an antibody or antigen-binding fragment selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, an scFv, an scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody.

[0058] In some embodiments, the anti-CD3 binding domain comprises an Fv fragment that binds to CD3ε (herein referred to as an anti-CD3ε Fv fragment).

[0059] In some embodiments, the anti-CD3ε Fv antibody fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81, 191, 196-200, 211, and 212. In some embodiments, the anti-CD3ε Fv antibody fragment comprises an 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, 191, 196-200, 211, and 212. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of an amino acid sequence selected from the group of SEQ ID NOs: 32-62, 196-198, and 211 with an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81, 191, 199, 200, and 212. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of an 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, 196-198, and 211, and an 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, 191, 199, 200, and 212.

[0060] In some embodiments, the anti-CD3ε Fv antibody fragment is a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv).

[0061] In some embodiments, the first component comprises one or more copies of an antigen-binding domain. In certain embodiments, the first component contains at least two antigen-binding domains, such as two antigen-binding domains. In some embodiments, at least two antigen-binding domains of the first component bind to the same TAA. In some cases, at least two antigen-binding domains of the first component bind to different epitopes of the same TAA. In some cases, at least two antigen-binding domains of the first component bind to the same epitope of the same TAA. In some embodiments, at least two antigen-binding domains of the first component bind to different TAAs.

[0062] In some embodiments, the second component comprises one or more copies of an antigen-binding domain. In certain embodiments, the second component contains at least two antigen-binding domains, such as two antigen-binding domains. In some embodiments, at least two antigen-binding domains of the second component bind to the same TAA. In some cases, at least two antigen-binding domains of the second component bind to different epitopes of the same TAA. In some cases, at least two antigen-binding domains of the second component bind to the same epitope of the same TAA. In some embodiments, at least two antigen-binding domains of the second component bind to different TAAs.

[0063] In some embodiments, the first component contains a first antigen-binding domain, and the antigen-binding domain of the second component is a second antigen-binding domain. In some embodiments, the multispecific 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. In some cases, the first antigen-binding domain and the second antigen-binding domain bind to different epitopes of the same TAA. In some cases, the first antigen-binding domain and the second antigen-binding domain bind to the same epitope of the same TAA. In some embodiments, the multispecific 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.

[0064] In some embodiments, the antigen-binding domain and CD3-binding region of the second component (which in some cases is the second antigen-binding domain) are operably linked via one or more additional amino acid linkers (referred to herein as intracomponent linkers). The intracomponent peptide linker of the second component (also referred to as LP2) can be a peptide linker such as any of those described in Section II.3. The intracomponent linker present in the second component, i.e., linking the CD3-binding region and the antigen-binding domain, can be of various lengths, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the intracomponent linker of the second component is composed primarily of the amino acids glycine and serine, and is referred to herein as a GS linker. In some embodiments, the GS linker has the amino acid sequence TIFF0007824455000007.tif19160.

[0065] Provided herein are multispecific polypeptide constructs comprising a first component comprising a heterodimeric Fc region and a second component comprising an anti-CD3 antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprising the anti-CD3 antibody or antigen-binding fragment are linked to opposing polypeptides of the heterodimeric Fc; the first and second components are coupled by a linker, and the heterodimeric Fc region is positioned N-terminal to the anti-CD3 antibody or antigen-binding fragment; and one or both of the first and second components comprises an antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0066] In some embodiments, the linker is a polypeptide up to 50 amino acids in length. In some embodiments, the linker is a polypeptide up to 25 amino acids in length. In some embodiments, the linker is a polypeptide up to 15 amino acids in length.

[0067] In any of the provided embodiments, the one or more antigen-binding domains that bind to the TAA provide monovalent, bivalent, trivalent, or tetravalent binding to the TAA. In some embodiments, the one or more antigen-binding domains that bind to the TAA are independently selected from an sdAb, an scFv, or an Fab. In some embodiments, the one or more antigen-binding domains that bind to the TAA are single-chain molecules such as single-chain antibody fragments, e.g., sdAb or scFv, containing a VH and a VL. In some embodiments, the one or more antigen-binding domains that bind to the TAA are VH and VL. H H or VH NAR In some embodiments, at least one of the antigen-binding domains is a Fab comprising a first chain comprising VH-CH1(Fd) and a second chain comprising VL-CL.

[0068] In some embodiments, the antigen binding domain that binds to the TAA is attached to the VH of the anti-CD3 binding domain. In some embodiments, the antigen binding domain that binds to the TAA is attached to the same side (e.g., knob or hole) of the heterodimeric Fc to which the VH of the anti-CD3 binding domain is attached. In some embodiments, the antigen binding domain that binds to the TAA is an sdAb attached to the VH of the anti-CD3 binding domain. In some embodiments, the antigen binding domain that binds to the TAA is an sdAb attached to the same side (e.g., knob or hole) of the heterodimeric Fc domain to which the VH of the anti-CD3 binding domain is attached. In some embodiments, the antigen binding domain that binds to the TAA is an sdAb attached to the VH of the anti-CD3 binding domain. H H or VH NAR In some embodiments, the antigen binding domain that binds to the TAA is a V that is attached to the same side (e.g., knob or hole) of the heterodimeric Fc domain to which the VH of the anti-CD3 binding domain is attached. H H or VH NAR In some embodiments, the antigen binding domain that binds to the TAA is a V that is attached to the V of the anti-CD3 binding domain. H In some embodiments, the antigen binding domain that binds to the TAA is a VH attached to the same side (e.g., knob or hole) of the heterodimeric Fc domain to which the VH of the anti-CD3 binding domain is attached. H In some embodiments, the antigen binding domain that binds to the TAA is a VH attached to the VH of the anti-CD3 binding domain. NAR In some embodiments, the antigen binding domain that binds to the TAA is a VH that is attached to the same side (e.g., knob or hole) of the Fc domain as the VH of the anti-CD3 binding domain is attached. NAR is.

[0069] In some embodiments, the multispecific polypeptide construct comprises at least (i) a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and a VH 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, and a VL domain of an anti-CD3 antibody or antigen-binding fragment, wherein one or both of the first and second polypeptides comprises at least one antigen-binding domain that binds to a tumor-associated antigen (TAA). In some cases, only one of the first or second polypeptides comprises at least one antigen-binding domain that binds to a TAA.

[0070] In some embodiments, at least one of the antigen-binding domains is a Fab. In some embodiments, 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, wherein the first and / or second polypeptide further comprises the other of the VH-CH1(Fd) or VL-CL of the Fab antibody fragment. In some cases, only one of the first or second polypeptide comprises the other of the VH-CH1(Fd) or VL-CL of the Fab antibody fragment. In some embodiments, both the first or second polypeptide comprise the other of the VH-CH1(Fd) or VL-CL of the Fab antibody fragment. In some cases, 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. In some embodiments, 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 polypeptide or the second polypeptide.

[0071] In some embodiments, at least one antigen-binding domain 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. In some cases, at least one 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 specific embodiments of the provided multispecific polypeptide constructs, at least one antigen-binding domain is positioned carboxy-terminal to the CD3-binding region of the multispecific construct. In some embodiments, at least one antigen-binding domain is an sdAb. In some embodiments, at least one antigen-binding domain that is an sdAb is positioned carboxy-terminal to the CD3-binding region of the multispecific construct. In some embodiments, at least one antigen-binding domain that is an sdAb is positioned amino-terminal to the Fc region of the multispecific construct. In some embodiments, at least one antigen-binding domain is a V H H. In some embodiments, V H At least one antigen binding domain that is H is positioned carboxy-terminal to the CD3 binding region of the multispecific construct. H At least one antigen binding domain that is H is positioned amino-terminal to the Fc region of the multispecific construct.

[0072] In some embodiments, the multispecific polypeptide construct comprises a first connecting peptide (LP1) between the first antigen-binding domain and the immunoglobulin Fc polypeptide region (Fc region). In some embodiments, the multispecific polypeptide construct comprises a second connecting peptide (LP2) between the anti-CD3 binding domain (CD3 binding region) and the second antigen-binding domain. In some embodiments, the multispecific polypeptide construct comprises a first connecting peptide (LP1) between the first antigen-binding domain and the immunoglobulin Fc polypeptide region (Fc region) and a second connecting peptide (LP2) between the anti-CD3 binding domain (CD3 binding region) and the second antigen-binding domain.

[0073] In some embodiments, the multispecific polypeptide construct has the following structural arrangement from N-terminus to C-terminus: first antigen binding domain - LP1 - immunoglobulin Fc polypeptide linker region (Fc region) - linker - anti-CD3 binding domain - LP2 - second antigen binding domain. In some embodiments, the multispecific polypeptide construct has the following structural arrangement from N-terminus to C-terminus: second antigen binding domain - LP2 - immunoglobulin Fc polypeptide linker region (Fc region) - linker - anti-CD3 binding domain (CD3 binding region) - LP1 - first antigen binding domain.

[0074] In some embodiments, the two connecting peptides, LP1 and LP2, are not identical to one another. In some cases, LP1 or LP2 are independently peptides of about 1 to 20 amino acids in length. In some examples, LP1 or LP2 independently comprise a peptide that is or includes a Gly-Ser linker set forth in SEQ ID NOs: 10-13, 119, 135, 147, or 149.

[0075] In some embodiments, the multispecific construct is a construct having any of the structural configurations shown in Figure 1. In some embodiments, the construct is a bispecific construct having the following structural configuration, from N- to C-terminus: The N-terminus of the bispecific construct comprises a first antigen-binding domain that binds to a tumor-associated antigen (TAA). The first binding domain binds to a first epitope of the TAA target. The first antigen-binding domain is coupled to a central immunoglobulin Fc polypeptide region that controls FcγR and / or FcRn interactions. In some embodiments, the central immunoglobulin Fc polypeptide region is heterodimeric. The immunoglobulin Fc polypeptide region is coupled to a linker located C-terminal to the end of the immunoglobulin Fc polypeptide region. The linker is attached to an anti-CD3 binding sequence located C-terminal to the Fc region, in some cases at the distal end of the second component. The C-terminus of the bispecific construct comprises a second antigen-binding domain that binds to a TAA. In some embodiments, the second antigen-binding domain binds to the same TAA as the first antigen-binding domain located on the first component. In some embodiments, the second antigen-binding domain binds to a second epitope on the TAA, where the second epitope does not compete with the first epitope on the TAA. In some embodiments, the second antigen-binding domain binds to a different TAA than that of the first antigen-binding domain.

[0076] In any of the provided embodiments, the anti-CD3 antibody or antigen-binding fragment is an Fv antibody fragment. In some embodiments, the Fv antibody fragment comprises a disulfide-stabilized anti-CD3-binding Fv fragment (dsFv). In some embodiments, the anti-CD3-binding sequence is an Fv antibody fragment that has been modified to include a disulfide bond between the heavy chain variable region (VH) and the light chain variable region (VL), thereby creating a disulfide-stabilized anti-CD3-binding Fv fragment (dsFv). In some embodiments, the VH and VL domains comprising the anti-CD3 Fv are operably linked to opposing members of a heterodimeric Fc region. In these embodiments, the anti-CD3 Fv binds to CD3 monovalently. In provided aspects, the anti-CD3 dsFv does not engage CD3 unless the multispecific polypeptide construct binds to its cognate antigen.

[0077] In some embodiments, each of the first and second antigen-binding domains of the bispecific construct comprises one or more copies of an antibody or antigen-binding fragment thereof. In some embodiments, each of the first and second antigen-binding domains of the bispecific construct comprises one or more copies of an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, an F(ab')2 fragment, an Fv fragment, an scFv, an scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. In some embodiments, the antigen-binding domain, or each of the antigen-binding domains, independently, is an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, an F(ab')2 fragment, an Fv fragment, an scFv, an scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. In some embodiments, each of the first and second antigen-binding domains of the bispecific construct comprises one or more single-domain antibody (sdAb) fragments, e.g., V H H, V NAR , modified V H domain, or modified V K Contains one or more copies of the domain. V HH can be generated from natural camelid heavy chain-only antibodies, genetically modified rodents producing heavy chain-only antibodies, or from naive / synthetic camelid or humanized camelid single domain antibody libraries. NAR can be generated from cartilaginous fish heavy chain-only antibodies. Various methods, including interface engineering and specific germline family selection, have been used to generate V-heterodimeric antibodies. H Domains and V K This has been done to generate monomeric sdAbs from the domains.

[0078] In some embodiments, the antibody or antigen-binding fragment is an sdAb. In some cases, the sdAb is a human or humanized sdAb. In some aspects, the sdAb is a VHH, VNAR, modified VH domain, or modified VK domain. In some examples, the antibody or antigen-binding fragment thereof is an scFv. In some cases, the antibody or antigen-binding fragment thereof is a Fab.

[0079] In any of the provided embodiments, the anti-CD3 antibody or antigen-binding fragment comprises a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO:16), an amino acid sequence VH CD2 containing TIFF0007824455000008.tif4128; amino acid sequence VH CDR3 containing TIFF0007824455000009.tif4128; amino acid sequence TIFF0007824455000010.tif4128; VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO:20); and VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO:21).

[0080] In some of the provided embodiments, the anti-CD3 antibody or antigen-binding fragment comprises a VH CDR1 comprising the amino acid sequence GFTFNTYAMN (SEQ ID NO:211); a VH CD2 comprising the amino acid sequence RIRSKYNNYATY (SEQ ID NO:212); VH CDR3 containing TIFF0007824455000011.tif4128; amino acid sequence TIFF0007824455000012.tif4128; VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO:20); and VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO:21).

[0081] In some of the provided embodiments, the anti-CD3 antibody or antigen-binding fragment comprises a VH CDR1 sequence comprising at least the amino acid sequence GFTFNTYAMN (SEQ ID NO:211); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATY (SEQ ID NO:212); VH CDR3 sequence containing at least TIFF0007824455000013.tif4128; amino acid sequence TIFF0007824455000014.tif4128; a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO:230); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNHWV (SEQ ID NO:225).

[0082] In some provided embodiments, the anti-CD3 antibody or antigen-binding fragment comprises a VH having the amino acid sequence of any of SEQ ID NOs: 14, 32-62, 196-198, and 211, 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, 196-198, and 211; and a VH having the amino acid sequence of any of SEQ ID NOs: 15, 63-81, 191, 199, 200, and 212, 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, 196-198, and 211; The present invention also includes a VL having a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of NOs:15, 63-81, 191, 199, 200, and 212.

[0083] In some embodiments, the anti-CD3 antibody or antigen-binding fragment is an Fv. In some embodiments, the anti-CD3 Fv comprises a VH having an amino acid sequence of any of SEQ ID NOs: 14, 32-43, 45-47, 48, 196, and 211, 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, 32-43, 45-47, 48, 196, and 211; and an VH having an amino acid sequence of any of SEQ ID NOs: 15, 63, 65-71, 73, 75, 77, and 199, 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, 32-43, 45-47, 48, 196, and 211; In some cases, the anti-CD3 dsFv comprises a VL having 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, 65-71, 73, 75, 77, and 199. In some cases, the anti-CD3 dsFv 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: 196 and the amino acid sequence of SEQ ID NO: 199.

[0084] In some embodiments, the VH chain region and VL chain region of the CD3-binding domain each independently comprise at least one amino acid modification. In some embodiments, the at least one amino acid modification in the VH chain region and VL chain region of the CD3-binding domain increases the stability of the CD3-binding domain. In some embodiments, the at least one amino acid modification in the VH chain region and VL chain region of the CD3-binding domain increases the ability of the CD3-binding domain to bind to CD3. In some embodiments, the at least one amino acid modification in the VH chain region and VL chain region of the CD3-binding domain increases the stability of the CD3-binding domain by creating a disulfide bond between the VH chain region and the VL chain region.

[0085] In some embodiments, the CD3 binding region has a disulfide-stabilized linkage between the VH and VL regions. 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 Cys mutation at position 44 and an anti-CD3 VL containing a Cys mutation at position 100, according to Kabat numbering. In some embodiments, the disulfide-stabilized anti-CD3 Fv comprises an anti-CD3 VH containing a G44C mutation and an anti-CD3 VL containing a G100C mutation, according to Kabat numbering. In some embodiments, the disulfide-stabilized anti-CD3 Fv comprises an anti-CD3 VH containing a Cys mutation at position 105 and an anti-CD3 VL containing a Cys mutation at position 43, according to Kabat numbering.

[0086] In some embodiments, the anti-CD3 dsFv comprises a VH having the amino acid sequence of any of SEQ ID NOs: 44, 49-62, 197, and 198, 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: 44, 49-62, 197, and 198; and a VH having the amino acid sequence of any of SEQ ID NOs: 64, 72, 74, 76, 78-81, 191, 200, and 212, 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: 64, 72, 74, 76, 78-81, 191, 200, and 212. In some embodiments, the anti-CD3 dsFv comprises a VL having 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:64, 72, 74, 76, 78-81, 191, 200, and 212. In some cases, 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:198 and the amino acid sequence of SEQ ID NO:200. In some embodiments, the anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO:197 and the amino acid sequence of SEQ ID NO:200.

[0087] In some embodiments, the multispecific construct also comprises an agent conjugated to the multispecific construct. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent. In some embodiments, the agent is conjugated to the multispecific construct via a linker. In some embodiments, the linker is a non-cleavable linker.

[0088] In some embodiments, the multispecific constructs described herein are used in conjunction with one or more additional agents or combinations of additional agents. Suitable additional agents include, for example, current pharmaceutical and / or surgical treatments for the intended application, such as cancer. For example, the multispecific constructs can be used in conjunction with additional chemotherapy or anti-tumor agents.

[0089] In some embodiments, the multispecific construct and the additional agent are formulated into a single therapeutic composition, and the multispecific construct and the additional agent are administered simultaneously. In some embodiments, the multispecific construct and the additional agent are separate from one another, e.g., each formulated into a separate therapeutic composition, and the multispecific construct and the additional agent are administered simultaneously, or the multispecific construct and the additional agent are administered at different times during the treatment regimen. For example, the multispecific construct is administered before the administration of the additional agent, the multispecific construct is administered after the administration of the additional agent, or the multispecific construct and the additional agent are administered alternately. As described herein, the multispecific construct and the additional agent are administered once or multiple times.

[0090] In some embodiments, the multispecific construct naturally contains one or more disulfide bonds. In some embodiments, the multispecific construct may be modified to contain one or more disulfide bonds.

[0091] The present disclosure also provides isolated nucleic acid molecules or polynucleotides encoding at least a portion of the multispecific constructs described herein, and / or one or more nucleic acid molecules encoding the multispecific constructs described herein, such as, for example, at least a first nucleic acid encoding at least a portion of a first component of the multispecific construct and a second nucleic acid encoding at least a portion of a second component of the multispecific construct, as well as vectors comprising these isolated nucleic acid sequences.

[0092] The provided embodiments include polynucleotides encoding any of the provided multispecific polypeptide constructs. Polynucleotides encoding any of the polypeptide chains of the provided multispecific polypeptide constructs are also provided. Further provided are polynucleotides comprising a first nucleic acid sequence encoding a first polypeptide of any of the provided multispecific constructs 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) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping. In some cases, 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 comprises a third polypeptide chain, and the polynucleotide further comprises 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 polypeptide 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 sequence. In some examples, the nucleic acid encoding the self-cleaving peptide or the peptide that causes ribosome skipping is selected from T2A, P2A, E2A, or F2A (encoded by the sequences set forth in SEQ ID NOs:159-164, or SEQ ID NO:165).

[0093] Provided herein is a vector comprising any of the provided polynucleotides. In some embodiments, the vector is an expression vector. In some examples, the vector is a viral vector or a eukaryotic vector, and optionally, the eukaryotic vector is a mammalian vector.

[0094] Cells containing any of the provided polynucleotides or vectors are provided. In some cases, the cells are recombinant or isolated. In some examples, the cells are mammalian cells. In some examples, the cells are HEK293 cells or CHO cells.

[0095] The present disclosure provides methods of making multispecific constructs by culturing cells containing such nucleic acid sequences under conditions that result in expression of the multispecific construct. In some embodiments, the cells contain such vectors.

[0096] Provided herein are methods for producing a multispecific polypeptide construct, comprising introducing any of the provided polynucleotides or vectors into cells and culturing the cells under conditions resulting in expression of the multispecific construct to produce the multispecific polypeptide construct. Also provided are methods for producing a multispecific polypeptide construct, comprising culturing any of the provided cells under conditions in which the multispecific polypeptide is expressed or produced by the cells. In some cases, the cells are mammalian cells. In some examples, the cells are HEK293 cells or CHO cells. In some embodiments, the method further comprises isolating or purifying the multispecific polypeptide construct from the cells. In some cases, the multispecific polypeptide construct is a heterodimer.

[0097] Provided herein are multispecific polypeptide constructs produced by any of the provided methods.

[0098] Provided herein are methods for stimulating or inducing an immune response, comprising contacting target cells and T cells with any of the provided multispecific polypeptide constructs or pharmaceutical compositions, wherein the target cells express a tumor-associated antigen recognized by the multispecific polypeptide construct. In some embodiments, the target cells are tumor cells that express a tumor-associated antigen (TAA).

[0099] In some embodiments, the contacting is performed ex vivo or in vitro. In some embodiments, the contacting is performed in vivo in a subject.

[0100] Methods are provided for stimulating or inducing an immune response in a subject, comprising administering a therapeutically effective amount of any of the provided multispecific conjugates or pharmaceutical compositions to a subject in need thereof. In some cases, the methods increase cellular immunity. In some embodiments, the methods increase T cell activity. In some embodiments, the methods increase cytolytic T cell (CTL) activity. In some examples, the immune response against tumors or cancer is increased. In some embodiments, the methods treat a disease or condition in the subject.

[0101] The present disclosure also provides methods for treating, preventing, slowing the progression of, or otherwise ameliorating the symptoms of, one or more pathologies, or alleviating the symptoms associated with such pathologies, by administering a multispecific polypeptide construct of the present disclosure to a subject in whom such treatment or prevention is desired. Provided herein are methods for treating a disease or condition in a subject, comprising administering a therapeutically effective amount of any of the provided multispecific conjugates or pharmaceutical compositions to a subject in need thereof. In some embodiments, the disease or condition is tumor or cancer.

[0102] In some embodiments of any of the provided methods, the subject, such as the subject being treated, is, for example, a human or other mammal. In some embodiments of any of the provided methods, the subject is a human. In some embodiments, the subject is a non-human mammal, such as a non-human primate, a pet (e.g., a cat, dog, horse), livestock, a laboratory animal, or a zoo animal. In some embodiments, the subject is a rodent.

[0103] The multispecific polypeptide constructs of the present disclosure used in any of these methods and embodiments of use can be administered at any stage of the disease. For example, such multispecific polypeptide constructs can be administered to patients with cancer at any stage from early to metastatic. The terms subject and patient are used interchangeably herein.

[0104] The multispecific polypeptide constructs of the present disclosure used in any of these methods and embodiments of use may be used in treatment regimens including neoadjuvant therapy.

[0105] The multispecific polypeptide constructs of the present disclosure used in any of these methods and use embodiments may be administered alone or in combination with one or more additional agents, including small molecule inhibitors, other antibody-based therapies, polypeptide- or peptide-based therapies, nucleic acid-based therapies, and / or other biologics. In some embodiments, the multispecific polypeptide constructs are administered in combination with one or more additional agents, such as chemotherapeutic agents, including, but not limited to, alkylating agents, antimetabolites, anti-microtubule agents, topoisomerase inhibitors, cytotoxic antibiotics, and other nucleic acid damaging agents. In some embodiments, the additional agent is a taxane, such as paclitaxel (e.g., Abraxane®). In some embodiments, the additional agent is an antimetabolite, such as gemcitabine. In some embodiments, the additional agent is an alkylating agent, such as a platinum-based chemotherapy, such as carboplatin or cisplatin. In some embodiments, the additional agent is a targeted agent, such as a kinase inhibitor, e.g., sorafenib or erlotinib. In some embodiments, the additional agent is a targeting agent such as another antibody, e.g., a monoclonal antibody (e.g., bevacizumab), a bispecific antibody, or a multispecific antibody. In some embodiments, the additional agent is a proteasome inhibitor such as bortezomib or carfilzomib. In some embodiments, the additional agent is an immunomodulatory agent such as lenalidomide or IL-2. In some embodiments, the additional agent is radiation. In some embodiments, the additional agent is an agent considered standard of care by those of skill in the art. In some embodiments, the additional agent is a chemotherapeutic agent well known to those of skill in the art. In some embodiments, the multispecific polypeptide construct and the additional agent are formulated into a single composition. In some embodiments, the multispecific polypeptide construct and the additional agent are administered as two or more separate compositions. In some embodiments, the multispecific polypeptide construct and the additional agent are administered simultaneously.In some embodiments, the multispecific polypeptide construct and the additional agent are administered sequentially.

[0106] In some embodiments, the additional agent is a chemotherapeutic agent such as a chemotherapeutic agent selected from the group consisting of docetaxel, paclitaxel, abraxane (i.e., paclitaxel conjugated to albumin), doxorubicin, oxaliplatin, carboplatin, cisplatin, irinotecan, and gemcitabine.

[0107] In some embodiments, the additional agent is a checkpoint inhibitor, a kinase inhibitor, an agent targeting an inhibitor of the tumor microenvironment, and / or a T cell or NK agonist. In some embodiments, the additional agent is radiation therapy, alone or in combination with another additional agent, such as a chemotherapeutic or anti-tumor agent. In some embodiments, the additional agent is a vaccine, an oncovirus, and / or a DC activator, such as, by way of non-limiting example, a Toll-like receptor (TLR) agonist and / or αCD40. In some embodiments, the additional agent is a tumor-targeting antibody designed to kill tumors via ADCC or via direct conjugation with a toxin (e.g., an antibody-drug conjugate (ADC)).

[0108] In some embodiments, the checkpoint inhibitor is an inhibitor of a target selected from the group consisting of CTLA-4, LAG-3, PD-1, PDL1, TIGIT, TIM-3, B7H3, B7H4, and Vista. In some embodiments, the kinase inhibitor is selected from the group consisting of B-RAFi, MEKi, and a Btk inhibitor such as ibrutinib. In some embodiments, the kinase inhibitor is crizotinib. In some embodiments, the tumor microenvironment inhibitor is selected from the group consisting of an IDO inhibitor, an αCSF1R inhibitor, an αCCR4 inhibitor, TGFβ, a myeloid-derived suppressor cell, or a T regulatory cell. In some embodiments, the agonist is selected from the group consisting of OX40, GITR, CD137, CD28, ICOS, CD27, and HVEM. In some embodiments, the checkpoint inhibitor is an antibody that binds to a target selected from CTLA-4, PD-1, and / or PD-L1. In some embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody, an anti-PD-1 antibody, and an anti-PD-L1 antibody, and / or a combination thereof. In some embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody, such as, for example, Yervoy™. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody, such as, for example, Opdivo™ and / or Keytruda™.

[0109] In some embodiments, the inhibitor is a CTLA-4 inhibitor. In some embodiments, the inhibitor is a LAG-3 inhibitor. In some embodiments, the inhibitor is a PD-1 inhibitor. In some embodiments, the inhibitor is a PDL1 inhibitor. In some embodiments, the inhibitor is a TIGIT inhibitor. In some embodiments, the inhibitor is a TIM-3 inhibitor. In some embodiments, the inhibitor is a B7H3 inhibitor. In some embodiments, the inhibitor is a B7H4 inhibitor. In some embodiments, the inhibitor is a Vista inhibitor. In some embodiments, the inhibitor is a B-RAFi inhibitor. In some embodiments, the inhibitor is a MEKi inhibitor. In some embodiments, the inhibitor is a Btk inhibitor. In some embodiments, the inhibitor is ibrutinib. In some embodiments, the inhibitor is crizotinib. In some embodiments, the inhibitor is an IDO inhibitor. In some embodiments, the inhibitor is an αCSF1R inhibitor. In some embodiments, the inhibitor is an αCCR4 inhibitor. In some embodiments, the inhibitor is TGFβ. In some embodiments, the inhibitor is a myeloid-derived suppressor cell. In some embodiments, the inhibitor is a T regulatory cell.

[0110] In some embodiments, the agonist is OX40. In some embodiments, the agonist is GITR. In some embodiments, the agonist is CD137. In some embodiments, the agonist is CD28. In some embodiments, the agonist is ICOS. In some embodiments, the agonist is CD27. In some embodiments, the agonist is HVEM.

[0111] In some embodiments, the multispecific polypeptide construct is administered in combination with one or more additional agents, such as, for example, chemotherapeutic agents, anti-inflammatory agents, and / or immunosuppressants, during and / or after treatment. In some embodiments, the multispecific polypeptide construct and the additional agent are formulated into a single therapeutic composition, and the multispecific polypeptide construct and the additional agent are administered simultaneously. Alternatively, the multispecific polypeptide construct and the additional agent are separate from each other, e.g., each is formulated into a separate therapeutic composition, and the multispecific polypeptide construct and the additional agent are administered simultaneously, or the multispecific polypeptide construct and the additional agent are administered at different times during the treatment regimen. For example, the multispecific polypeptide construct is administered before the administration of the additional agent, the multispecific polypeptide construct is administered after the administration of the additional agent, or the multispecific polypeptide construct and the additional agent are administered alternately. As described herein, the multispecific polypeptide construct and the additional agent are administered once or multiple times.

[0112] In some embodiments, the multispecific polypeptide construct and the additional agent are administered simultaneously. For example, the multispecific polypeptide construct and the additional agent may be formulated into a single composition or may be administered as two or more separate compositions. In some embodiments, the multispecific polypeptide construct and the additional agent are administered sequentially, or the multispecific polypeptide construct and the additional agent are administered at different times during a treatment regimen.

[0113] In addition to the above elements, the multispecific polypeptide construct may contain additional elements, such as an amino acid sequence at the N- or C-terminus of the multispecific polypeptide construct. For example, the multispecific polypeptide construct may include a targeting moiety that facilitates delivery to a cell or tissue of interest. The multispecific polypeptide construct may be conjugated to an agent such as a therapeutic agent, a detectable moiety, or a diagnostic agent. Examples of agents are disclosed herein.

[0114] The multispecific polypeptide construct may also include a multispecific polypeptide construct of the present disclosure along with a conjugated agent, linker, and any of the other components described herein.

[0115] The present disclosure also relates to immunoconjugates comprising multispecific polypeptide constructs conjugated to a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin, or fragment thereof, derived from bacteria, fungi, plants, or animals), or a radioisotope (i.e., a radioconjugate). Suitable cytotoxic agents for use in targeting diseased T cells, such as in T cell-derived lymphomas, include, for example, dolastatin and its derivatives (e.g., auristatin E, AFP, MMAD, MMAF, MMAE). In some embodiments, the agent is a dolastatin. In some embodiments, the agent is an auristatin or a derivative thereof. In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine.

[0116] In some embodiments, the linker between the multispecific polypeptide construct and the cytotoxic agent is cleavable. In some embodiments, the linker is non-cleavable. In some embodiments, there are two or more linkers. The two or more linkers are all the same, e.g., cleavable or non-cleavable, or the two or more linkers are different, e.g., at least one is cleavable and at least one is non-cleavable.

[0117] The multispecific polypeptide constructs and conjugates thereof are useful in methods for treating a variety of disorders and / or diseases. Non-limiting examples of diseases include all types of cancer (breast cancer, lung cancer, colorectal cancer, prostate cancer, melanoma, head and neck cancer, pancreatic cancer, etc.), rheumatoid arthritis, Crohn's disease, SLE, cardiovascular disorders, ischemia, etc. For example, indications may include leukemias, including T-cell acute lymphoblastic leukemia (T-ALL), lymphoblastic diseases, including multiple myeloma, and solid tumors, including lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, and breast cancer, including triple-negative breast cancer. For example, indications include bone disease or metastasis of cancer regardless of primary tumor origin; breast cancer, including, but not limited to, ER / PR+ breast cancer, Her2+ breast cancer, and triple-negative breast cancer; colorectal cancer; endometrial cancer; gastric cancer; glioblastoma; head and neck cancer, such as esophageal cancer; lung cancer, including, but not limited to, non-small cell lung cancer; multiple myeloma; ovarian cancer; pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; kidney cancer, including, but not limited to, renal cell carcinoma; and / or skin cancer, including, but not limited to, squamous cell carcinoma, basal cell carcinoma, or melanoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is cutaneous squamous cell carcinoma. In some embodiments, the cancer is esophageal squamous cell carcinoma. In some embodiments, the cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is lung squamous cell carcinoma.

[0118] Pharmaceutical compositions are provided that include any of the multispecific polypeptide constructs provided herein and a pharmaceutically acceptable carrier. In some cases, the pharmaceutical composition is sterile. Pharmaceutical compositions according to the present disclosure may include a multispecific polypeptide construct of the present disclosure and a carrier. These pharmaceutical compositions may be included in a kit, such as a diagnostic kit.

[0119] Those skilled 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 can also be used as reagents or diagnostic tools in diagnostic kits, or they can be used in competitive assays to generate therapeutic reagents. [The present invention 1001] 1. A multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, the antigen-binding fragment, wherein the CD3-binding region is an anti-CD3 antibody or an Fv antibody fragment comprising a variable heavy chain region (VH) and a variable light chain region (VL); the Fc is a heterodimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide, wherein the VH and VL of the anti-CD3 antibody or antigen-binding fragment are linked to opposing polypeptides of the heterodimeric Fc; the first and second components are coupled by a non-cleavable linker, and the Fc region is positioned N-terminal to the CD3 binding region; and 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); Multispecific polypeptide constructs. [The present invention 1002] 1001. A multispecific polypeptide construct of the present invention, wherein the CD3 binding region binds to CD3 (CD3ε). [The present invention 1003] 1001 or 1002. The multispecific polypeptide construct of invention 1001 or 1002, 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. [The present invention 1004] From the N-terminus to the C-terminus, a first antigen-binding domain that binds to a tumor-associated antigen (TAA); immunoglobulin Fc region; non-cleavable linkers; a CD3-binding region that binds to CD3 (CD3ε); and a second antigen-binding domain that binds to a tumor-associated antigen (TAA) The multispecific polypeptide construct of any of claims 1001 to 1003, comprising: [The present invention 1005] 1. A multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, an antigen-binding fragment in which the CD3-binding region is an anti-CD3 antibody or a disulfide-stabilized Fv antibody fragment (dsFv) comprising a variable heavy chain (VH) and a variable light chain (VL); the Fc is a heterodimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide, wherein the VH and VL of the anti-CD3 antibody or antigen-binding fragment are linked to opposing polypeptides of the heterodimeric Fc; the first and second components are coupled by a non-cleavable linker, and the Fc region is positioned N-terminal to the CD3 binding region; and One or both of the first and second components comprises an antigen-binding domain that binds to a tumor-associated antigen (TAA); Multispecific polypeptide constructs. [The present invention 1006] 1. A multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, the antigen-binding fragment, wherein the CD3-binding region is an anti-CD3 antibody or an Fv antibody fragment comprising a variable heavy chain (VH) and a variable light chain (VL); the Fc is a heterodimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide, wherein the VH and VL of the anti-CD3 antibody or antigen-binding fragment are linked to opposing polypeptides of the heterodimeric Fc; the first and second components are coupled by a non-cleavable linker, and the Fc region is positioned N-terminal to the CD3 binding region; and one or both of the first and second components comprises an antigen-binding domain that binds to a tumor-associated antigen (TAA), and the antigen-binding domain is a single-chain antibody fragment; Multispecific polypeptide constructs. [The present invention 1007] 1006. The multispecific polypeptide construct of the present invention, wherein the single-chain antibody fragment is a single-domain antibody or a single-chain variable fragment (scFv). [The present invention 1008] 1008. The multispecific polypeptide construct of any of claims 1005 to 1007, wherein the CD3 binding region binds to CD3 (CD3ε). [The present invention 1009] From the N-terminus to the C-terminus, a first antigen-binding domain that binds to a tumor-associated antigen (TAA); immunoglobulin Fc region; non-cleavable linkers; a CD3-binding region that binds to CD3 (CD3ε); and a second antigen-binding domain that binds to a tumor-associated antigen (TAA) 9. The multispecific polypeptide construct of any one of claims 1005 to 1008, comprising: [The present invention 1010] From the N-terminus to the C-terminus, immunoglobulin Fc region; non-cleavable linkers; a CD3-binding region that binds to CD3 (CD3ε); and An antigen-binding domain that binds to a tumor-associated antigen (TAA) 9. The multispecific polypeptide construct of any one of claims 1005 to 1008, comprising: [The present invention 1011] From the N-terminus to the C-terminus, an antigen-binding domain that binds to a tumor-associated antigen (TAA); immunoglobulin Fc region; a non-cleavable linker; and CD3 binding domain that binds to CD3 (CD3ε) 9. The multispecific polypeptide construct of any one of claims 1005 to 1008, comprising: [The present invention 1012] 10. The multispecific polypeptide construct of any of claims 1001 to 1011, wherein one or both of the first and second Fc polypeptides of the heterodimeric Fc region are variant Fc polypeptides comprising at least one modification to induce heterodimerization compared to the Fc region of human IgG1, human IgG2, or human IgG4. [The present invention 1013] 10. The multispecific polypeptide construct of any of claims 1001 to 1012, wherein one or both of the first and second Fc polypeptides of the heterodimeric Fc region are variant Fc polypeptides comprising at least one modification to induce heterodimerization compared to the Fc region of human IgG1, and optionally compared to the Fc polypeptide set forth in SEQ ID NO: 1 or an immunologically active fragment thereof. [The present invention 1014] The multispecific polypeptide construct of invention 1012 or invention 1013, wherein each of the Fc polypeptides of the heterodimeric Fc independently comprises at least one amino acid modification. [The present invention 1015] 10. The multispecific polypeptide construct of any of claims 1012 to 1014, wherein at least one modification is selected from a steric modification, a knob-into-hole modification, a charge mutation to increase the electrostatic complementarity of the polypeptide, a modification to alter the isoelectric point (pI variant), or a combination thereof. [The present invention 1016] 1016. The multispecific polypeptide construct of any of claims 1001 to 1015, wherein a first Fc polypeptide of the heterodimeric Fc comprises a modification selected from Thr366Ser, Leu368Ala, Tyr407Val, and a combination thereof, and a second Fc polypeptide of the heterodimeric Fc comprises the modification T366W. [The present invention 1017] 1016. The multispecific polypeptide construct of the invention, wherein the first and second Fc polypeptides further comprise a modification of a non-cysteine ​​residue to a cysteine ​​residue, wherein the modification in the first polypeptide is at one of positions Ser354 and Y349 and the modification in the second Fc polypeptide is at the other of positions Ser354 and Y349. [The present invention 1018] 10. The multispecific polypeptide construct of any of claims 1001 to 1017, wherein the first Fc polypeptide comprises the modifications T366W / S354C and the second Fc polypeptide comprises the modifications T366S / L368A / Y407V / Y349C. [The present invention 1019] 1016. The multispecific polypeptide construct of any of claims 1001 to 1015, wherein the first Fc polypeptide comprises the modifications L368D / K370S and the second Fc polypeptide comprises the modifications S364K / E357Q. [The present invention 1020] 1016. The multispecific polypeptide construct of any of claims 1001 to 1015, wherein at least one of the first and second polypeptides comprises the modifications Q295E / N384D / Q418E / N421D. [The present invention 1021] one of the first or second Fc polypeptides of the heterodimeric Fc further comprises a modification at residue Ile253, optionally, the modification is Ile253Arg; and / or one of the first or second Fc polypeptides of the heterodimeric Fc further comprises a modification at residue His435, optionally wherein the modification is His35Arg; A multispecific polypeptide construct according to any one of claims 1001 to 1020. [The present invention 1022] 1022. The multispecific polypeptide construct of any of claims 1001 to 1021, wherein one or both of the first and second Fc polypeptides comprises a polypeptide lacking Lys447. [The present invention 1023] The multispecific polypeptide construct of any of claims 1001 to 1022, wherein the first polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NOs: 82, 86, or 201, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NOs: 83, 87, 90, 92, 202, or 205. [The present invention 1024] The multispecific polypeptide construct of any of claims 1001 to 1023, wherein the first Fc polypeptide and the second Fc polypeptide comprise a sequence selected from the group consisting of SEQ ID NOs: 82 and 83, respectively; SEQ ID NOs: 86 and 87, respectively; SEQ ID NOs: 201 and 202, respectively; SEQ ID NOs: 82 and 90, respectively; SEQ ID NOs: 86 and 92, respectively; and SEQ ID NOs: 201 and 205, respectively. [The present invention 1025] 10. The multispecific polypeptide construct of any of claims 1001 to 1022, wherein the Fc region comprises a polypeptide comprising at least one modification to enhance FcRn binding. [The present invention 1026] 1025. The multispecific fusion polypeptide construct of the invention, wherein the modification is at a position selected from the group consisting of Met252Y, Ser254T, Thr256E, Met428L, Met428V, Asn434S, and combinations thereof. [The present invention 1027] 1027. The multispecific fusion polypeptide construct of claim 1025 or 1026, wherein the modifications are Met252Y and Met428L or Met252Y and Met428V. [The present invention 1028] The multispecific polypeptide construct of any of claims 1001 to 1022 and 1025 to 1027, wherein the first polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NOs: 94, 96, or 207, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NOs: 98, 100, or 209. [The present invention 1029] 1028. The multispecific polypeptide construct of any of claims 1001 to 1028, wherein the first Fc polypeptide and the second Fc polypeptide comprise a sequence selected from the group consisting of SEQ ID NOs: 94 and 98, respectively; SEQ ID NOs: 96 and 100, respectively; and SEQ ID NOs: 207 and 209, respectively. [The present invention 1030] The Fc region Polypeptides comprising at least one amino acid modification that reduces effector function and / or reduces binding to an effector molecule selected from an Fcγ receptor or C1q The multispecific polypeptide construct of any of claims 1001 to 1022 and 1025 to 1027, comprising: [The present invention 1031] 1030. The multispecific polypeptide construct of the invention, wherein the one or more amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235. [The present invention 1032] The multispecific polypeptide construct of any of claims 1001 to 1022, 1025 to 1027, 1030, and 1031, wherein the first polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NOs: 84, 88, 95, 97, 203, or 208, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NOs: 85, 89, 91, 93, 99, 101, 204, 206, or 210. [The present invention 1033] The multispecific polypeptide construct of any of claims 1001 to 1022, 1025 to 1027, and 1030 to 1032, wherein the first Fc polypeptide and the second Fc polypeptide comprise a sequence selected from the group consisting of SEQ ID NOs: 84 and 85, respectively; SEQ ID NOs: 88 and 89, respectively; SEQ ID NOs: 203 and 204, respectively; SEQ ID NOs: 95 and 99, respectively; SEQ ID NOs: 97 and 101, respectively; SEQ ID NOs: 208 and 210, respectively; SEQ ID NOs: 84 and 91, respectively; SEQ ID NOs: 88 and 93, respectively; and SEQ ID NOs: 203 and 206, respectively. [The present invention 1034] 4. The multispecific polypeptide construct of any of claims 1001 to 1033, wherein the CD3 binding region is incapable or substantially incapable of binding to or engaging with CD3 unless at least one of the antigen binding domains is bound to its TAA. [This invention 1035] 4. The multispecific polypeptide construct of any of claims 1001 to 1033, wherein the CD3 binding region is incapable or substantially incapable of binding to or engaging with CD3 unless at least two of the antigen binding domains are bound to a TAA. [The present invention 1036] 1036. The multispecific polypeptide construct of any of claims 1001 to 1035, wherein the linker is a polypeptide linker. [This invention 1037] 1036. The multispecific polypeptide construct of the invention, wherein the linker is a polypeptide of up to 25 amino acids in length. [The present invention 1038] The linker may be 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, 1 A polypeptide of 0 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 a polypeptide of about 2 to 24 amino acids, about 2 to 20 amino acids, about 2 to 18 amino acids, about 2 to 14 amino acids, about 2 to 12 amino acids, about 2 to 10 amino acids, about 2 to 8 amino acids, about 2 to 6 amino acids, about 6 to 24 amino acids, about 6 to 20 amino acids, about 6 to 18 amino acids, about 6 to 14 amino acids, about 6 to 12 amino acids, about 6 to 10 amino acids, about 6 to 8 amino acids, about 8 to 24 amino acids, about 8 to 20 amino acids, about 8 to 18 amino acids, about 8 to 14 amino acids, about 8 to 12 amino acids, about 8 to 10 amino acids, about 10 to 24 amino acids, about 10 to 20 amino acids The multispecific polypeptide construct of the present invention 1036 or 1037, which is a polypeptide of about 10 to 18 amino acids, about 10 to 14 amino acids, about 10 to 12 amino acids, about 12 to 24 amino acids, about 12 to 20 amino acids, about 12 to 18 amino acids, about 12 to 14 amino acids, about 14 to 24 amino acids, about 14 to 20 amino acids, about 14 to 18 amino acids, about 18 to 24 amino acids, about 18 to 20 amino acids, or about 20 to 24 amino acids. [This invention 1039] 9. The multispecific polypeptide construct of any one of claims 1036 to 1038, 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. [The present invention 1040] 1039. The multispecific polypeptide construct of any of claims 1036 to 1039, wherein the linker is a polypeptide having a length of 3 to 18 amino acids. [This invention 1041] The multispecific polypeptide construct of any of claims 1036 to 1040, wherein the linker is a polypeptide having a length of 12 to 18 amino acids. [The present invention 1042] The multispecific polypeptide construct of any of claims 1036 to 1040, wherein the linker is a polypeptide having a length of 15 to 18 amino acids. [This invention 1043] The non-cleavable linker A multispecific polypeptide construct of any of claims 1001 to 1042, including TIFF0007824455000015.tif4128 and combinations thereof. [This invention 1044] 1044. The multispecific polypeptide construct of any of claims 1001 to 1043, wherein the non-cleavable linker comprises (GGS)n, where n is 1 to 10. [This invention 1045] 1046. The multispecific polypeptide construct of any of claims 1001 to 1044, wherein the non-cleavable linker comprises (GGGGS)n (SEQ ID NO:173), wherein n is 1 to 10. [The present invention 1046] 104. The multispecific polypeptide construct of any of claims 1001 to 1044, wherein the non-cleavable linker comprises (GGGGGS)n (SEQ ID NO:172), wherein n is 1 to 4. [This invention 1047] The multispecific polypeptide construct of any of claims 1001 to 1044, wherein the non-cleavable linker is or comprises GGS. [This invention 1048] The multispecific polypeptide construct of any of claims 1001 to 1043 and 1045, wherein the non-cleavable linker is or comprises GGGGS (SEQ ID NO: 149). [This invention 1049] The multispecific polypeptide construct of any of claims 1001 to 1043 and 1046, wherein the non-cleavable linker is or comprises GGGGGS (SEQ ID NO: 135). [The present invention 1050] The multispecific polypeptide construct of any of claims 1001 to 1044, wherein the non-cleavable linker is or comprises (GGS)2 (SEQ ID NO:10). [This invention 1051] The multispecific polypeptide construct of any of claims 1001 to 1044, wherein the non-cleavable linker is or comprises GGSGGSGGS (SEQ ID NO: 11). [This invention 1052] The multispecific polypeptide construct of any of claims 1001 to 1044, wherein the non-cleavable linker is or comprises GGSGGSGGSGGS (SEQ ID NO: 12). [This invention 1053] The non-cleavable linker is A multispecific polypeptide construct of any of claims 1001 to 1044 of the present invention, which is or comprises TIFF0007824455000016.tif4128. [This invention 1054] The non-cleavable linker is A multispecific polypeptide construct of any of the present inventions 1001 to 1043 and 1046, which is or comprises TIFF0007824455000017.tif4128. [This invention 1055] The non-cleavable linker is A multispecific polypeptide construct of any of claims 1001 to 1049 of the present invention, which is or comprises TIFF0007824455000018.tif4128. [This invention 1056] The non-cleavable linker is A multispecific polypeptide construct of any of the present inventions 1001 to 1043 and 1045, which is or comprises TIFF0007824455000019.tif4128. [This invention 1057] (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 thereof; and (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 thereof; 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); A multispecific polypeptide construct according to any one of claims 1001 to 1056. [This invention 1058] 10. The multispecific polypeptide construct of any of claims 1001 to 1057, wherein the VH of the anti-CD3 antibody or antigen-binding fragment is on the same polypeptide as at least one antigen-binding domain that binds to a tumor-associated antigen (TAA). [This invention 1059] 1058. The multispecific polypeptide construct of the present invention, wherein the polypeptide comprising the VL of the anti-CD3 antibody or antigen-binding fragment does not contain at least one antigen-binding domain that binds to a tumor-associated antigen (TAA). [The present invention 1060] The multispecific polypeptide construct of any of claims 1001 to 1004, 1009, and 1012 to 1059, which exhibits bivalent, trivalent, or tetravalent binding to a TAA. [This invention 1061] 10. The multispecific polypeptide construct of any of claims 1005 to 1008 and 1010 to 1059, which exhibits monovalent, divalent, trivalent, or tetravalent binding to a TAA. [This invention 1062] 1062. The multispecific polypeptide construct of any of claims 1001 to 1061, wherein only one of the first and second polypeptides comprises at least one antigen-binding domain that binds to a TAA. [This invention 1063] 1063. The multispecific polypeptide construct of any of claims 1001 to 1062, wherein 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 is positioned carboxy-terminal to a CD3-binding region of one of the first or second polypeptides of the multispecific polypeptide construct. [The present invention 1064] 1063. The multispecific polypeptide construct of any of claims 1001 to 1062, wherein 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. [This invention 1065] 10. The multispecific polypeptide construct of any of claims 1001 to 1064, wherein 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 a variant thereof that exhibits binding activity with the TAA. [The present invention 1066] The multispecific polypeptide construct of any of claims 1001 to 1005 and 1008 to 1065, wherein the antigen-binding domain, or each of the 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, an scFv, an scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. [This invention 1067] 1066. The multispecific polypeptide construct of the present invention, wherein the antibody or antigen-binding fragment thereof is an Fv, scFv, Fab, or single domain antibody (sdAb). [The present invention 1068] The multispecific polypeptide construct of any of claims 1001 to 1067, wherein the antibody or antigen-binding fragment thereof is an sdAb. [This invention 1069] 1068. The multispecific polypeptide construct of the invention, wherein the sdAb is a human sdAb or a humanized sdAb. [The present invention 1070] The multispecific polypeptide construct of invention 1068 or invention 1069, wherein the sdAb is a VHH, a VNAR, a modified VH domain, or a modified VK domain. [This invention 1071] The multispecific polypeptide construct of any of claims 1001 to 1067, wherein the antibody or antigen-binding fragment thereof is an scFv. [This invention 1072] The multispecific polypeptide construct of any of claims 1001 to 1005 and 1008 to 1067, wherein the antibody or antigen-binding fragment thereof is a Fab. [This invention 1073] (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 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; A multispecific polypeptide construct of the present invention 1072. [This invention 1074] 1073. A multispecific polypeptide construct of the invention, wherein only one of the first and second polypeptides comprises the other of said VH-CH1(Fd) or VL-CL of a Fab antibody fragment. [This invention 1075] 1073. The multispecific polypeptide construct of the present invention, wherein both the first and second polypeptides comprise the other of said VH-CH1(Fd) or VL-CL of a Fab antibody fragment. [This invention 1076] The multispecific polypeptide construct of invention 1074 or invention 1075, 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 one of the first and second polypeptides of the multispecific polypeptide construct, and / or carboxy-terminal to the CD3 binding region of one of the first and second polypeptides of the multispecific polypeptide construct. [This invention 1077] 1076. The multispecific polypeptide construct of any of claims 1074 to 1076, 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. [This invention 1078] The antigen-binding domain, or each antigen-binding domain independently, may be: 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, 10. The multispecific polypeptide construct of any of claims 1001 to 1077, 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. [This invention 1079] comprising at least a first antigen-binding domain and a second antigen-binding domain; the first antigen-binding domain and the second antigen-binding domain bind to the same TAA; A multispecific polypeptide construct according to any one of claims 1001 to 1078. [The present invention 1080] 1079. The multispecific polypeptide construct of the present invention, wherein the first antigen-binding domain and the second antigen-binding domain bind to different epitopes of the same TAA. [This invention 1081] 1079. The multispecific polypeptide construct of the present invention, wherein the first antigen-binding domain and the second antigen-binding domain bind to the same epitope of the same TAA. [This invention 1082] comprising at least a first antigen-binding domain and a second antigen-binding domain; the first antigen-binding domain and the second antigen-binding domain bind to different TAAs; A multispecific polypeptide construct according to any one of claims 1001 to 1078. [This invention 1083] The anti-CD3 antibody or antigen-binding fragment thereof VH CDR1 comprising the amino acid sequence GFTFNTYAMN (SEQ ID NO:211); VH CD2 comprising the amino acid sequence RIRSKYNNYATY (SEQ ID NO:212); Amino acid sequence VH CDR3 containing TIFF0007824455000020.tif4128; Amino acid sequence VL CDR1 containing TIFF0007824455000021.tif4128; VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO:20); and VL CDR3 containing the amino acid sequence ALWYSNLWV (SEQ ID NO:21) Contains; or The anti-CD3 antibody or antigen-binding fragment is a VH CDR1 sequence comprising at least the amino acid sequence GFTFNTYAMN (SEQ ID NO:211); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATY (SEQ ID NO:212); Amino acid sequence VH CDR3 sequence containing at least TIFF0007824455000022.tif4128; Amino acid sequence VL CDR1 sequence containing at least TIFF0007824455000023.tif4128; a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO:230); and VL CDR3 sequence containing at least the amino acid sequence ALWYSNHWV (SEQ ID NO: 225) Including, A multispecific polypeptide construct according to any one of claims 1001 to 1082. [This invention 1084] Anti-CD3 Fv, a VH having the amino acid sequence of any of SEQ ID NOs: 14, 32-43, 45-47, 48, 196, and 211, 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: 14, 32-43, 45-47, 48, 196, and 211; and VL having an amino acid sequence of any of SEQ ID NOs: 15, 63, 65 to 71, 73, 75, 77, and 199, 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, 65 to 71, 73, 75, 77, and 199. A multispecific polypeptide construct according to any one of claims 1001 to 1004 and 1006 to 1083, comprising: [This invention 1085] The multispecific polypeptide construct of any of claims 1001 to 1004 and 1006 to 1084, 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. [The present invention 1086] The multispecific polypeptide construct of any of claims 1001 to 1004 and 1006 to 1084, wherein the anti-CD3 Fv comprises the amino acid sequence of SEQ ID NO:196 and the amino acid sequence of SEQ ID NO:199. [This invention 1087] The multispecific polypeptide construct of any of claims 1001 to 1004 and 1006 to 1083, wherein the Fv antibody fragment comprises a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv). [This invention 1088] The multispecific polypeptide construct of the invention 1005 or 1087, wherein the disulfide-stabilized anti-CD3 binding Fv fragment (dsFv) comprises a VH chain comprising the mutation G44C and a VL chain comprising the mutation G100C according to the Kabat numbering. [This invention 1089] Anti-CD3 Fv, a VH having the amino acid sequence of any of SEQ ID NOs: 44, 49-62, 197, and 198, 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: 44, 49-62, 197, and 198; and a VL having an amino acid sequence of any of SEQ ID NOs: 64, 72, 74, 76, 78-81, 191, 200, and 212, 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: 64, 72, 74, 76, 78-81, 191, 200, and 212; The multispecific polypeptide construct of any of claims 1005, 1087 and 1088, comprising: [The present invention 1090] The multispecific polypeptide construct of any of claims 1005 and 1087 to 1089, 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. [This invention 1091] A multispecific polypeptide construct of any of claims 1005 and 1087 to 1089, wherein the anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO:198 and the amino acid sequence of SEQ ID NO:200, or the amino acid sequence of SEQ ID NO:197 and the amino acid sequence of SEQ ID NO:200. [This invention 1092] 10. The multispecific polypeptide construct of any one of claims 1001 to 1091, conjugated to a drug. [This invention 1093] The multispecific polypeptide construct of the present invention 1092, wherein the agent is a therapeutic agent, an anti-tumor agent, a toxin or fragment thereof, a detectable moiety, or a diagnostic agent. [This invention 1094] The multispecific polypeptide construct of the present invention 1093, wherein the drug is conjugated to the multispecific polypeptide construct via a linker. [This invention 1095] A polynucleotide encoding any one of the multispecific polypeptide constructs of the present inventions 1001 to 1094. [This invention 1096] A polynucleotide encoding any one of the polypeptide chains of the multispecific polypeptide constructs of the present invention. [This invention 1097] A polynucleotide comprising a first nucleic acid sequence encoding a first polypeptide of the multispecific polypeptide construct of any one of claims 1001 to 1094 and a second nucleic acid sequence encoding a second polypeptide of the multispecific polypeptide 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. [This invention 1098] The polynucleotide of claim 1097, wherein the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter. [This invention 1099] The polynucleotide of claim 1097 or claim 1098, 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 polypeptide construct. [The present invention 1100] The polynucleotide of the present invention 1099, wherein 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 present invention 1101] The polynucleotide of any one of 1097 to 1100, wherein the nucleic acid encoding the self-cleaving peptide or the peptide that causes ribosome skipping is selected from T2A, P2A, E2A, and F2A. [The present invention 1102] A vector comprising any one of the polynucleotides 1095 to 1101 of the present invention. [The present invention 1103] The vector of the present invention 1102, which is an expression vector. [The present invention 1104] a viral vector or a eukaryotic vector, Optionally, the eukaryotic vector is a mammalian vector. The vector of the present invention 1102 or 1103. [This invention 1105] A cell comprising one or more polynucleotides according to any one of 1095 to 1101 of the present invention, or one or more vectors according to any one of 1102 to 1104 of the present invention. [The present invention 1106] The cell of the present invention 1105, which is recombinant or isolated. [This invention 1107] The cell of the present invention 1105 or 1106, which is a mammalian cell. [This invention 1108] The cell of the present invention 1107, which is a HEK293 cell or a CHO cell. [This invention 1109] A step of introducing one or more polynucleotides according to any one of 1095 to 1101 of the present invention or one or more vectors according to any one of 1102 to 1104 of the present invention into a cell; culturing said cells under conditions such that the multispecific polypeptide construct is produced. 1. A method for producing a multispecific polypeptide construct, comprising: [The present invention 1110] Culturing the cells of any of claims 1105 to 1108 under conditions in which the multispecific polypeptide is produced by said cells. 1. A method for producing a multispecific polypeptide construct, comprising: [The present invention 1111] The method of claim 1109 or claim 1110, further comprising the step of isolating or purifying the multispecific polypeptide construct from the cells. [The present invention 1112] 111. The method of any of claims 1109 to 1111, wherein the multispecific polypeptide construct is a heterodimer. [The present invention 1113] A multispecific polypeptide construct produced by any of the methods of the present invention 1109 to 1112. [This invention 1114] A pharmaceutical composition comprising any one of the multispecific polypeptide constructs of the present inventions 1001 to 1094 and 1113, and a pharmaceutically acceptable carrier. [This invention 1115] The pharmaceutical composition of the present invention 1114, which is sterile. [The present invention 1116] contacting target cells and T cells with any of the multispecific polypeptide constructs of the present inventions 1001 to 1094 or 1113, or the pharmaceutical composition of the present invention 1114 or 1115; the target cells express a tumor-associated antigen recognized by the multispecific polypeptide construct; A method of stimulating or inducing an immune response. [This invention 1117] The method of claim 1116, wherein the target cells are tumor cells expressing a tumor-associated antigen (TAA). [This invention 1118] The method of claim 1116 or claim 1117, wherein the contacting step is carried out ex vivo or in vitro. [This invention 1119] The method of any one of claims 1116 to 1118, wherein the contacting step is carried out in vivo in a subject. [The present invention 1120] Administering a therapeutically effective amount of any of the multispecific polypeptide constructs of the present inventions 1001 to 1094 or 1113 or the pharmaceutical composition of the present invention 1114 or 1115 to a subject in need thereof. 20. A method of stimulating or inducing an immune response in a subject, comprising: [This invention 1121] Any of the methods of the present inventions 1116 to 1120, which increase cellular immunity. [This invention 1122] Any of the methods of the present inventions 1116 to 1121, which increase T cell activity. [This invention 1123] The method of any one of claims 1116 to 1122, which increases cytolytic T cell (CTL) activity. [This invention 1124] The method of any of claims 1116 to 1123, wherein the immune response against the tumor or cancer is increased. [This invention 1125] The method of any of claims 1116 to 1124 for treating a disease or condition in a subject. [Invention 1126] Administering a therapeutically effective amount of any of the multispecific conjugates of the present inventions 1001 to 1094 or 1113, or the pharmaceutical composition of the present invention 1114 or 1115, to a subject in need thereof. 20. A method of treating a disease or condition in a subject, comprising: [This invention 1127] The method of claim 1125 or claim 1126, wherein the disease or condition is a tumor or cancer. [This invention 1128] The method of any one of claims 1119 to 1127, wherein the subject is a human. [Brief explanation of the drawings]

[0120] [Figure 1] Figure 1 is a schematic diagram of the basic components of a multispecific polypeptide construct of the present disclosure with constrained CD3 binding. The antigen-binding domain is located at the amino-terminus and / or carboxy-terminus. An Fc region, such as a heterodimeric Fc region, is located N-terminal to the CD3-binding region. This positioning of the Fc in close proximity to the CD3-binding region prevents CD3 binding. The linker can be a non-cleavable linker as provided herein. [Figure 2]Figures 2A and 2B are schematic diagrams of various constrained CD3 constructs targeting FRα, composed of two polypeptides, chain 1 and chain 2. As shown in Figure 2A, chain 1 contains an FRα sdAb linked to a heterodimeric Fc "hole" linked via a non-cleavable linker (ranging from 3 amino acids at cx1356 to 18 amino acids at cx681) to an anti-CD3 VL domain linked to a second FRα sdAb (antigen-binding domain); chain 2 contains an FRα sdAb linked to a complementary heterodimeric Fc "knob" linked via the same non-cleavable linker to an anti-CD3 VH domain linked to a second FRα sdAb. When co-expressed, the CD3-binding domains are properly assembled via VL:VH association at the hole and knob, respectively (Figure 2B). An exemplary anti-CD3 is a disulfide-stabilized Fv (dsFv) containing a variable light chain (VL) containing the mutation G100C and a variable heavy chain (VH) containing the mutation G44C. [Figure 3A]Figure 3A is a schematic diagram of various constrained CD3 constructs targeting B7H3, composed of two polypeptides, chain 1 and chain 2. Chain 1 contains either a heterodimeric Fc "hole" linked via a non-cleavable linker to an anti-CD3 VL domain modified at G100C (top), a B7H3-targeting sdAb linked to a heterodimeric Fc "hole" linked via a non-cleavable linker to an anti-CD3 VL domain (middle), or a B7H3-targeting sdAb linked to a heterodimeric Fc "hole" linked via a non-cleavable linker to an anti-CD3 VL domain modified at G100C (bottom). Chain 2 contains either a B7H3-targeting sdAb linked to a complementary heterodimeric Fc "knob" linked via a linker to an anti-CD3 VH domain modified at G44C, which is linked to a second B7H3 sdAb (top); a B7H3-targeting sdAb linked to a complementary heterodimeric Fc "knob" linked via a linker to an anti-CD3 VH domain (middle); or a B7H3-targeting sdAb linked to a complementary heterodimeric Fc "knob" linked via a linker to an anti-CD3 VH domain modified by G44C (bottom). When coexpressed, the CD3-binding domains assemble properly via VL:VH association at the hole and knob, respectively. Where indicated, the VH:VL interaction is stabilized by an engineered disulfide bond between modified residues G44C in the VH domain and G100C in the VL domain. [Figure 3B]Figure 3B is a schematic diagram of various constrained CD3 constructs targeting B7H3, composed of two polypeptides, chain 1 and chain 2. Chain 1 contains a heterodimeric Fc "hole" linked via a non-cleavable linker to an anti-CD3 VL domain modified at G100C, which is linked to an sdAb targeting a costimulatory receptor. Chain 2 contains either a B7H3-targeting sdAb linked to a complementary heterodimeric Fc "knob" linked via a linker to an anti-CD3 VH domain modified at G44C, which is linked to a second B7H3-targeting sdAb (top); a heterodimeric Fc "knob" linked via a linker to an anti-CD3 VH domain modified at G44C, which is linked to a B7H3-targeting sdAb (middle); or a B7H3-targeting sdAb linked to a complementary heterodimeric Fc "knob" linked via a linker to an anti-CD3 VH domain modified by G44C (bottom). When coexpressed, the CD3-binding domains are properly assembled via VL:VH association at the hole and knob, respectively. The VH:VL interaction is stabilized by an engineered disulfide bond between modified residues G44C in the VH domain and G100C in the VL domain. The resulting constructs engage B7H3 either bivalently (top) or monovalently (middle and bottom). All constructs here contain sdAbs that target costimulatory receptors. [Figure 3C]Figure 3C is a schematic diagram of various B7H3-targeting constrained CD3 constructs composed of three polypeptides, chain 1, chain 2, and chain 3, in which the B7H3 targeting domain is FAB. Chain 1 contains a BH73-targeting VH, IgG constant heavy chain 1 (CH1), linked via a hinge to a first member of a heterodimeric Fc (Fc-Het-1), which is linked via a linker to an anti-CD3 VL domain lacking (top) or containing (bottom) the G100C modification. Chain 2 contains a BH73-targeting VH, IgG constant heavy chain 1 (CH1), linked via a hinge to a second member of a heterodimeric Fc (Fc-Het-2), which is linked via a linker to an anti-CD3 VH domain lacking (top) or containing (bottom) the G44C modification. Chain 3 contains a complementary B7H3-targeting VL domain linked to a human Ig constant light (CL) region. When coexpressed, the CD3-binding domain properly assembles through VL:VH association in the complementary heterodimeric Fc region. Where shown, the VH:VL interaction is stabilized by an engineered disulfide bond between modified residues G44C in the VH domain and G100C in the VL domain. [Figure 4A]Figure 4A is a schematic diagram of various constrained CD3 constructs targeting DLL3, composed of two polypeptides, chain 1 and chain 2. Chain 1 contains a heterodimeric Fc "hole" linked via a non-cleavable linker to an anti-CD3 VL domain modified at G100C, which is linked to an sdAb targeting a costimulatory receptor. Chain 2 contains either a DLL3-targeting sdAb linked to a complementary heterodimeric Fc "knob" linked via a linker to an anti-CD3 VH domain modified at G44C, which is linked to a second DLL3-targeting sdAb (top); a heterodimeric Fc "knob" linked via a linker to an anti-CD3 VH domain modified at G44C, which is linked to a DLL3-targeting sdAb (middle); or a DLL3-targeting sdAb linked to a complementary heterodimeric Fc "knob" linked via a linker to an anti-CD3 VH domain modified by G44C (bottom). When coexpressed, the CD3-binding domains are properly assembled via VL:VH association at the hole and knob, respectively. The VH:VL interaction is stabilized by an engineered disulfide bond between modified residues G44C in the VH domain and G100C in the VL domain. The resulting constructs engage DLL3 either bivalently (top) or monovalently (middle and bottom). All constructs here contain sdAbs that target costimulatory receptors. [Figure 4B] Figure 4B is a schematic diagram of the DLL3-targeting constrained CD3 construct cx5499, composed of two polypeptides, chain 1 and chain 2. cx5499 is identical to cx5352 shown in Figure 4A (top), but lacks the costimulatory receptor-targeting sdAb at the C-terminus of chain 1. When coexpressed, the CD3-binding domains assemble properly through VL:VH association at the hole and knob, respectively. The VH:VL interaction is stabilized by an engineered disulfide bond between modified residues G44C in the VH domain and G100C in the VL domain. The resulting construct engages DLL3 either bivalently (top) or monovalently (middle and bottom). [Figure 5-1]Figures 5A-D illustrate cell binding by representative constrained CD3-engaging constructs cx1356 and cx681 targeting FRα. Figures 5A and 5C show binding to Ovcar5 cells (an FRα-positive ovarian cancer cell line). Figures 5B and 5D illustrate the lack of binding to T cells. Figures 5A and 5B show histograms of normalized cell number versus fluorescence at 100 nM for each construct. In Figures 5A and 5B, the secondary anti-human APC antibody-only control is shown as a solid black trace, the positive control anti-CD3 binding is shown as a white trace, and cx1356 and cx681 are shown as gray-shaded traces. A complete titration of each construct in various cell types is shown in Figures 5C and 5D. [Figure 5-2] See description of Figure 5-1. [Figure 6-1] Figures 6A-F illustrate cell binding by a representative restricted CD3-engaging construct targeting B7H3. Figures 6A, C, and E show binding to A375 cells (a B7H3-positive human melanoma cell line). Figures 6B, D, and F show the lack of binding to isolated T cells. [Figure 6-2] See description of Figure 6-1. [Figure 7-1] Figures 7A-F illustrate the effect of linker length on CD3 agonism in the presence of FRα-positive IGROV1 cells (Figures 7A, 7C, 7E) or FRα-negative NCI-H460 cells (Figures 7B, 7D, 7F). Figures 7A-B show the kinetics of CD3 signaling by various constructs at 2 nM in antigen-positive and antigen-negative cells, respectively. Figures 7C-D show the magnitude of CD3 agonism by various constructs at 2 nM in antigen-positive and antigen-negative cells, respectively. Figures 7E-F show the CD3 agonism potency of various constructs with different linker lengths in antigen-positive and antigen-negative cells, respectively. The Jurkat CD3 NFAT-GFP reporter cell line was used to assess CD3 signaling. The constrained CD3-binding protein effectively engages and clusters CD3 on T cells only upon binding to a second antigen on the target cells. [Figure 7-2]See description of Figure 7-1. [Figure 7-3] See description of Figure 7-1. [Figure 8] Figures 8A-D illustrate the ability of representative B7H3-targeted constrained CD3-engaging constructs to agonize CD3 in a target-dependent manner. Figures 8A and 8C illustrate their ability to mediate CD3 signaling in the presence of B7H3-positive A375 cells, while Figures 8B and 8D show their lack of ability to mediate CD3 signaling in the presence of B7H3-negative CCRF-CEM cells. The Jurkat CD3 NFAT-GFP reporter cell line was used to assess CD3 agonism. [Figure 9] Figure 9A illustrates the ability of a representative B7H3-targeting constrained CD3-engaging construct (cx3072) to induce T cell-mediated cytotoxicity in a target-dependent manner. Target cells were labeled with cytoID Red, and dead cells were visualized by adding caspase 3 / 7 green reagent. Cytotoxicity was assessed by determining the overlap area between red target cells and green dead cells. A B7H3-negative A375 cell line generated by CRISPR technology was used to test antigen-specific T cell-mediated cytotoxicity. cx3072 was unable to induce T cell-mediated cytotoxicity in these B7H3-deficient cells. Figure 9B shows that cx5952 can induce T cell-mediated cytotoxicity in the presence of B7H3-positive A375 cells, but not in the presence of B7H3-negative CCRF-CEM cells. [Figure 10]Figures 10A and 10B illustrate the ability of representative restricted CD3-engaged constructs targeting B7H3 to induce T cell-mediated cytotoxicity in a target-dependent manner. Figure 10A illustrates the ability of these constructs to induce T cell-mediated cytotoxicity in the presence of B7H3-positive A375 cells, and Figure 10B illustrates the ability of these constructs to induce T cell-mediated cytotoxicity in the presence of B7H3-negative CCRF-CEM cells. Cytotoxicity was assessed by determining the overlap area between red target cells and green dead cells. Figures 10C and 10D illustrate the ability of representative restricted CD3-engaged constructs targeting B7H3 to induce T cell-mediated cytotoxicity in a target-dependent manner. Figure 10C illustrates the ability of these constructs to induce T cell-mediated cytotoxicity in the presence of B7H3-positive A375 cells, and Figure 10D illustrates the ability of these constructs to induce T cell-mediated cytotoxicity in the presence of B7H3-negative CCRF-CEM cells. Cytotoxicity was assessed by determining the overlap area between red target cells and green dead cells. [Figure 11-1] Figures 11A-C illustrate the ability of a representative restricted CD3-engaging construct (cx5952) targeting B7H3 to induce T cell-mediated activation in a target-dependent manner. T cell activation of CD4+ or CD8+ T cells was assessed by expression of the T cell activation markers CD25 (Figure 11A), CD69 (Figure 11B), and CD71 (Figure 11C). Figures 11D-11K illustrate the ability of a representative restricted CD3-engaging construct targeting B7H3 to induce T cell activation in a target-dependent manner. B7H3 target-dependent CD4+ T cell activation is indicated by expression of the T cell activation markers CD25 (Figure 11D) and CD71 (Figure 11F). B7H3 target-dependent CD8+ T cell activation is indicated by expression of the T cell activation markers CD25 (Figure 11H) and CD71 (Figure 11J). T cell activation was not observed in the absence of B7H3-positive cells, based on the T cell activation marker CD25 expressed in CD4+ T cells (FIG. 11E) or CD8+ T cells (FIG. 11I), or based on the T cell activation marker CD71 expressed in CD4+ T cells (FIG. 11G) or CD8+ T cells (FIG. 11K). [Figure 11-2]See description of Figure 11-1. [Figure 11-3] See description of Figure 11-1. [Figure 11-4] See description of Figure 11-1. [Figure 11-5] See description of Figure 11-1. [Figure 12A] Figure 12A illustrates the ability of representative constrained CD3-engaging constructs targeting B7H3 to induce IFNγ production in a target-dependent manner. Figure 12A shows IFNγ production from T cells cultured in the presence of B7H3-positive A375 cells and B7H3-negative CCRF-CEM cells in the presence of representative CD3-engaging constructs targeting B7H3. [Figure 12B] Figure 12B illustrates the ability of representative constrained CD3-engaging constructs targeting B7H3 to induce IFNγ production in a target-dependent manner. Figure 12B shows IFNγ production from T cells cultured in the presence of B7H3-positive A375 cells and B7H3-negative CCRF-CEM cells in the presence of representative CD3-engaging constructs targeting B7H3. [Figure 13-1] Figures 13A and 13B illustrate cell binding of representative constrained CD3-engaging constructs targeting B7H3. cx5187 and cx5823 each contain two B7H3-binding domains, while the conjugates cx5873 and cx5965 each contain one B7H3-binding domain. Figure 13A shows binding to B7H3-positive A375 cells. Figure 13B shows the lack of binding to B7H3-negative CCRF-CEM cells and isolated T cells. [Figure 13-2]Figures 13C and 13D illustrate the ability of representative constrained CD3 engagement constructs targeting B7H3 to agonize CD3 in a target-dependent manner. Figure 13C shows that engagement of B7H3-positive A375 cells with a bivalent, biepitopic construct for B7H3 (cx5187) induced stronger CD3 signaling than constructs monovalent for B7H3 (cx5873 and cx5965). Figure 13D shows the lack of T cell activation in the presence of B7H3-negative CCRF-CEM cells. The Jurkat CD3 NFAT-GFP reporter cell line was used to assess CD3 agonism. [Figure 14] Figures 14A and 14B illustrate the ability of representative constrained CD3-engaging constructs targeting B7H3 to induce T cell-mediated cytotoxicity in a target-dependent manner. Figure 14A shows that targeting B7H3-positive A375 cells with a bivalent, biepitopic construct (cx5187) against B7H3 induced more potent T cell-mediated cytotoxicity than monovalent constructs (cx5873 and cx5965) against B7H3. Figure 14B illustrates the lack of T cell-mediated cytotoxicity against B7H3-negative CCRF-CEM cells. [Figure 15] Figures 15A-D illustrate that a representative restricted CD3-engaging molecule targeting B7H3 can activate T cells in the presence of B7H3-positive A375 cells but not in the presence of B7H3-negative CCRF-CEM cells. Figures 15A and 15B show that targeting B7H3-positive A375 cells with a bivalent, biepitopic construct (cx5187) against B7H3 induced CD25 expression in CD4+ and CD8+ T cells more potently than monovalent constructs (cx5873 and cx5965) against B7H3. Figures 15C and 15D show the lack of CD25 expression in CD4+ and CD8+ T cells in the presence of B7H3-negative CCRF-CEM cells. [Figure 16-1]Figures 16A and 16B demonstrate that a representative restricted CD3-engaging construct targeting B7H3 can induce T cell-mediated cytotoxicity in the presence of B7H3-positive A375 cells (Figure 16A), but not in the presence of CCRF-CEM B7H3-negative cells (Figure 16B). Figures 16C-16J demonstrate that a representative restricted CD3-engaging construct targeting B7H3 can induce T cell activation in the presence of B7H3-positive A375 cells, but not in the presence of CCRF-CEM B7H3-negative cells, as assessed by CD25 expression in CD4+ T cells (Figures 16C and 16D, respectively), CD25 expression in CD8+ T cells (Figures 16E and 16F, respectively), CD71 expression in CD4+ T cells (Figures 16G and 16H, respectively), and CD71 expression in CD8+ T cells (Figures 16I and 16J, respectively). Figures 16K and 16L demonstrate that a representative restricted CD3-engaging construct targeting B7H3 can induce T cell cytokine production in the presence of B7H3-positive A375 cells (Figure 16K), but not in the presence of CCRF-CEM B7H3-negative cells (Figure 16L). [Figure 16-2] See description of Figure 16-1. [Figure 16-3] See description of Figure 16-1. [Figure 16-4] See description of Figure 16-1. [Figure 17-1] Figures 17A and 17B demonstrate that representative restricted CD3-engaging constructs targeting monovalent (cx5800 and cx5801) and bivalent (cx5352) DLL3 bind to the DLL3-expressing cell line SHP-77 (Figure 17A) but not to isolated T cells (Figure 17B). Binding was assessed by flow cytometry. [Figure 17-2]Figure 17C illustrates the ability of representative constrained CD3-engaging constructs targeting DLL3 to drive CD3 signaling in the presence of DLL3-positive SHP-77 cells. Engagement of DLL3-positive cells with a bivalent, biepitopic construct for DLL3 (cx5352) induced more potent T cell activation than constructs monovalent for DLL3 (cx5800 and cx5801). A Jurkat CD3 NFAT luciferase reporter cell line was used to assess CD3 signaling. [Figure 18-1] Figures 18A-18E demonstrate the ability of a representative restricted CD3-engaged construct targeting DLL3, cx5499, to induce T cell-mediated cytotoxicity and T cell activation in the presence of DLL3-positive SHP-77 cells. Figure 18A demonstrates the ability of a representative restricted CD3-engaged construct targeting DLL3 to induce T cell-mediated cytotoxicity in the presence of DLL3-positive SHP-77 cells. Figures 18C-18D demonstrate the ability of a representative restricted CD3-engaged construct targeting DLL3 to induce T cell activation in the presence of DLL3-positive SHP-77 cells, as assessed by CD25 expression in CD4+ T cells (Figure 18B), CD69 expression in CD4+ T cells (Figure 18C), CD25 expression in CD8+ T cells (Figure 18D), and CD69 expression in CD8+ T cells (Figure 18E). [Figure 18-2] See description of Figure 18-1. DETAILED DESCRIPTION OF THE INVENTION

[0121] Detailed Description The present disclosure provides constrained T cell-engaging fusion proteins in the form of multispecific polypeptide constructs that bind at least to CD3 and a second antigen. The multispecific polypeptide constructs provided herein include at least a first component comprising one or more copies of an antigen-binding domain that binds to the antigen operably linked to an immunoglobulin Fc region, a second component comprising one or more copies of a binding domain that binds at least to CD3 (referred to herein as an anti-CD3 binding domain or CD3 binding region, terms used interchangeably herein), and a linker, such as a polypeptide linker, joining the first component and the second component. In some embodiments, the antigen is a tumor-associated antigen (TAA). In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker does not contain a substrate recognition site that is specifically recognized by a protease, such as a protease that is granzyme B, an MMP, or matriptase.

[0122] The provided multispecific polypeptide constructs include 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 C-terminal to the end of the Fc region. In some embodiments, the antigen-binding domain is located in the amino-terminal (N-terminal) region of the multispecific polypeptide construct. In some embodiments, the antigen-binding domain is located in the carboxy-terminal (C-terminal) region of the multispecific polypeptide construct. In some embodiments, the antigen-binding domain is located in both the N-terminal and C-terminal regions of the multispecific polypeptide construct. Various arrangements of the multispecific polypeptide constructs provided herein are shown in Figure 1.

[0123] Because the provided multispecific polypeptide constructs substantially bind to CD3 only after antigen is bound via the antigen-binding domain, such constructs exhibit restricted T cell-engaging activity. This is exemplified in the examples and figures provided herein, which demonstrate that restricted CD3-engaging proteins can efficiently bind to TAA-positive cells but exhibit little to no binding to T cells. This unique property allows the restricted CD3-engaging proteins to distribute to sites where TAAs are present without binding to peripheral T cells. This format differs from other CD3-engaging multispecific constructs in that constitutive CD3 binding is not permitted or eliminated, providing a significant advantage by avoiding peripheral T cell binding and allowing preferential distribution to sites where the antigen recognized by the antigen-binding domain is present. Furthermore, other CD3-engaging constructs mediate antigen-dependent T cell activation. However, the multispecific polypeptide constructs provided herein mediate both antigen-dependent T cell binding and activation.

[0124] The constrained T cell-engaging activity of the provided multispecific polypeptide constructs is, in some aspects, due to the positioning of an Fc region N-terminal to the CD3-binding region. In some embodiments, such positioning reduces, attenuates, interferes with, and / or prevents CD3 binding by the CD3-binding region. In the absence of antigen binding by the antigen-binding domain, the multispecific polypeptide constructs provided herein exhibit reduced or eliminated CD3 binding and T cell activation capabilities. In some embodiments, in the presence of an antigen binding event mediated by the antigen-binding domain of the multispecific polypeptide construct, the ability of the CD3-binding region to bind to CD3 is greatly enhanced. In some 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 some embodiments, the provided multispecific polypeptide constructs can be used to increase an immune response, for example, to enhance T cell activity, including cytolytic (or cytotoxic) T cell activity. Modulation of the immune response can, in some aspects, treat a disease or condition in a subject.

[0125] In some embodiments, one or more antigen-binding domains bind to antigens of tumor cells or cells in the tumor microenvironment. In some aspects, the provided multispecific polypeptide constructs can be used to increase immune responses, such as T cell activity, e.g., cytotoxic activity, against tumors or cancer. In some embodiments, the provided multispecific polypeptide constructs can be used to treat tumors or cancer in a subject.

[0126] In some embodiments, the CD3-binding region of the multispecific polypeptide constructs of the present disclosure is constrained or otherwise blocked and / or inhibited by the presence of an Fc region, thereby ensuring that T cell binding via CD3 in the peripheral blood does not occur. Thus, the multispecific polypeptide constructs of the present disclosure provide numerous advantages. In some aspects, these constructs limit the sink effect caused by binding of all T cells. In some aspects, these constructs reduce systemic toxicity.

[0127] In some embodiments, the multispecific polypeptide constructs provided herein allow for controlled biodistribution to desired sites in a subject, such as, for example, sites of tumor-associated antigen (TAA) expression, including, for example, the tumor and surrounding tumor microenvironment.

[0128] In some embodiments, the multispecific polypeptide constructs of the present disclosure exhibit specificity for CD3 and one or more other antigens. In some embodiments, the multispecific polypeptide constructs may contain multiple antigen-binding domains capable of binding to one or more TAAs, such as two, three, or four antigen-binding domains. See, for example, FIG. 1. In some embodiments, one or more antigen-binding domains bind to the same antigen. In some embodiments, the multispecific polypeptide constructs comprise multiple antigen-binding domains that bind to different epitopes of the same antigen. In some embodiments, the multispecific polypeptide constructs comprise multiple antigen-binding domains that bind to one or more different antigens. In some embodiments, the multispecific polypeptide constructs comprise multiple antigen-binding domains that bind to different epitopes of the same antigen, and also comprise additional antigen-binding domains that bind to one or more different antigens. In some aspects, the provided multispecific polypeptide constructs are bispecific polypeptide constructs that can bind to CD3 and another antigen, such as a TAA, through binding of the antigen-binding domains of the multispecific polypeptide construct. In some instances, the provided multispecific polypeptide constructs are bispecific polypeptide constructs that provide for multivalent engagement of one or more TAAs through the use of a first antigen-binding domain and a second antigen-binding domain. For example, in some embodiments, the bispecific polypeptide construct comprises a first antigen-binding single domain antibody (sdAb) and a second antigen-binding sdAb.

[0129] 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 the absence of binding of any or all of the antigen binding domains, such that CD3 binding is constrained and T cell interaction is abolished, and (2) an "active" state occurs upon antigen binding by any or all of the antigen binding domains, such that the CD3 binding region can bind CD3 and T cell interaction is possible.

[0130] In some embodiments, the Fc region is linked to the CD3 binding domain via a linker, hi some embodiments, the Fc region is linked to the CD3 binding domain via one or more non-cleavable linkers such as any of those described.

[0131] 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 cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the Fc region of the multispecific polypeptide construct can interact with complement proteins, 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.

[0132] In some embodiments, the multispecific polypeptide constructs of the present disclosure allow for simultaneous T cell- and NK cell-mediated cytotoxicity. In some cases, such activity can occur in a multispecific polypeptide construct that contains 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.

[0133] It is contemplated that constrained CD3-engaging constructs can be used with any TAA-binding domain to avoid interactions with peripheral T cells and mediate potent TAA-dependent T cell cytotoxicity, thereby allowing better therapeutic exposure in the tumor or tumor microenvironment. In some embodiments, the second portion or component contains a CD3-binding region that is monovalent for CD3, such that T cell activation does not occur unless the TAA is present.

[0134] In some aspects, the multispecific polypeptide constructs of the present disclosure offer numerous advantages over 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 may allow for better target, e.g., tumor, penetration. In some aspects, the overall size of the multispecific polypeptide construct provides a long half-life of the construct. In some aspects, because CD3 binding by the CD3 binding region relies on TAA engagement before CD3 engagement occurs, the multispecific polypeptide constructs of the present disclosure exhibit reduced systemic toxicity or toxicity in areas outside the tumor and / or tumor microenvironment.

[0135] All publications and patent documents cited herein are incorporated by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute an admission as to the contents or date thereof. While the invention has been described by way of illustration, those skilled in the art will recognize that the invention can be embodied in a variety of embodiments, and that the foregoing description and following examples are intended to illustrate, but not limit, the scope of the claims which follow.

[0136] I. Definition Unless otherwise defined, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. The term "a" entity or "an" entity refers to one or more of that entity. For example, a compound refers to one or more compounds. Thus, the terms "(a)," "(an)," "one or more," and "at least one" may be used interchangeably. Furthermore, unless otherwise required by context, the singular shall include the plural, and the plural shall include the singular. Generally, the nomenclature utilized in connection with, and techniques of, cell and tissue culture, molecular biology, protein and oligonucleotide or polynucleotide chemistry, and hybridization described herein are those 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). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The above techniques and procedures are generally carried out according to conventional methods well known in the art, as described in the various general and more specific references cited and described herein. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The nomenclature utilized in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the laboratory procedures and techniques thereof, are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0137] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0138] As used herein, the term "antibody" refers to immunoglobulin molecules and antigen-binding portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts" or "immunospecifically binds" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with, or with a much lower affinity (K d >10 -6 ) means that the antibody binds to the antigen of interest. Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, fully human, domain antibodies, single chain, Fab and F(ab')2 fragments, Fv, scFv, and Fab expression libraries. Typically, an "antigen-binding fragment" contains at least one CDR of an immunoglobulin heavy and / or light chain that binds to at least one epitope of an antigen of interest. In this regard, an antigen-binding fragment may contain one, two, three, four, five, or all six CDRs of the variable heavy (VH) and variable light (VL) chain sequences derived from an antibody that binds to the antigen, generally six CDRs ("CDR1," "CDR2," and "CDR3" for each of the heavy and light chains) for antibodies containing a VH and VL, or three CDRs for antibodies containing a single variable domain. Antigen-binding fragments include single domain antibodies, e.g., those containing only a VH, or those containing only a VL, e.g., a V H H, V NAR , modified V H domain, or modified V K Contains the domain.

[0139] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 amino acids or more primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Generally, antibody molecules obtained from humans belong to one of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other depending on the nature of the heavy chain present in the molecule. Certain classes also have subclasses, such as IgG1, IgG2, IgG3, IgG4, and others. Furthermore, in humans, light chains can be kappa or lambda chains.

[0140] The term "monoclonal antibody" (mAb) or "monoclonal antibody composition," as used herein, refers to a population of antibody molecules containing only one molecular species of antibody molecule, consisting of a unique light chain gene product and a unique heavy chain gene product. Specifically, the complementarity-determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. MAbs contain an antigen-binding site capable of immunoreacting with a particular epitope of an antigen, characterized by a unique binding affinity.

[0141] The term "antigen-binding site" or "binding portion" refers to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues from the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly diverse stretches within the V regions of the heavy and light chains, called "hypervariable regions," are interposed between adjacent, more conserved stretches known as "framework regions" or "FRs." Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions of immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity-determining regions" or "CDRs." The assignment of amino acids to each domain follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk J. Mol. Biol. 196:901-917(1987), Chothia et al. Nature 342: 878-883(1989).

[0142] As used herein, the term "epitope" includes the specific portion of an antigen 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 capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants generally consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and generally have specific three-dimensional structural and charge characteristics. For example, antibodies can be raised against N-terminal, central, or C-terminal peptides of a polypeptide. Furthermore, antibodies can be raised against linear or discontinuous epitopes of a polypeptide. An antibody is said to specifically bind to an antigen when the dissociation constant is ≦1 μM, e.g., in some embodiments ≦100 nM, and in some embodiments ≦10 nM, and does not exhibit binding to other proteins, whether closely related or distinct.

[0143] As used herein, the terms "specific binding," "immunological binding," and "immunological binding characteristics" refer to noncovalent interactions of the type that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction is determined by the dissociation constant (K d ) and smaller K d represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of formation and dissociation of the antigen-binding site / antigen complex, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, the "association rate constant" (K on ) and "dissociation rate constant" (K off ) can be determined by calculation of the concentration and the actual rates of association and dissociation (see Nature 361:186-87 (1993)). off / Kon The ratio of α to β allows for the cancellation of all parameters unrelated to affinity, and the dissociation constant K d (See generally Davies et al. (1990) Annual Rev Biochem 59 parameter 73). The antibodies of the present disclosure may have a binding constant (K) as measured by an assay such as a radioligand binding assay or similar assay known to those of skill in the art. d ) is ≦1 μM, for example, in some embodiments, ≦100 nM, in some embodiments, ≦10 nM, and in some embodiments, ≦100 pM to about 1 pM.

[0144] The term "isolated polynucleotide," as used herein, is intended to mean a polynucleotide of genomic, cDNA, or synthetic origin, or a combination thereof, and because of its origin, an "isolated polynucleotide" is not associated with all or a portion of a polynucleotide with which it is found in nature, (2) it is operably linked to a polynucleotide with which it is not naturally linked, or (3) it is not naturally occurring as part of a larger sequence. Polynucleotides according to the present disclosure include nucleic acid molecules encoding heavy chain immunoglobulin molecules as set forth herein, and nucleic acid molecules encoding light chain immunoglobulin molecules as set forth herein.

[0145] The term "isolated protein" as referred to herein means a protein derived from cDNA, recombinant RNA, or synthetically, or some combination thereof, and because of its origin or derivation, an "isolated protein" is (1) not related to proteins found in nature, (2) free from other proteins from the same source, e.g., free from mouse proteins, (3) expressed by cells from a different species, or (4) not naturally occurring.

[0146] The term "polypeptide" is used herein as a general term to refer to a native protein, a fragment, or an analog of a polypeptide sequence. Thus, fragments and analogs of a native protein are species of polypeptides. Polypeptides according to the present disclosure include heavy chain immunoglobulin molecules as set forth herein, and light chain immunoglobulin molecules as set forth herein, including antibody molecules formed by combinations comprising heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof.

[0147] The term "naturally-occurring," as used herein, as applied to a substance, refers to the fact that the substance can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified artificially in a laboratory or otherwise is naturally occurring.

[0148] The term "operably linked," as used herein, refers to the positioning of the components so described being in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0149] The term "control sequences," as used herein, refers to polynucleotide sequences necessary to effect expression and processing of ligated coding sequences. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include a promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, such control sequences generally include a promoter and transcription termination sequence. The term "control sequences" is intended to include at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. The term "polynucleotide," as referred to herein, means a nucleotide of at least 10 bases in length, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide. The term includes single- and double-stranded forms of DNA.

[0150] The term "oligonucleotide" as used herein includes naturally occurring nucleotides and modified nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide linkages. Oligonucleotides are generally a polynucleotide subset containing a length of 200 bases or less. In some embodiments, oligonucleotides are 10-60 bases long, for example, in some embodiments, 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 bases long. Oligonucleotides are generally single-stranded, for example, for probes, but oligonucleotides may also be double-stranded, for example, for use in constructing gene mutants. The oligonucleotides of the present disclosure are either sense or antisense oligonucleotides.

[0151] The term "naturally occurring nucleotides" referred to herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" referred to herein includes nucleotides having modified or substituted sugar groups, etc. The term "oligonucleotide linkage" referred to herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselerloate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoronmidate, etc. See, for example, LaPlanche et al. Nucl. Acids Res. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984); Stein et al. Nucl. Acids Res. 16:3209 (1988); Zon et al. Anticancer Drug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al., U.S. Pat. No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990). Optionally, the oligonucleotide may contain a label for detection.

[0152] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)). Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), α,α-disubstituted amino acids, N-alkylamino acids, lactic acid, and other unconventional amino acids such as unnatural amino acids may also be suitable components for the polypeptides of the present disclosure. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0153] Similarly, unless otherwise specified, the left-hand end of a single-stranded polynucleotide sequence is the 5'-end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction. The direction of 5' to 3' addition of a nascent RNA transcript is referred to as the transcription direction, and the region of the DNA strand that has the same sequence as the RNA and is 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence," and the region of the DNA strand that has the same sequence as the RNA and is 3' to the 3' end of the RNA transcript is referred to as the "downstream sequence."

[0154] The term "substantial identity" as applied to polypeptides means that two peptide sequences, when optimally aligned using default gap weights, such as by the programs GAP or BESTFIT, share at least 80 percent sequence identity, e.g., 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.

[0155] In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

[0156] As described herein, minor variations in the amino acid sequence of an antibody or immunoglobulin molecule are contemplated as being encompassed by the present disclosure, so long as the variation in the amino acid sequence is at least 75%, e.g., in some embodiments, at least 80%, 90%, 95%, and in some embodiments, 99%. Specifically, conservative amino acid substitutions are contemplated. Conservative substitutions are those that occur within a family of amino acids whose side chains are related. 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 amino acid families include: (i) the aliphatic hydroxy family, serine and threonine; (ii) the amide-containing family, asparagine and glutamine; (iii) the aliphatic family, alanine, valine, leucine, and isoleucine; and (iv) the aromatic family, phenylalanine, tryptophan, and tyrosine. For example, 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 between structurally related amino acids will not have a significant effect on the binding or properties of the resulting molecule, especially when the substitution does not involve an amino acid within a framework region. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative, which assays are described in detail herein.Fragments or analogs of antibodies or immunoglobulin molecules can be easily prepared by those skilled in the art. In some embodiments, the amino and carboxy termini of the fragments or analogs are located near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data with public or proprietary sequence databases. Computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains present in other proteins with known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known. Bowie et al. Science 253:164 (1991). Thus, the above examples demonstrate that those skilled in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains according to the present disclosure.

[0157] In some embodiments, amino acid substitutions confer or modify the following properties of such analogs: (1) reducing their susceptibility to proteolysis; (2) reducing their susceptibility to oxidation; (3) altering their binding affinity for forming protein complexes; (4) altering their binding affinity; and (4) imparting other physicochemical or functional properties. Analogs can include various muteins of sequences other than naturally occurring peptide sequences. For example, one or more amino acid substitutions (e.g., conservative amino acid substitutions) can be made in the naturally occurring sequence, for example, in a portion of the polypeptide outside the intermolecular contact-forming domain. Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., the replacement amino acid should not tend to disrupt the helix present in the parent sequence or other types of secondary structure that characterize the parent sequence). Examples of art-recognized secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354:105(1991).

[0158] The term "polypeptide fragment," as used herein, refers to a polypeptide having an amino- and / or carboxy-terminal deletion and / or one or more intrasequence deletions, where the remaining amino acid sequence is identical to the corresponding positions in a naturally occurring sequence, e.g., as deduced from a full-length cDNA sequence. Fragments are typically at least 5, 6, 8, or 10 amino acids in length, e.g., in some embodiments, at least 14 amino acids in length, in some embodiments, at least 20 amino acids in length, generally at least 50 amino acids in length, and in some embodiments, at least 70 amino acids in length. The term "analog," as used herein, refers to a polypeptide comprised of a segment of at least 25 amino acids that has substantial identity to a portion of the deduced amino acid sequence and has specific binding to EGFR under appropriate binding conditions. Typically, polypeptide analogs contain conservative amino acid substitutions (or additions or deletions) relative to the naturally occurring sequence. Analogs are typically at least 20 amino acids in length, e.g., in some embodiments, at least 50 amino acids in length or longer, and can often be as long as a full-length naturally occurring polypeptide.

[0159] The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological material.

[0160] As used herein, the term "label" or "labeled" refers to the incorporation of a detectable marker, for example, by incorporation of a radiolabeled amino acid into a polypeptide or by attachment of a biotinyl moiety that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In certain circumstances, the label or marker may also be therapeutic. A variety of methods of 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, rhodamines, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labels are attached by spacer arms of various lengths to reduce potential steric hindrance. The term "pharmaceutical agent or drug," as used herein, refers to a chemical compound or composition capable of inducing a desired therapeutic effect when appropriately administered to a patient.

[0161] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells, which may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0162] 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.

[0163] The terms "treating," "treatment," or "therapy" of a disease or disorder, as used herein, refer to the slowing, halting, or reversal of the progression of a disease or disorder, as evidenced by a reduction, arrest, or elimination of clinical or diagnostic symptoms, by administering a pharmaceutical composition of the present disclosure, alone or in combination with another compound described herein. "Treating," "treatment," or "therapy" also refers to a reduction in the severity of symptoms or a reduction in the rate of recurrence in acute or chronic diseases or disorders. As used herein, the terms "treating," or "inhibiting," "inhibiting," or "inhibition" of cancer, with respect to cancer, refer to at least one of the following: a statistically significant reduction in the rate of tumor growth, a halt in tumor growth, or a decrease in tumor size, mass, metabolic activity, or volume, or a statistically significant increase in progression-free survival (PFS) or overall survival (OS), as measured by standard criteria, such as, but not limited to, Response Evaluation Criteria for Solid Tumors (RECIST). "Preventing," "prophylaxis," or "prevention" of a disease or disorder refers to the administration of a pharmaceutical composition, alone or in combination with another compound, to prevent the occurrence or development of a disease or disorder, or some or all of the symptoms of a disease or disorder, or to reduce the likelihood of developing a disease or disorder.

[0164] The term "effective amount" or "therapeutically effective amount" refers to an 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, provides a statistically significant reduction in disease progression, e.g., by ameliorating or eliminating the symptoms and / or causes of the disease. An effective amount can be an amount that relieves, reduces, or alleviates at least one symptom or biological response or effect associated with a disease or disorder, prevents the progression of a disease or disorder, or improves the patient's physical function.

[0165] As used herein, "substantially pure" means that a species is the predominant species present (i.e., it is more abundant than other individual species in the composition on a molar basis), and a substantially purified fraction is a composition in which a species constitutes at least about 50 percent (on a molar basis) of all macromolecular species.

[0166] Generally, a substantially pure composition will comprise greater than about 80 percent of all macromolecular species present in the composition, e.g., in some embodiments, greater than about 85%, 90%, 95%, and 99%. In some embodiments, the species is purified to essential homogeneity, such that the composition consists essentially of a single macromolecular species (contaminating species cannot be detected in the composition by conventional detection methods).

[0167] The term patient includes human and veterinary subjects.

[0168] Other chemical terms herein are used in accordance with conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

[0169] The term "about" as used herein refers to a general error range for each value, which is readily known to those skilled in the art. Reference to a value or parameter with "about" herein includes (describes) aspects related to the value or parameter itself. For example, a description of "about X" includes the description of "X."

[0170] II. Multispecific Polypeptide Constructs Provided herein is 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 located N-terminal to the CD3-binding region; one or both of the first and second components comprises an antigen-binding domain that binds to a tumor-associated antigen (TAA). In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker does not contain a substrate recognition domain that is specifically recognized for cleavage by a protease.

[0171] In some embodiments, the multispecific polypeptide construct contains, from N-terminus to 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). In some embodiments, the multispecific polypeptide construct contains, from N-terminus to C-terminus, an antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker; and a CD3-binding region that binds to CD3 (CD3ε). 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, from N-terminus to C-terminus, a first 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 second antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0172] Each of the components of the multispecific polypeptide constructs of the present disclosure is described in more detail below.

[0173] 1. Anti-CD3 binding domain: The multispecific polypeptide constructs of the present disclosure comprise one or more copies of an anti-CD3 binding domain. The anti-CD3 binding domain of the present disclosure activates T cells through engagement of CD3ε on T cells. The anti-CD3 binding domain of the present disclosure induces, stimulates, activates, and / or otherwise enhances CD3-mediated T cell activation. The biological activity of CD3 includes, for example, T cell activation and other signaling through the interaction between CD3 and the antigen-binding subunit of the T cell receptor (TCR). For example, the anti-CD3 binding domain of the present disclosure activates T cells fully or partially through engagement of CD3ε on T cells by partially or fully modulating CD3-mediated T cell activation, e.g., by inducing, stimulating, activating, or otherwise enhancing it.

[0174] In preferred embodiments, the anti-CD3 binding domain of the present disclosure specifically binds to the ε chain of CD3, also known as CD3ε. The anti-CD3ε binding domain of the present disclosure activates T cells through engagement of CD3ε on T cells. The anti-CD3ε binding domain of the present disclosure includes monoclonal antibodies, such as mammalian monoclonal antibodies, primate monoclonal antibodies, fully human monoclonal antibodies, as well as humanized monoclonal antibodies and chimeric antibodies, and antigen-binding fragments thereof. In some embodiments, the anti-CD3ε binding domain comprises one or more copies of an antibody or its antigen-binding fragment.

[0175] 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, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence comprises at least the amino acid sequence TYAMN (SEQ ID NO: 16); VH CD2 sequence containing at least TIFF0007824455000024.tif4128; and amino acid sequence TIFF0007824455000025.tif4128. 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, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence comprises a VH CDR1 sequence comprising at least the amino acid sequence GFTFNTYAMN (SEQ ID NO:211); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATY (SEQ ID NO:212); and an amino acid sequence The VH CDR3 sequence is selected from VH CDR3 sequences containing at least TIFF0007824455000026.tif4128.

[0176] 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, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence is the amino acid sequence a VL CDR1 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).

[0177] 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); VH CDR2 sequence containing at least TIFF0007824455000028.tif4128; amino acid sequence VH CDR3 sequence containing at least TIFF0007824455000029.tif4128; amino acid sequence TIFF0007824455000030.tif4128; 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).

[0178] In some embodiments, the anti-CD3ε binding domain comprises a VH CDR1 sequence comprising at least the amino acid sequence GFTFNTYAMN (SEQ ID NO:211); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATY (SEQ ID NO:212); VH CDR3 sequence containing at least TIFF0007824455000031.tif4128; amino acid sequence TIFF0007824455000032.tif4128; 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).

[0179] In some embodiments, the anti-CD3ε binding domain comprises a VH CDR1 sequence comprising at least the amino acid sequence GFTFNTYAMN (SEQ ID NO:211); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATY (SEQ ID NO:212); VH CDR3 sequence containing at least TIFF0007824455000033.tif4128; amino acid sequence TIFF0007824455000034.tif4128; a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO:230); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNHWV (SEQ ID NO:225).

[0180] 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, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence comprises 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 comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007824455000035.tif4128; and an amino acid sequence TIFF0007824455000036.tif4128, or a VH CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007824455000036.tif4128.

[0181] 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, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence comprises a VH CDR1 sequence that comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence GFTFNTYAMN (SEQ ID NO:211); a VH CDR2 sequence that comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RIRSKYNNYATY (SEQ ID NO:212); and an amino acid sequence TIFF0007824455000037.tif4128, and VH CDR3 sequences containing sequences at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007824455000037.tif4128.

[0182] 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, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence is the amino acid sequence a VL CDR1 sequence comprising a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007824455000038.tif4128; a VL CDR2 sequence comprising a sequence 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 comprising a sequence 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).

[0183] 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); a VH CD2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007824455000039.tif4128; an amino acid sequence A VH CDR3 sequence, amino acid sequence, that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007824455000040.tif4128 TIFF0007824455000041.tif4128; 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).

[0184] In some embodiments, the anti-CD3 epsilon binding domain comprises a VH CDR1 sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence GFTFNTYAMN (SEQ ID NO:211); a VH CDR2 sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RIRSKYNNYATY (SEQ ID NO:212); an amino acid sequence a VH CDR3 sequence, amino acid sequence, that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007824455000042.tif4128 TIFF0007824455000043.tif4128; 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).

[0185] In some embodiments, the anti-CD3 epsilon binding domain comprises a VH CDR1 sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence GFTFNTYAMN (SEQ ID NO:211); a VH CDR2 sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RIRSKYNNYATY (SEQ ID NO:212); an amino acid sequence A VH CDR3 sequence, amino acid sequence, that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007824455000044.tif4128 TIFF0007824455000045.tif4128; 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:230); 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:225).

[0186] In some embodiments, the anti-CD3ε binding domain comprises a VH CDR1 sequence comprising at least the amino acid sequence GFTFSTYAMN (SEQ ID NO:227); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATY (SEQ ID NO:228); VH CDR3 sequence containing at least TIFF0007824455000046.tif4128, amino acid sequence TIFF0007824455000047.tif4128; a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO:230); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNHWV (SEQ ID NO:225).

[0187] In some embodiments, the anti-CD3 epsilon binding domain comprises a VH CDR1 sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence GFTFSTYAMN (SEQ ID NO:227); a VH CDR2 sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RIRSKYNNYATY (SEQ ID NO:228); an amino acid sequence A VH CDR3 sequence, amino acid sequence, that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007824455000048.tif4128 TIFF0007824455000049.tif4128; 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:230); 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:225).

[0188] In some embodiments, the anti-CD3ε binding domain comprises a CDR3 comprising at least the amino acids VLWYSNRWV (SEQ ID NO:226). In some embodiments, the anti-CD3ε binding domain comprises a CDR3 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acids VLWYSNRWV (SEQ ID NO:226).

[0189] In some embodiments, the anti-CD3ε binding domain comprises one or more copies of an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, an scFv, a scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. In some embodiments, the anti-CD3ε binding domain comprises an Fv antibody fragment 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.

[0190] In some embodiments, the CD3 binding region is not a single chain antibody, e.g., in some aspects, the CD3 binding region is not a single chain variable fragment (scFv).

[0191] In some embodiments, the CD3 binding region is an Fv antibody fragment containing a variable heavy chain (Hv, also referred to as VH) and a variable light chain (Lv, also referred to as VL), such as any of those described. In aspects of such embodiments, the immunoglobulin Fc region is a heterodimeric Fc region containing two different Fc polypeptides, such as any of those described in Section II.2, that are capable of heterodimeric association between both polypeptides of the Fc heterodimer. In such embodiments, the variable heavy chain (VH) and variable light chain (VL) of the CD3 binding region are linked to opposite chains of the heterodimeric Fc.

[0192] In some embodiments, the anti-CD3ε Fv antibody fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81. In some embodiments, the anti-CD3ε Fv antibody fragment comprises an 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. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of an 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 an 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.

[0193] In some embodiments, the anti-CD3ε binding domain comprises a combination of a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence comprising an amino acid sequence selected from the group of SEQ ID NOs: 32 to 81. In some embodiments, the anti-CD3ε binding domain comprises a combination of a heavy chain variable region amino acid sequence selected from the group of SEQ ID NOs: 32 to 62 and a light chain variable region amino acid sequence comprising an amino acid sequence selected from the group of SEQ ID NOs: 63 to 81.

[0194] In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence and a light chain variable amino acid sequence. In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence and a light chain variable amino acid sequence comprising an amino acid sequence 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, 191, 196-200, 211, and 212. In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence and a light chain variable amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 32-81, 191, 196-200, 211, and 212. In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence selected from the group of SEQ ID NOs: 14, 32-62, 196-198, and 211 and a light chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 63-81, 191, 199, 200, and 212. In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising 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: 14, 32-62, 196-198, and 211, in combination with 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: 15, 63-81, 191, 199, 200, and 212.

[0195] In some embodiments, the anti-CD3ε binding domain comprises a combination of a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence comprising an amino acid sequence selected from the group of SEQ ID NOs: 32-81, 191, 196-200, 211, and 212. In some embodiments, the anti-CD3ε binding domain comprises a combination of a heavy chain variable region amino acid sequence selected from the group of SEQ ID NOs: 32-62, 196-198, and 211, and a light chain variable region amino acid sequence comprising an amino acid sequence selected from the group of SEQ ID NOs: 63-81, 191, 199, 200, and 212.

[0196] In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of an 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, 32-43, 45-47, 48, 196, and 211, and an 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, 65-71, 73, 75, 77, and 199. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of an amino acid sequence selected from the group of SEQ ID NOs: 14, 32-43, 45-47, 48, 196, and 211 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 63, 65-71, 73, 75, 77, and 199.

[0197] In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence 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 (VL) comprising an amino acid sequence 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 (VH) comprising an amino acid sequence 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 (VL) comprising an amino acid sequence 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 (VH) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:14 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:15.

[0198] In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 196. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 199. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 196, and a variable light chain (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 199. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 196. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 199. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:196 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:199.

[0199] In a specific embodiment, Fv is V H -V LThe heterodimer 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 altered by mutations at framework positions of the VH and / or VL chains. In some embodiments, the disulfide-stabilized anti-CD3 Fv comprises an anti-CD3 VH containing a Cys mutation at position 44 and an anti-CD3 VL containing a Cys mutation at position 100, according to the Kabat numbering. For example, in some embodiments, the VH chain contains a G44C mutation and the VL chain contains a G100C mutation, each according to the Kabat numbering. In some embodiments, the disulfide-stabilized anti-CD3 Fv comprises an anti-CD3 VH containing a Cys mutation at position 105 and an anti-CD3 VL containing a Cys mutation at position 43, according to the Kabat numbering.

[0200] In some embodiments, the anti-CD3ε Fv comprises a combination of an 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: 44, 49-62, 197, and 198, and an 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: 64, 72, 74, 76, 78-81, 191, 200, and 212. In some such embodiments, the anti-CD3 Fv is a dsFv having a VH chain containing a G44C mutation and a VL chain containing a G100C mutation, each according to the Kabat numbering system. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of an amino acid sequence selected from the group of SEQ ID NOs: 44, 49-62, 197, and 198 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 72, 74, 76, 78-81, 191, 200, and 212.

[0201] In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence 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 (VL) comprising an amino acid sequence 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 (VH) comprising an amino acid sequence 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 (VL) comprising an amino acid sequence 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 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 according to 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:44. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 44 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 72.

[0202] In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 198. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:200. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:198, and a variable light chain (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:200. In some such embodiments, the anti-CD3 Fv is a dsFv having a VH chain containing the G44C mutation and a VL chain containing the G100C mutation, each according to 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:198. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 200. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 198 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 200.

[0203] In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 197. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:200. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:197, and a variable light chain (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:200. In some such embodiments, the anti-CD3 Fv is a dsFv having a VH chain containing the G44C mutation and a VL chain containing the G100C mutation, each according to 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:197. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 200. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 197 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 200.

[0204] 2. Immunoglobulin Fc Polypeptides: The first component of the multispecific polypeptide construct of the present disclosure comprises 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 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 the amino acid sequence of any of SEQ ID NOs: 1-6, or an immunologically active fragment thereof.

[0205] In some embodiments, the multispecific polypeptide construct is a dimer formed by polypeptides each containing an Fc. In some specific embodiments, identical or substantially identical polypeptides will dimerize to create a homodimer. In some embodiments, the dimer is a homodimer in which the two polypeptides 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 to promote heterodimerization are known and include the following:

[0206] Generally, the Fc region is responsible for effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in addition to antigen-binding ability, which is the primary function of immunoglobulins. Furthermore, the FcRn sequence present in the Fc region plays a role in regulating serum IgG levels by increasing in vivo half-life through conjugation with the in vivo FcRn receptor. In some embodiments, such functions may be altered, for example, reduced or enhanced, in the Fc for use with the provided multispecific polypeptide constructs.

[0207] In some embodiments, the Fc region of the provided multispecific polypeptide constructs 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. Thus, in some embodiments in which the multispecific polypeptide construct contains a cleavable linker, cleavage of the linker can produce two components, each with biological activity: a CD3-binding region capable of binding and engaging CD3 on T cells, and an Fc region linked to a TAA antigen-binding domain capable of exhibiting target-specific effector function. In certain embodiments provided herein, the multispecific polypeptide constructs contain a non-cleavable linker and, in some aspects, may not exhibit independent Fc-mediated effector function.

[0208] 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 in Fc polypeptides to alter, e.g., reduce, effector function are known and include those described below. In some embodiments, unless described with reference to a particular SEQ ID NO, references to amino acid substitutions in the Fc region are in accordance with 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, EA et al. Sequences of Proteins of Immunological interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0209] In some embodiments, provided multispecific polypeptide constructs containing an Fc region exhibiting reduced effector function may be desirable candidates for applications in which constrained CD3 binding is desired, but certain effector functions (such as CDC and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the multispecific polypeptide construct and / or truncated components thereof lack FcγR binding (and thus likely lack ADCC activity) but retain FcRn binding ability. The primary cells for mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063(1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502(1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361(1987)). Alternatively, non-radioactive assay methods may be 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.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the multispecific polypeptide construct or a truncated component thereof is unable to bind C1q and therefore lacks CDC activity. See, for example, the C1q binding ELISA and C3c binding ELISA of WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163(1996); Cragg, MS et al., Blood 101:1045-1052(2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743(2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769(2006)).

[0210] In some embodiments, the immunoglobulin Fc region or immunologically active fragment thereof is of the IgG isotype. For example, the immunoglobulin Fc region of the fusion protein is of the human IgG1 isotype, having the following amino acid sequence: TIFF0007824455000050.tif34138

[0211] In some embodiments, the immunoglobulin Fc region or immunologically active fragment thereof comprises a human IgG1 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:1.

[0212] In some embodiments, an IgG1 Fc polypeptide or variant thereof, such as any of those described below, can be made with the G1 m1 or G1 m3 allotype. In some embodiments, the Fc region may contain amino acids of the human G1 m1 allotype, such as residues containing Asp (D) and Leu (L) at positions 356 and 358, as shown in SEQ ID NO:1. In some cases, the Fc polypeptide may contain amino acid substitutions E356D and M358L to reconstitute residues of allotype G1 m1. In other embodiments, the Fc region may contain amino acids of the human G1 m3 allotype, such as residues Glu (E) and Met (M) at positions 356 and 358, according to EU numbering, as shown in SEQ ID NOs:194 and 195. In some cases, the Fc polypeptide may contain amino acid substitutions D356E and L358M to reconstitute residues of allotype G1 m3. In some embodiments, the human IgG1 Fc region is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). For example, Natsume et al., 2008 Cancer Res,68(10): 3863-72;Idusogie et al., 2001 J Immunol, 166 (4):2571-5;Moore et al., 2010 mAbs, 2 (2): 181-189;Lazar et al., 2006 PNAS, 103 (11): 4005-4010, Shields et al., 2001 JBC, 276 (9): 6591-6604;Stavenhagen et al., 2007 Cancer Res,67(18): 8882-8890;Stavenhagen et al.,2008 Advan. Enzyme Regul., 48: 152-164;Alegre et al, 1992 J Immunol, 148: 3461-3468; and amino acid modifications reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11.the contents of each of which are incorporated herein by reference in their entirety.

[0213] In some embodiments, an Fc region, such as a human IgG1 Fc region, is modified to enhance ADCC 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)).

[0214] In some embodiments, the human IgG1 Fc region fusion protein of the present disclosure lacks or has reduced fucose attached to the N-linked glycan chain at N297. Numerous methods exist to prevent fucosylation, including, but not limited to, production in a FUT8-deficient cell line; 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.

[0215] In some embodiments, the Fc region is modified to provide reduced Fc-mediated effector function, e.g., through reduced Fc receptor binding, e.g., FcγR binding rather than the usual FcRn binding. In some embodiments, the Fc region of the fusion protein is altered at one or more of the following positions to reduce Fc receptor binding: Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325), 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 interactions (e.g., Leu235Glu (L235E) or Leu235Ala (L235A)). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (in the box of SEQ ID NO:1, Kabat numbering) to alter Fc receptor interactions (e.g., Leu234Ala (L234A)). In some embodiments, the Fc region of the fusion protein is altered at both amino acids 234 and 235 (e.g., Leu234Ala and Leu235Ala (L234A / L235A) or Leu234Val and Leu235Ala (L234V / L235A)). In preferred embodiments, modifications within the Fc region reduce binding to the Fc receptor gamma receptor but have minimal effect on binding to the neonatal Fc receptor (FcRn).

[0216] In some embodiments, the human IgG Fc region is modified to enhance FcRn binding. Examples of Fc mutations that enhance FcRn binding 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) (Kabat et al. 1991 EU Index of Sequences of Proteins of Immunological Interest). In some embodiments, the variant or modified Fc polypeptide comprises the following mutations, using the Kabat numbering system: Met252Tyr and Met428Leu or Met252Tyr and Met428Val (M252Y, M428L or M252Y, M428V).

[0217] In some embodiments, the Fc region of the fusion protein lacks amino acids 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. TIFF0007824455000051.tif32138

[0218] In some embodiments, the Fc region is mutated at one or more of the following positions to reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (L235). The one or more mutations may include E233P, L234V, and / or L235A.

[0219] In some embodiments, the Fc region of the fusion protein is altered 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, a human IgG1 Fc region is modified at amino acid Gly236 (e.g., Gly236Ala (G236A)) to enhance interaction with CD32A.

[0220] In specific embodiments, mutations in the Fc region to reduce Fc effector function, e.g., via reduced binding of the Fc receptor to FcγR, include any of the following 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.

[0221] In some embodiments, the human IgG1 Fc region lacks Lys447 (Kabat et al 1991 EU Index of Sequences of Proteins of Immunological Interest).

[0222] In some embodiments, the fusion or immunologically active fragment thereof comprises a human IgG2 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:2.

[0223] In some embodiments, the immunoglobulin Fc region of the fusion protein, or an immunologically active fragment thereof, is of the human IgG2 isotype, having the following amino acid sequence: TIFF0007824455000052.tif33138

[0224] In some embodiments, the fusion or immunologically active fragment thereof comprises a human IgG2 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:3.

[0225] In some embodiments, the human IgG2 Fc region is modified at amino acid Asn297 (intrabox, to prevent glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D)). In some embodiments, the human IgG2 Fc region lacks Lys447 (Kabat et al 1991 Sequences of Proteins of Immunological Interest, EU Index).

[0226] In some embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG3 isotype, having the following amino acid sequence: TIFF0007824455000053.tif34138

[0227] In some embodiments, the antibody or immunologically active fragment thereof comprises a human IgG3 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:4.

[0228] In some embodiments, the human IgG3 Fc region is modified at amino acid Asn297 (boxed, Kabat numbering) to prevent glycosylation of the antibody (e.g., Asn297Ala (N297A) or Asn297Asp (N297D)). In some embodiments, the human IgG3 Fc region is modified at amino acid 435 (e.g., Arg435His (R435H)) to extend half-life. In some embodiments, the human IgG3 Fc region lacks Lys447 (Kabat et al 1991 Sequences of Proteins of Immunological Interest, EU Index).

[0229] 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: TIFF0007824455000054.tif34138

[0230] In some embodiments, the antibody or immunologically active fragment thereof comprises a human IgG4 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:5.

[0231] 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: TIFF0007824455000055.tif33138

[0232] In some embodiments, the antibody or immunologically active fragment thereof comprises a human IgG4 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:6.

[0233] 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) to prevent glycosylation of the antibody (e.g., Asn297Ala (N297A) or Asn297Asp (N297D)). In some embodiments, the human IgG4 Fc region lacks Lys447 (Kabat et al 1991 Sequences of Proteins of Immunological Interest EU Index).

[0234] 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 Tyr349 to Cys (Y349C) (S354C / Y349C).

[0235] In certain embodiments of the multispecific polypeptide constructs provided herein, the human IgG Fc region is modified to induce heterodimerization. Various methods are known for promoting heterodimerization of complementary Fc polypeptides. For example, 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 No. WO 1998 / 050431, WO2009 / 089004, WO2011143545, WO2014 / 067011, WO2012 / 058768, WO2018027025; published U.S. patent application numbers US20140363426, US20150307628, US20180016354, US20150239991; and U.S. Patent Nos. 5,731,168, 7,183,076, 9,701,759, 9,605,084, and 9,650,446. Methods for promoting heterodimerization of the Fc chain include mutagenesis of the Fc region, for example, by including a set of "knob-into-hole" mutations or mutations to achieve electrostatic steering of the Fc to favor attractive interactions between different polypeptide chains.For example, in some embodiments, heterodimeric Fc polypeptides contain mutations to alter the charge polarity at the Fc dimer interface so that coexpression of electrostatically matched Fc chains favors favorable attractive interactions, thereby promoting the formation of desired Fc heterodimers, and unfavorable repulsive charge interactions suppress the formation of undesired Fc homodimers (Guneskaran et al., (2010) JBC, 285:19637-19646). When coexpressed in cells, interchain association is possible, but due to charge repulsion, the chains do not substantially self-associate. Another strategy for generating heterodimeric Fc involves mixing CH3 domain segments from human IgG and IgA to create heterodimers of complementary CH3s, referred to as SEED Fc.

[0236] Heterodimerization methods and variants also include those described in published International PCT application WO2014 / 145806, including "knobs and holes" mutations (also called "skew" variants), mutations involving "electrostatic steering" or "charge pairing," and pI variants. Heterodimer variants also include any described in published U.S. application numbers US2012 / 0149876 or US2018 / 011883.

[0237] In some embodiments, both polypeptides of the Fc heterodimer contain paired or complementary amino acid modifications to promote heterodimerization. Exemplary pairs of amino acid modifications for polypeptides of Fc fusions are shown in Table 1.

[0238] Table 1. Amino acid pairs of heterodimeric Fc TIFF0007824455000056.tif51145

[0239] In some embodiments, the modification comprises introducing a protuberance (knob) into a first Fc polypeptide and a cavity (hole) into a second Fc polypeptide, such that the protuberance 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 protuberance or cavity in the polypeptide are typically interface amino acids that interact with or contact one or more amino acids at the interface of the second polypeptide.

[0240] In some embodiments, the first Fc polypeptide modified to contain a protuberance (hole) amino acid comprises a substitution of a native or initial amino acid with an amino acid having at least one side chain that protrudes from the interface of the first Fc polypeptide and thus is positioned in a compensatory cavity (hole) in the adjacent interface of the second polypeptide. The replacement amino acid most often has a side chain volume larger than that of the initial amino acid residue. Methods for determining and / or evaluating the properties of amino acid residues to identify ideal replacement amino acids for creating a protuberance are known to those skilled in the art. In some embodiments, the replacement residue for forming the protuberance is a naturally occurring amino acid residue, including, for example, arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). In some examples, the initial 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.

[0241] In some embodiments, the second Fc polypeptide to be modified to contain a cavity (hole) is recessed from the interface of the second polypeptide and thus includes a substitution of the native or initial amino acid with an amino acid having at least one side chain that can accommodate a corresponding protrusion from the interface of the first polypeptide. The substituted amino acid most often has a smaller side chain volume than the initial amino acid residue. Methods for determining and / or evaluating the characteristics of amino acid residues to identify ideal replacement residues for cavity formation are known to those skilled in the art. Generally, replacement 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 initial amino acid identified for substitution is an amino acid with a large side chain, such as, for example, tyrosine, arginine, phenylalanine, or tryptophan.

[0242] The CH3 interface of human IgG1, for example, comprises 16 residues from each domain located on four antiparallel β-strands buried 1090 Å 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). Modifications of the CH3 domain to create protuberances or cavities are 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 instances, modifications of the CH3 domain to create a bulge or cavity are typically targeted to residues located in the two central antiparallel β-strands, with the goal being to minimize the risk that the resulting bulge will be accommodated by protruding into the surrounding solvent rather than being accommodated in a compensatory cavity in the partner CH3 domain.

[0243] For example, in some embodiments, the heterodimeric Fc comprises a polypeptide having an amino acid modification at Thr366 in a CH3 domain, which, when replaced with a bulkier amino acid, e.g., Try (T366W), can preferentially pair with a second CH3 domain having an amino acid modification at positions Thr366, Leu368, and Tyr407 to a less bulky amino acid, e.g., Ser, Ala, or Val (T366S / L368A / Y407V). Heterodimerization via CH3 modifications can be further stabilized by the introduction of disulfide bonds, e.g., by changing Ser354 to Cys (S354C) and Tyr349 to Cys (Y349C) in the opposing CH3 domain (reviewed in Carter, 2001 Journal of Immunological Methods, 248:7-15).

[0244] In specific embodiments, the multispecific polypeptide construct contains a first and a second Fc capable of mediating Fc heterodimerization, wherein 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 an Fc polypeptide comprising the sequence set forth in SEQ ID NO:201 or 207, and the second Fc polypeptide is selected from an Fc polypeptide comprising the sequence set forth in SEQ ID NO:202, 205, or 209. In some embodiments, the first Fc polypeptide is or comprises the sequence of amino acids set forth in any of SEQ ID NOs:82, 86, 94, or 96, and the second Fc polypeptide is or comprises the sequence of amino acids set forth in any of SEQ ID NOs:83, 87, 90, 92, 98, or 100.

[0245] In some embodiments, the Fc polypeptide exhibits features that provide Fc-mediated effector functions. In a specific example, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:201, and the second Fc polypeptide is or comprises SEQ ID NO:202 or 205. In some embodiments, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:82, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:83 or 90. In some embodiments, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:86, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:87 or 92. The first and second Fc polypeptides may be formatted in either polypeptide chain of the construct.

[0246] 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 function include any of those described. In some embodiments, the modification may 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 an Fc polypeptide comprising a sequence set forth in SEQ ID NO:203 or 208, and the second Fc polypeptide is selected from an Fc polypeptide comprising a sequence set forth in SEQ ID NO:204, 206, or 210. In some embodiments, the first Fc polypeptide is or comprises the sequence of amino acids set forth in any of SEQ ID NOs:84, 88, 95, or 97, and the second Fc polypeptide is or comprises the sequence of amino acids set forth in any of SEQ ID NOs:85, 89, 91, 93, 99, or 101.

[0247] In a specific example, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:203, and the second Fc polypeptide is or comprises SEQ ID NO:204 or 206. In some embodiments, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:84, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:85 or 91. In some embodiments, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:88, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:89 or 93. The first and second Fc polypeptides may be formatted in either polypeptide chain of the construct.

[0248] In some embodiments, the first Fc polypeptide or the second Fc polypeptide further comprises the mutation M252Y and / or M428V. In a specific example, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:207, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:209. In some embodiments, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:94, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:98. In some embodiments, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:96, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:100. In other examples, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:208, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:210. In some embodiments, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:95, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:99. In some embodiments, the first Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:97, and the second Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:101. The first and second Fc polypeptides may be formatted in either polypeptide chain of the construct.

[0249] Additional examples of variants that can facilitate the promotion of heterodimers include S364K / E357Q and L368D / K370S; L368D / K370S and S364K; L368E / K370S and S364K; T411T / E360E / Q362E and D401K; L368D / K370S and S364K / E357L, K370S and S364K / E357Q and and T366S / L368A / Y407V and T366W, or 366S / L368A / Y407V / Y349C and T366W / S354C (each pair representing mutations in the first and second Fc polypeptides). In a specific embodiment, the provided constructs contain first and second Fc polypeptides containing the mutation pairs L368D / K370S and S364K and E357Q.

[0250] An additional mechanism that can be used in generating heterodimers 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 "charge pairing." In this embodiment, electrostatics are used to bias formation toward heterodimerization. As one skilled in the art will appreciate, these may also have an effect on pI, and therefore purification, and therefore, in some cases, may also be considered pI variants. However, because these were generated to force heterodimerization and not used as purification tools, they are classified as "steric variants." In one embodiment, the first Fc polypeptide may contain the mutations D221E / P228E / L368E, and the second Fc polypeptide may contain the mutations D221R / P228R / K409R. In another embodiment, the first Fc polypeptide may contain the mutations C220E / P228E / 368E and the second Fc polypeptide may contain the mutations C220R / E224R / P228R / K409R.

[0251] In some embodiments, heterodimerization can be facilitated by pI variants. In some aspects, pI variants can include those that increase the pI of the protein (basic changes). In other aspects, pI variants can include those that decrease the pI of the protein (acidic changes). In some cases, all combinations of these variants can be made, including combinations in which one Fc polypeptide can be wild-type or a variant that does not exhibit a pI significantly different from wild-type, and the other Fc polypeptide can be more basic or more acidic. Alternatively, one Fc polypeptide can be made more basic and the other more acidic. In some embodiments, at least one Fc polypeptide is a negative pI variant Fc containing the mutations Q295E / N384D / Q418E / N421D.

[0252] In some embodiments, a combination of steric heterodimerization variants (eg, knobs and holes) and pI variants or charge pair variants may be used.

[0253] In specific embodiments, the provided constructs contain (a) a first Fc polypeptide comprising the scubariant S364K / E357Q; and (b) a second Fc polypeptide containing the scubariant L368D / K370S and pI variants 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 capable of mediating Fc heterodimerization include the sequences set forth in SEQ ID NOs: 194 and 195. The first and second Fc polypeptides may be formatted in either polypeptide chain of the construct.

[0254] The resulting multispecific polypeptide construct can be purified by an appropriate method, such as affinity chromatography on a Protein A or Protein G column. When two nucleic acid molecules encoding different polypeptides are transformed into a cell, homodimers and heterodimers will form. Expression conditions can be adjusted so that heterodimer formation is favored over homodimer formation.

[0255] Techniques for recovering heterodimers from homodimers based on the differential affinity of the heterodimers for affinity reagents are known. In some aspects, such techniques involve designing 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 eliminate or reduce the affinity of one of the polypeptides of the Fc heterodimer for the Protein A reagent. See, e.g., WO2017134440, WO2010151792, and Jendeberg et al. (Jendeberg et al., (1997) J. Immunol. Methods, 201(1): 25-34). In some of these embodiments, the Fc region may be modified in the Protein A binding site of one member of the heterodimer to prevent Protein A binding, thereby allowing for more efficient purification of the heterodimeric fusion protein. An exemplary modification at this binding site is 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 can contain a modification (pA+ / pG-) that allows it to bind to Protein A but not Protein G. Exemplary pA+ / pG- amino acid modifications include, for human IgG1, an Fc containing serine at position 428, serine at position 434, and optionally, histidine at position 436, or those residues at the corresponding positions in human IgG2, 3, or 4. In some aspects, such amino acid modifications at positions 428, 434, and optionally, at position 436 of an IgG Fc polypeptide reduce or prevent Protein G binding and enhance protein purification.

[0256] In some embodiments, any of these modifications that confer differential affinity to affinity reagents can be combined with one or more of the other amino acid modifications described above. For example, the I253R modification can be combined with either the T366S / L368A / Y407V or 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 T366W modified Fc. Thus, in some embodiments, the I253R modification is combined with the T366S / L368A / Y407V modified Fc to disallow purification of any homodimeric Fc that may have formed. A similar modification can be utilized by combining T366S / L368A / Y407V with H453R.

[0257] In some embodiments, the Fc region of the heterodimeric molecule may further comprise one or more other Fc mutations, such as any of those described above, hi some embodiments, the heterodimeric molecule comprises an Fc region comprising a mutation that reduces effector function.

[0258] In some embodiments, one Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NO:201 (e.g., SEQ ID NO:82), 86, 207 (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 any of SEQ ID NO:201 (e.g., SEQ ID NO:83), 87, 205 (e.g., SEQ ID NO:90), 92, 209 (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 any of SEQ ID NO:203 (e.g., SEQ ID NO:84), 88, 208 (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 any of SEQ ID NO:204 (e.g., SEQ ID NO:85), 89, 206 (e.g., SEQ ID NO:91), 93, 210 (e.g., SEQ ID NO:99), or 101.

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

[0260] In some embodiments, the Fc region of the fusion protein is altered at one or more of the following positions to reduce Fc receptor binding: Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325), or Ala327 (A327). For example, Leu234Ala (L234A), Leu235Ala (L235A), Asp265Asn (D265N), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Asn325Glu (N325E), or Ala327Ser (A327S). In preferred embodiments, modifications within the Fc region reduce binding to the Fc receptor gamma receptor but have minimal effect on binding to the neonatal Fc receptor (FcRn).

[0261] In some embodiments, the fusion protein contains a polypeptide derived from an immunoglobulin hinge region. The hinge region can be selected from any of the human IgG subclasses. For example, the fusion protein can contain a modified IgG1 hinge having the sequence EPKSSDKTHTCPPC (SEQ ID NO:7), in which Cys220, which forms a disulfide with the C-terminal cysteine ​​of the light chain, is mutated to serine (e.g., Cys220Ser (C220S)). In other embodiments, the fusion protein contains a shortened hinge having the sequence DKTHTCPPC (SEQ ID NO:8).

[0262] In some embodiments, the fusion protein has a modified hinge derived from IgG4 modified to prevent or reduce strand exchange (e.g., Ser228Pro (S228P)), having 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 hinge polypeptide.

[0263] 3. Linker The provided multispecific polypeptide constructs contain a linker that joins or couples a first component containing an immunoglobulin Fc region to a second component containing a CD3-binding region. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker does not contain a substrate recognition site that is specifically recognized for cleavage by a protease. Thus, the linker in the provided multispecific polypeptide constructs does not contain an amino acid sequence that can serve as a substrate for a protease, such as an extracellular protease. For example, a non-cleavable linker does not contain a cleavage sequence containing at least one peptide bond present within a protease-cleavable peptide sequence.

[0264] 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 N-terminal to the CD3 binding region. Because the provided multispecific polypeptide constructs are multimers, such as dimers, the provided constructs include a linker joining a first Fc polypeptide and a first domain (e.g., VH) of the CD3 binding region of a first polypeptide to a second domain (e.g., VL) of the CD3 binding region of a second polypeptide. 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 to the opposing polypeptide of the Fc, such as a heterodimeric Fc, via a linker, such as the same linker.

[0265] 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).

[0266] In some embodiments, the linker is selected so that, when the CD3-binding region is conjugated to the Fc region of the multispecific polypeptide conjugate, the CD3-binding region is constrained and is unable or substantially unable to bind or engage with CD3 on the surface of cells, e.g., T cells, when the multispecific polypeptide construct contacts the cells. Various assays can be used to evaluate CD3 binding or engagement by the multispecific polypeptide construct, including assays to assess T cell binding, NFAT activation using reporter systems, cytolytic T cell activity, cytokine production, and / or expression of T cell activation markers. Exemplary assays are shown in the provided examples. Typically, the linker ensures correct folding of the polypeptide construct, does not exhibit charges inconsistent with the activity or function of the linked polypeptide, and does not form bonds or other interactions with amino acid residues in one or more of the domains that would weaken or alter the activity of the linked polypeptide. In some embodiments, the linker is a polypeptide linker. The polypeptide linker can be a flexible linker or a non-flexible linker, or a combination of both.

[0267] In some aspects, the linker is a short, medium, or long linker. In some embodiments, the linker is up to 40 amino acids in length. In some embodiments, it is up to 25 amino acids in length. In some embodiments, the linker is at least about 2 amino acids in length. In some aspects, a suitable length is, for example, at least 1 amino acid residue, typically less than about 40 amino acid residues, e.g., 2 to 25 amino acid residues, 5 to 20 amino acid residues, 5 to 15 amino acid residues, or 8 to 12 amino acids in length. In some embodiments, the linker is 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. 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.

[0268] In certain aspects, the longer the linker length, the greater the CD3 binding when the multispecific polypeptide conjugate is bound to its antigen, e.g., a TAA. Thus, in some aspects, the linker is greater than 12 amino acids in length, e.g., greater than 13, 14, 15, 16, 17, or 18 amino acids in length. 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.

[0269] The linker may be naturally occurring, synthetic, or a combination of both. Particularly suitable linker polypeptides primarily comprise amino acid residues selected from glycine (Gly), serine (Ser), alanine (Ala), and threonine (Thr). For example, the linker may contain at least 75%, e.g., at least 80%, at least 85%, or at least 90% (calculated based on the total number of residues present in the peptide linker) of amino acid residues selected from Gly, Ser, Ala, and Thr. The linker may consist exclusively of Gly, Ser, Ala, and / or Thr residues. 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 comprises only glycine residues. In some embodiments, the peptide linker comprises only glycine and serine residues.

[0270] In some embodiments, these linkers are composed primarily of the amino acids glycine and serine, and are referred to herein as GS linkers. In some embodiments, the linker contains (GGS)n (n is 1-10, e.g., 1-5, e.g., 1-3), e.g., GGS(GGS)n (SEQ ID NO:171) (n is 0-10). In a specific embodiment, the linker contains the sequence (GGGGS)n (SEQ ID NO:173) (n is 1-10 or n is 1-5, e.g., 1-3). In a further embodiment, the linker contains (GGGGGS)n (SEQ ID NO:172) (n is 1-4, e.g., 1-3). The linker may include any combination of the above, e.g., 2, 3, 4, or 5 repeats of the GS linker, GGS linker, GGGGS linker, and / or GGGGGS linker may be combined. In some embodiments, such linkers are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids in length.

[0271] In some embodiments, the linker is as follows (single letter amino acid code): GGS, GGGGS (SEQ ID NO: 149), or GGGGGS (SEQ ID NO: 135). In some embodiments, the GS linker is TIFF0007824455000057.tif41158. In some embodiments, the linker is GGGG (SEQ ID NO: 103). In some embodiments, the linker is GGGGG (SEQ ID NO: 192). In some of the foregoing examples, serine may be substituted with alanine (e.g., (Gly4Ala) or (Gly3Ala)).

[0272] In some embodiments, the linker comprises the amino acid sequence Gly x -Xaa-Gly y -Xaa-Gly z(SEQ ID NO:174) wherein each Xaa is independently selected from alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp), proline (Pro), glycine (Gly), serine (Ser), threonine (Thr), cysteine ​​(Cys), tyrosine (Tyr), asparagine (Asn), glutamine (Gln), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), and glutamic acid (Glu); and wherein x, y, and z are each integers ranging from 1 to 5. In some embodiments, each Xaa is independently selected from the group consisting of Ser, Ala, and Thr. In a particular variation, x, y, and z each equal 3, thus providing a peptide linker having the amino acid sequence Gly-Gly-Gly-Xaa-Gly-Gly-Gly-Xaa-Gly-Gly-Gly (SEQ ID NO:175), where each Xaa is selected as described above.

[0273] In some embodiments, the linker is a serine-rich linker based on repeating (SSSSG)n (SEQ ID NO:185) motifs, where n is at least 1, but can also be 2, 3, 4, 5, 6, 7, 8, and 9.

[0274] In some cases, it may be desirable to provide a peptide linker with a degree of immobility. This can be achieved by including proline residues in the amino acid sequence of the peptide linker. Thus, in some embodiments, the linker includes at least one proline residue within the amino acid sequence of the peptide linker. For example, the peptide linker may have an amino acid sequence in which at least 25% (e.g., at least 50% or at least 75%) of the amino acid residues are proline residues. In one specific embodiment, the peptide linker includes only proline residues.

[0275] In some aspects, the peptide linker comprises at least one cysteine ​​residue, e.g., one cysteine ​​residue. For example, in some embodiments, the linker comprises at least one cysteine ​​residue and an amino acid residue selected from the group consisting of Gly, Ser, Ala, and Thr. In some such embodiments, the linker comprises only glycine and cysteine ​​residues, e.g., only glycine and cysteine ​​residues. Typically, only one cysteine ​​residue is included in each peptide linker. An example of a specific linker containing a cysteine ​​residue is the amino acid sequence Gly m -Cys-Gly n (n and m are each an integer from 1 to 12, e.g., 3 to 9, 4 to 8, or 4 to 7.) In a particular variation, such a peptide linker has the amino acid sequence GGGGG-C-GGGGG (SEQ ID NO:177).

[0276] In some embodiments, the linker of the fusion protein is a structured or constrained linker. In specific embodiments, the structured linker contains the sequence (AP)n or (EAAAK)n (SEQ ID NO:178) (n is 2 to 20, preferably 4 to 10), such as, but not limited to, AS-(AP)n-GT (SEQ ID NO:179) or AS-(EAAAK)n-GT (SEQ ID NO:180) (n is 2 to 20, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). In other embodiments, the linker contains the sequence TIFF0007824455000058.tif19159 (n is 2 to 20). In some embodiments, the linker TIFF0007824455000059.tif11159. In some embodiments, such linkers, due to their structure, may be more resistant to proteolytic cleavage and therefore may offer advantages when injected in vivo.

[0277] In some embodiments, the linker is not a cleavable linker (used interchangeably with non-cleavable linker). In some embodiments, the linker is not cleavable by a protease. In some embodiments, linkers that are not cleavable or non-cleavable by a protease are generally stable for in vivo delivery or recombinant production. In some aspects, linkers that are not cleavable by a protease include those that do not contain at least one peptide bond that is preferably located within a cleavable peptide sequence or recognition site of a protease. In specific embodiments, a non-cleavable linker is not a target substrate of a protease and therefore is not preferentially or specifically cleaved by a protease compared to a linker that contains a substrate recognition site of the same protease.

[0278] In some embodiments, the linker does not contain a substrate recognition site or cleavage site for a specific protease, which is a sequence recognized by the active site of the protease and cleaved by the protease. Typically, for example, the cleavage sequence for a serine protease is composed of P1-P4 amino acids and P1'-P4' amino acids in the substrate, with cleavage occurring after the P1 position. Typically, the cleavage sequence for a serine protease is six residues long to match the extended substrate specificity of many proteases, but may be longer or shorter depending on the protease. Typically, the linker does not contain a releasable P1-P1' bond sequence recognized by the protease.

[0279] In some aspects, a non-cleavable linker, or a linker that does not contain a substrate recognition site specifically recognized for cleavage by a protease, is one that is cleaved by the protease substantially less than the cleavage of the target substrate of the protease. Typically, a protease exhibits specificity or preference for cleavage of a particular target substrate compared to other non-target substrates. The degree of such specificity can be determined based on the rate constant of cleavage of a sequence, e.g., a linker sequence, which is a measure of the preference of the protease for that substrate and the efficiency of the enzyme. Any method that determines the rate of increase in cleavage over time in the presence of various concentrations of substrate can be used to calculate the specificity constant. For example, the substrate is linked to a fluorogenic moiety, which is released upon cleavage by the protease. By determining the rate of cleavage at different protease concentrations, the specificity constant for cleavage (k cat / K m ) can be determined for a particular protease for a particular linker. In some embodiments, a non-cleavable linker, or a linker that does not contain a substrate recognition site that is specifically recognized for cleavage by a protease, may be cleaved, if at all, with a yield of less than 1×10 4 M -1 S -1 Less than 5 x 10 3 M -1 Less than S, 1 x 10 3 M -1 Less than S or 1 x 10 2 M -1 It is a linker that is cleaved by a protease at a rate less than S or slower.

[0280] In some embodiments, the linkers in the multispecific constructs provided herein do not contain substrate recognition sites for proteases, including, for example, matrix metalloproteinases (MMPs), cysteine ​​proteases, serine proteases, and plasmin activators. In specific embodiments, the linkers do not contain substrate recognition sites for proteases that are produced by tumors, by activated immune effector cells (e.g., T cells or NK cells), or by cells in the tumor microenvironment.

[0281] In some embodiments, the linker does not contain a substrate recognition site that is specifically recognized by one or more of the following enzymes or proteases: ADAMS, ADAMTS, e.g., ADAM8; ADAM9; ADAM10; ADAM12; ADAM15; ADAM17 / TACE; ADAMDECl; ADAMTS1; ADAMTS4; ADAMTS5; aspartic proteases, e.g., BACE or renin; aspartic cathepsins, e.g., cathepsin D or cathepsin E; caspases, e.g., , caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, or caspase 14; cysteine ​​cathepsins, for example, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P; cysteine ​​proteinases, for example, cruzipain; legumain; otubain 2; KLKs, for example, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10 , KLK11, KLK13, or KLK14; metalloproteinases, e.g., meprin; neprilysin; PSMA; BMP-1; MMPs, e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, or MMP27, serine proteases, e.g., activated protein C, cathepsin A, cathepsin G, chymase, coagulation factor proteases (e.g., FVII a, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, guanidinobenzoatase, HtrA1, human neutrophil elastase, lactoferrin, marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA; type II transmembrane serine proteases (TTSPs), such as DESC1, DPP-4, FAP, hepsin, matriptase 2, matriptase, TMPRSS2, TMPRSS3, or TMPRSS4;and any combination thereof. In some embodiments, the linker does not contain a substrate recognition site that is specifically recognized by an MMP, such as granzyme B, matriptase, or MMP-2.

[0282] In some embodiments, the linker does not contain an amino acid that is a substrate for granzyme B. In some embodiments, the linker does not contain an amino acid sequence having the general formula P4 P3 P2 P1↓P1′ (SEQ ID NO:150) (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 linker does not contain an amino acid sequence having the general formula P4 P3 P2 P1↓P1′ (SEQ ID NO:151) (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).

[0283] In some embodiments, the linker comprises the amino acid sequence In some embodiments, the linker does not contain the amino acid sequence Does not contain TIFF0007824455000061.tif26155.

[0284] In some embodiments, the linker does not contain an amino acid that is a substrate for matriptase. In some embodiments, the linker does not contain the sequence P1QAR↓(A / V) (SEQ ID NO:154) (P1 is any amino acid). In some embodiments, the linker does not contain the sequence RQAR(A / V) (SEQ ID NO:155). In some embodiments, the linker does not contain the amino acid sequence RQAR (SEQ ID NO:23). In some embodiments, the linker does not contain the amino acid sequence RQARV (SEQ ID NO:156).

[0285] In some embodiments, the linker does not contain an amino acid that is a substrate for one or more matrix metalloproteinases (MMPs). In some embodiments, the MMP is MMP-2. In some embodiments, the linker does not contain a sequence having the general formula P3 P2 P1↓P1′ (SEQ ID NO:157) (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 linker does not contain the general formula P3 P2 P1↓P1′ (SEQ ID NO:158) (P3 is P; P2 is Q or D; P1 is A or N; P1′ is L or I). In some embodiments, the linker does not contain the amino acid sequence PAGL (SEQ ID NO:24).

[0286] In some embodiments, the linker is It is not a linker containing the amino acid sequence shown as TIFF0007824455000062.tif99159.

[0287] 4. Antigen-binding domain: The multispecific polypeptide constructs of the present disclosure comprise at least one antigen-binding domain, e.g., at least a first antigen-binding domain and a second antigen-binding domain. In some aspects, the antigen-binding domain, or each of the antigen-binding domains, is independently selected from an antibody or antigen-binding fragment, a natural cognate binding partner, anticalin (a modified lipocalin), darpin, fynomer, centirin (a modified fibronectin III domain), a cystine-knot domain, affilin, affibody, or a modified CH3 domain. In some embodiments, the natural cognate binding partner comprises the extracellular domain of the native cognate binding partner of the TAA or a binding fragment thereof, or a variant thereof that exhibits binding activity with the TAA.

[0288] In some embodiments, a TAA is a counter-structure that is present primarily 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; the proportion of cells bearing a particular mammalian tumor-associated antigen or the level of the tumor-associated antigen on the surface of the tumor is sufficiently high that it can be targeted by an anti-tumor therapeutic, such as a provided multispecific polypeptide construct, to provide prevention or treatment for the effects of the mammalian tumor. In some embodiments, in a random statistical sample of cells derived from a mammal with a tumor, at least 50% of cells exhibiting a TAA are cancer cells. In other embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of cells exhibiting a TAA are cancer cells.

[0289] In some embodiments, the antigen-binding domain, or each of the antigen-binding domains, such as the first and second antigen-binding domains, independently, comprises one or more copies of an antibody or antigen-binding fragment thereof. In some embodiments, the antigen-binding domain, or each of the antigen-binding domains, independently, comprises one or more copies of an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, an F(ab')2 fragment, an Fv fragment, an scFv, an scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. In some embodiments, the antigen-binding domain, or each of the antigen-binding domains, such as the first and second antigen-binding domains, independently, is a single-chain antibody. In some embodiments, the single chain is an scFv, an scAb, a single-domain heavy chain antibody, or a single-domain light chain antibody. In some embodiments, the first and second antigen-binding domains each comprise one or more single-domain antibody (sdAb) fragments, e.g., V H H, V NAR , modified V H domain, or modified V K Includes domain. V H H can be generated from natural camelid heavy chain-only antibodies, genetically modified rodents producing heavy chain-only antibodies, or from naive / synthetic camelid or humanized camelid single domain antibody libraries. NAR can be generated from cartilaginous fish heavy chain-only antibodies. Various methods, including interface engineering and specific germline family selection, have been used to generate conventional heterodimeric V H Domains and V K This has been done to generate monomeric sdAbs from the domains.

[0290] In some embodiments, the antigen-binding domain of the multispecific polypeptide construct, or each of the antigen-binding domains, such as the first antigen-binding domain and / or the second antigen-binding domain, independently contains at least one sdAb or scFv that binds to a TAA. In some embodiments, at least one scFv or sdAb that binds to a TAA is positioned amino-terminal to the Fc region and / or carboxy-terminal to the CD3-binding region of the multispecific polypeptide construct. In some embodiments, the multispecific polypeptide construct contains only one scFv or sdAb that binds to a TAA, which may be positioned either amino-terminal to the Fc region and / or carboxy-terminal to the CD3-binding region. In some embodiments, the multispecific polypeptide construct contains two scFvs or sdAbs that bind to a TAA, positioned amino-terminal to the Fc region and / or carboxy-terminal to the CD3-binding region. In some embodiments, the multispecific polypeptide construct contains three scFvs or sdAbs, two of which are positioned amino terminal to the Fc region or carboxy terminal to the CD3 binding region, and the third is positioned at the other end of the multispecific polypeptide construct.

[0291] In some embodiments, the multispecific polypeptide construct is formed from or comprises two polypeptides, including a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, a VH domain of an anti-CD3 antibody or antigen-binding fragment (e.g., Fv), and an scFv or sdAb that binds to a tumor-associated antigen; and a second polypeptide comprising a second Fc polypeptide of a heterodimeric Fc region, a linker, a VL domain of an anti-CD3 antibody or antigen-binding fragment (e.g., Fv), and optionally, an identical or different scFv or sdAb that binds to a tumor-associated antigen. The scFv or sdAb that binds to a TAA may be located amino-terminal to the Fc polypeptide of the heterodimeric Fc and / or carboxy-terminal to the VH chain or VL chain of the CD3 binding region. In some embodiments, the antigen-binding domain of the multispecific polypeptide construct, or each of the antigen-binding domains independently, contains a VH sequence and a VL sequence assembled as a FAB or scFv. In some embodiments, the antigen binding domain of the multispecific polypeptide construct, or each of the antigen binding domains independently, comprises a binding domain as a single domain antibody (sdAb).

[0292] In some embodiments, the antigen binding domain, or each of the antigen binding domains, such as the first and second antigen binding domains, independently, contains multiple chains. In some embodiments, the antigen binding domain, or each of the antigen binding domains, such as the first and / or second antigen binding domains, of the multispecific polypeptide construct independently contains a VH sequence and a VL sequence assembled as a FAB.

[0293] In some embodiments, the antigen-binding domain of the multispecific polypeptide construct, or each of the antigen-binding domains, such as the first antigen-binding domain and / or the second antigen-binding domain, independently contains the VH-CH1(Fd) and VL-CL of a Fab antibody that binds to a TAA. In some embodiments, the Fab antibody containing the VH-CH1(Fd) and VL-CL is positioned amino-terminal to the Fc region and / or carboxy-terminal to the CD3-binding region of the multispecific polypeptide construct. In some embodiments, the multispecific polypeptide construct contains only one Fab antibody containing a VH-CH1(Fd) and VL-CL that binds to a TAA, which may be positioned either amino-terminal to the Fc region and / or carboxy-terminal to the CD3-binding region. In some embodiments, the multispecific polypeptide construct contains two Fab antibody fragments, each containing a VH-CH1(Fd) and VL-CL that binds a TAA, one positioned amino-terminal to the Fc region and the other positioned carboxy-terminal to the CD3-binding region.

[0294] In some embodiments, the multispecific polypeptide construct is formed from or comprises three or more polypeptides, including a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and the VH-CH1(Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen; a second polypeptide comprising a second Fc polypeptide of a heterodimeric Fc region, a linker, and optionally the same VH-CH1(Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen; and a third polypeptide comprising the other of the VH-CH1(Fd) or VL-CL of the Fab antibody fragment that binds to a TAA.

[0295] In some embodiments, the antigen binding domain, or each of the antigen binding domains independently, is or comprises the extracellular domain of the native cognate binding partner of the TAA, or a binding fragment thereof, or a variant thereof that exhibits binding activity with the TAA.

[0296] In some embodiments, each of the antigen binding domains, such as each of the first and second antigen binding domains, binds to the same antigen. In some embodiments, each of the first and second antigen binding domains binds to different antigens. In some embodiments, each of the antigen binding domains, such as each of the first and second antigen binding domains, binds to the same tumor-associated antigen (TAA). In some embodiments, each of the antigen binding domains, such as each of the first and second antigen binding domains, binds to different TAAs. In some embodiments, each of the antigen binding domains, such as each of the first and second antigen binding domains, binds to different epitopes of the same TAA. In some embodiments, each of the antigen binding domains, such as each of the first and second antigen binding domains, binds to the same epitope of the same TAA.

[0297] In some embodiments, the antigen-binding domain provides monovalent, bivalent, trivalent, or tetravalent binding to the TAA. In some embodiments, bivalent binding to a TAA comprises two antigen-binding domains that bind to the same epitope of the same antigen (e.g., monoepitopic). In some embodiments, bivalent binding to a TAA comprises two antigen-binding domains that bind to different epitopes of the same antigen (e.g., biepitopic). In some embodiments, monovalent binding to a TAA comprises one antigen-binding domain that binds to one epitope of an antigen (e.g., monoepitopic).

[0298] In some embodiments, the TAA is 1-92-LFA-3, 5T4, alpha4 integrin, alphaV integrin, alpha4beta1 integrin, alpha4beta7 integrin, AGR2, anti-Lewis Y, apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD 20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD 64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD16 6, 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, EG FR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, 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, 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.

[0299] In some embodiments, at least one antigen-binding domain, or each antigen-binding domain independently, binds to the tumor-associated antigen (TAA), folate receptor alpha (FRα). For example, the antigen-binding domain contains a binding domain as an sdAb that binds to FRα. Exemplary FRα-binding sdAbs are shown in SEQ ID NOs: 120, 121, and 122.

[0300] In some embodiments, at least one antigen-binding domain, or each antigen-binding domain independently, binds to cMET, a tumor-associated antigen (TAA). For example, the antigen-binding domain contains a binding domain as an sdAb that binds to cMET. An exemplary cMET-binding sdAb is shown in SEQ ID NO: 123 (U.S. Patent No. 9,346,884).

[0301] In some embodiments, at least one antigen-binding domain, or each antigen-binding domain independently, binds to the tumor-associated antigen (TAA) B7H3. For example, the antigen-binding domain contains a binding domain as an scFv that binds to B7H3. An exemplary B7H3-binding scFv is set forth in SEQ ID NO:124. In some embodiments, the antigen-binding domain is an sdAb, such as a VHH. An exemplary B7H3-binding sdAb is set forth in any of SEQ ID NOs:214-218. In some embodiments, the antigen-binding domain is or contains a Fab antibody fragment comprising a VH-CH1 (Fd) or LC. An exemplary B7H3 Fd is set forth in SEQ ID NO:127, and an exemplary B7H3 LC is set forth in SEQ ID NO:128 (PCT Publication No. WO2017 / 030926).

[0302] In some embodiments, at least one antigen-binding domain, or each antigen-binding domain independently, binds to the tumor-associated antigen (TAA) CD20. In some embodiments, such antigen-binding domains contain a VH set forth in SEQ ID NO: 189 and a VL set forth in SEQ ID NO: 190, or a sequence exhibiting at least, or about, 85%, 90%, 95%, 96%, 97%, 98%, 98%, or 99% sequence identity with SEQ ID NO: 189 or SEQ ID NO: 190. For example, the antigen-binding domain contains a binding domain as an scFv that binds to CD20. Exemplary CD20-binding scFvs are set forth in SEQ ID NOs: 125 and 213 (U.S. Publication No. US2005 / 0123546).

[0303] In some embodiments, at least one antigen-binding domain, or each antigen-binding domain independently, each binds to the tumor-associated antigen (TAA) DLL3. For example, the antigen-binding domain contains a binding domain as an scFv that binds to DLL3. Exemplary DLL3-binding scFvs are set forth in SEQ ID NOs: 126 and 188 (U.S. Publication No. US2017 / 0037130). In some embodiments, the antigen-binding domain is an sdAb, such as a VHH. Exemplary DLL3-binding sdAbs are set forth in either the VH set forth in SEQ ID NO: 219 or SEQ ID NO: 220. In some embodiments, the antigen-binding domain is or contains a Fab antibody fragment comprising an Fd and LC that binds to DLL3. An exemplary DLL3 Fd is set forth in SEQ ID NO: 133, and an exemplary DLL3 LC is set forth in SEQ ID NO: 134 (U.S. Publication No. US8,044,178).

[0304] In some embodiments, at least one antigen binding domain, or each antigen binding domain independently, binds to the tumor-associated antigen (TAA), 5T4. An exemplary 5T4 Fd is set forth in SEQ ID NO: 129, and an exemplary 5T4 LC is set forth in SEQ ID NO: 130. In some embodiments, the antibody binding domain comprises a VH-CH1(Fd) or VL-CL set forth in SEQ ID NOs: 167 and 168 (U.S. Patent No. 8,044,178).

[0305] In some embodiments, at least one antigen-binding domain, or each antigen-binding domain independently, binds to the tumor-associated antigen (TAA) gpNMB. In some embodiments, the antigen-binding domain is or contains a Fab fragment comprising an Fd chain and an LC chain. An exemplary gpNMB Fd is set forth in SEQ ID NO:131, and an exemplary gpNMB LC is set forth in SEQ ID NO:132.

[0306] In some embodiments, the antigen-binding domain is linked to the Fc region and / or the CD3-binding region directly or indirectly via a linker. In some embodiments, the linkage is via a linker. In some embodiments, the linker is a connecting peptide (LP), which may include a flexible or non-flexible linker as described in Section II.3, although generally the peptide linking the antigen-binding domains is not a cleavable linker.

[0307] In some embodiments, the multispecific polypeptide construct comprises a first connecting peptide (LP1) between the first antigen-binding domain and the Fc region. In some embodiments, the multispecific polypeptide construct comprises a second connecting peptide (LP2) between the CD3-binding region and the second antigen-binding domain. In some embodiments, the multispecific polypeptide construct comprises a first connecting peptide (LP1) between the first antigen-binding domain and the Fc region and a second connecting peptide (LP2) between the CD3-binding region and the second antigen-binding domain. In some aspects, the multispecific polypeptide construct has the following structural arrangement from N-terminus to C-terminus: first antigen-binding domain - LP1 - Fc region - linker - CD3-binding region - LP2 - second antigen-binding domain. In some embodiments, the two connecting peptides are not identical to each other.

[0308] In some embodiments, LP1 or LP2 are independently peptides of about 1 to 20 amino acids in length. In some embodiments, LP1 or LP2 are independently peptides that are or include a Gly-Ser linker set forth in SEQ ID NOs: 10-13, 119, 135, 147, 149, or GGS.

[0309] III. Pharmaceutical Compositions Compositions of any of the provided multispecific polypeptide constructs are provided herein. It will be understood that administration of therapeutic entities according to the present disclosure will be administered with appropriate carriers, excipients, and other agents incorporated into the formulation to provide improved entry, delivery, tolerance, etc. Many suitable formulations can be found in the formulary known to all pharmacists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug and Seymour therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water emulsions, water-in-oil emulsions, carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the above mixtures may be suitable in treatments and therapies according to the present disclosure, provided that the active ingredients in the formulation are not inactivated by the formulation and the formulation is physiologically compatible and acceptable for the route of administration.For additional information regarding formulations, excipients, and carriers well known to pharmacists, see also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. Toxicol Pharmacol. 32(2):210-8 (2000); Wang W. "Lyophilization and development of solid protein pharmaceuticals." Int. J. Pharm. 203(1-2):1-60 (2000); Charman WN "Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts." J Pharm Sci. 89(8):967-78 (2000); Powell et al. "Compendium of excipients for parenteral formulations." PDA J Pharm Sci Technol. 52:238-311 (1998), and citations therein.

[0310] In some embodiments, the multispecific polypeptide constructs, conjugated multispecific polypeptide constructs, and compositions thereof, as well as derivatives, fragments, analogs, and homologs thereof, collectively referred to herein as therapeutic agents, can be incorporated into pharmaceutical compositions suitable for administration. The principles and considerations involved in the preparation of such compositions, as well as guidance in the selection of components, are provided, for example, in Remington's Pharmaceutical Sciences: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0311] Such compositions typically comprise the multispecific polypeptide construct or a conjugate thereof and a pharmaceutically acceptable carrier. When the multispecific polypeptide construct comprises an antibody fragment, the smallest fragment of the antibody that specifically binds to the target protein can be used. For example, based on the variable region sequence of the antibody, a peptide molecule can be designed that retains the antibody's ability to bind to the target protein sequence. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)).

[0312] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in this field, which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmacologically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated.

[0313] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0314] The pharmaceutical compositions of the present disclosure are formulated to be compatible with their intended route of administration. Examples of administration routes include parenteral administration, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium sulfite; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetic acid, citric acid, or phosphate, and an agent for adjusting osmolality such as sodium chloride or dextrose. pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0315] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (water soluble) or dispersions, or sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be appropriate to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0316] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent, optionally with one or a combination of the above-listed ingredients, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution. If the composition is lyophilized, sterilization using this method can be carried out either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. Furthermore, parenteral compositions are generally placed in a container with a sterile access port, such as an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle.

[0317] In some embodiments, the pharmaceutical composition is administered to a subject through any route, including orally, transdermally, by inhalation, intravenously, intraarterially, intramuscularly, directly to a wound site, applied to a surgical site, intraperitoneally, by suppository, subcutaneously, intradermally, transdermally, by spray, intrapleurally, intraventricularly, intraarticularly, intraocularly, or intraspinally.

[0318] Oral compositions generally contain inert diluents or edible carriers.They can be enclosed in gelatin capsules or compressed into tablets.For oral therapeutic administration, active compounds can be incorporated with excipients and used in the form of tablets, lozenges or capsules.Oral compositions can also be prepared using liquid carriers for use as mouthwash, where the compound in the liquid carrier is applied to the mouth, sipped, expectorated or swallowed.Pharmaceutically compatible binders and / or auxiliary materials can be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a flow aid such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavor.

[0319] For administration by inhalation, the multispecific polypeptide constructs are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0320] Systemic administration can also be via transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, for transmucosal administration, surfactants, bile acids, and fusidic acid derivatives.Transmucosal administration can be achieved throug...

Claims

1. A multispecific polypeptide construct comprising a first component comprising a heterodimeric immunoglobulin Fc region and a second component comprising a CD3 binding region, the CD3 binding region is a disulfide-stabilized anti-CD3 Fv antibody fragment (dsFv) comprising a heavy chain variable region (VH) and a light chain variable region (VL); the Fc is a heterodimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide, wherein the VH of the anti-CD3 dsFv is linked to the first Fc polypeptide of the heterodimeric Fc, and the VL of the anti-CD3 dsFv is linked to the second Fc polypeptide of the heterodimeric Fc; the Fc region and the CD3 binding region are linked by a non-cleavable linker that is a polypeptide 2 to 18 amino acids in length, and the Fc region is positioned N-terminal to the CD3 binding region; and one or both of the first and second components comprises at least one antigen-binding domain that binds to a tumor-associated antigen (TAA), the at least one antigen-binding domain being selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, and an scFv, and the at least one antigen-binding domain is linked by a linker to the N-terminus of the Fc region, the C-terminus of the CD3-binding region, or both; Multispecific polypeptide constructs.

2. In order from the N-terminus to the C-terminus, a first antigen-binding domain that binds to a TAA; immunoglobulin Fc region; a non-cleavable linker; a CD3 binding region that binds to CD3 (CD3ε); and A second antigen-binding domain that binds to the TAA 2. The multispecific polypeptide construct of claim 1, comprising:

3. In order from the N-terminus to the C-terminus, immunoglobulin Fc region; a non-cleavable linker; a CD3 binding region that binds to CD3 (CD3ε); and Antigen-binding domain that binds to TAA 2. The multispecific polypeptide construct of claim 1, comprising:

4. In order from the N-terminus to the C-terminus, an antigen-binding domain that binds to a TAA; immunoglobulin Fc region; a non-cleavable linker; and CD3-binding domain that binds to CD3 (CD3ε) 2. The multispecific polypeptide construct of claim 1, comprising:

5. A multispecific polypeptide construct described in any one of claims 1 to 4, wherein one or both of the first and second Fc polypeptides of the heterodimeric Fc region are variant Fc polypeptides comprising at least one modification to induce heterodimerization compared to the Fc region of human IgG1, human IgG2, or human IgG4.

6. A multispecific polypeptide construct as described in claim 5, wherein one or both of the first and second Fc polypeptides of the heterodimeric Fc region are variant Fc polypeptides comprising at least one modification to induce heterodimerization compared to the Fc region of human IgG1.

7. A multispecific polypeptide construct as described in claim 5 or 6, wherein the modification is compared to the Fc polypeptide or an immunologically active fragment thereof shown in SEQ ID NO:

1.

8. A multispecific polypeptide construct described in any one of claims 5 to 7, wherein at least one modification is selected from a steric modification, a knob-into-hole modification, a charge mutation to increase the electrostatic complementarity of the polypeptide, a modification to alter the isoelectric point (pI variant), or a combination thereof. (i) the Fc region comprises a polypeptide comprising at least one modification to enhance FcRn binding; and / or (ii) the Fc region is A polypeptide comprising at least one amino acid modification that reduces effector function and / or reduces binding to an effector molecule selected from an Fcγ receptor or C1q. Including, 9. The multispecific polypeptide construct of any one of claims 1 to 8.

10. A multispecific polypeptide construct described in any one of claims 1 to 9, wherein the linker is a polypeptide of about 2 to 14 amino acids, about 2 to 12 amino acids, about 2 to 10 amino acids, about 2 to 8 amino acids, about 2 to 6 amino acids, about 6 to 18 amino acids, about 6 to 14 amino acids, about 6 to 12 amino acids, about 6 to 10 amino acids, about 6 to 8 amino acids, about 8 to 18 amino acids, about 8 to 14 amino acids, about 8 to 12 amino acids, about 8 to 10 amino acids, about 10 to 18 amino acids, about 10 to 14 amino acids, about 10 to 12 amino acids, about 12 to 18 amino acids, about 12 to 14 amino acids, or about 14 to 18 amino acids.

11. (i) The linker is a polypeptide that is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids in length; (ii) the linker is a polypeptide that is 3 to 18 amino acids in length; and / or (iii) the linker is a polypeptide that is 15 to 18 amino acids in length; 11. The multispecific polypeptide construct of any one of claims 1 to 10.

12. A multispecific polypeptide construct described in any one of claims 1 to 11, wherein the non-cleavable linker comprises GS, GGS, GGGGS (SEQ ID NO:149), GGGGGS (SEQ ID NO:135), and combinations thereof.

13. The non-cleavable linker is (GGS)n, where n is 1 to 6; (GGGGS)n (SEQ ID NO:173), where n is 1 to 3; or (GGGGGS)n (SEQ ID NO:172), where n is 1 to 3 13. The multispecific polypeptide construct of any one of claims 1 to 12, comprising:

14. The multispecific polypeptide construct of any one of claims 1 to 13, wherein the non-cleavable linker is or comprises an amino acid sequence selected from GGS, GGGGS (SEQ ID NO:149), GGGGGS (SEQ ID NO:135), (GGS)2 (SEQ ID NO:10), GGSGGSGGS (SEQ ID NO:11), GGSGGSGGSGGS (SEQ ID NO:12), GGSGGSGSGGSGGGS (SEQ ID NO:13), GGGGGSGGGGGSGGGGGS (SEQ ID NO:119), GGSGGGGSGGGGSGGGGS (SEQ ID NO:147), and GGGSGGGGSGGGGGS (SEQ ID NO:170).

15. A multispecific polypeptide construct described in any one of claims 1 to 14, wherein the VH of the CD3 binding region is linked to the same Fc polypeptide as one of at least one antigen binding domain that binds to the TAA.

16. A multispecific polypeptide construct as described in claim 15, wherein the Fc polypeptide linked to the VL of the CD3 binding region does not contain an antigen binding domain that binds to a TAA.

17. A multispecific polypeptide construct described in any one of claims 1 to 16, wherein only one of the first and second components comprises at least one antigen-binding domain that binds to a TAA.

18. The antigen-binding domain, or each of the antigen-binding domains independently, is selected from the group consisting of 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, CD6, CD9, CD11a, C D19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD 56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132 (IL-2RG), CD133, CD137, CD138, C D166, 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 , 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. (i) comprising 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; or (ii) at least comprising 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; 19. The multispecific polypeptide construct of any one of claims 1 to 18.

20. A multispecific polypeptide construct described in any one of claims 1 to 19, wherein the disulfide-stabilized CD3 binding region comprises a VH chain containing the mutation G44C and a VL chain containing the mutation G100C according to the Kabat numbering.

21. The CD3 binding region of claim 1 （i） a VH having the amino acid sequence of any of SEQ ID NOs: 44, 49-62, 197, and 198, 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: 44, 49-62, 197, and 198; and a VL having the amino acid sequence of any of SEQ ID NOs: 64, 72, 74, 76, 78-81, 191, 200, and 212, 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: 64, 72, 74, 76, 78-81, 191, 200, and 212; (ii) the amino acid sequence of SEQ ID NO:44 and the amino acid sequence of SEQ ID NO:72; and / or (iii) the amino acid sequence of SEQ ID NO:198 and the amino acid sequence of SEQ ID NO:200, or the amino acid sequence of SEQ ID NO:197 and the amino acid sequence of SEQ ID NO:200 21. The multispecific polypeptide construct of claim 20, comprising:

22. A multispecific polypeptide construct as described in claim 19 or 20, wherein VH has the amino acid sequence of SEQ ID NO:44 or a sequence that shows at least 95% sequence identity with SEQ ID NO:44, and VL has the amino acid sequence of SEQ ID NO:72 or a sequence that shows at least 95% sequence identity with SEQ ID NO:

72.

23. The CD3 binding region of claim 1, a VH CDR1 sequence comprising at least the amino acid sequence GFTFNTYAMN (SEQ ID NO:211); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATY (SEQ ID NO:212); 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: 229); a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO:230); and VL CDR3 sequence containing at least the amino acid sequence ALWYSNHWV (SEQ ID NO: 225) Including, 23. The multispecific polypeptide construct of any one of claims 1 to 22.

24. A multispecific polypeptide construct described in any one of claims 1 to 23, conjugated to a drug.

25. The multispecific polypeptide construct of claim 24, wherein the agent is a therapeutic agent, an anti-tumor agent, a toxin or fragment thereof, a detectable moiety, or a diagnostic agent.

26. A polynucleotide encoding the multispecific polypeptide construct described in any one of claims 1 to 25.

27. ​​A vector comprising the polynucleotide of claim 26.

28. The vector of claim 27, which is an expression vector.

29. A cell comprising one or more polynucleotides according to claim 26, or one or more vectors according to claim 27 or 28.

30. A pharmaceutical composition comprising a multispecific polypeptide construct according to any one of claims 1 to 25 and a pharmaceutically acceptable carrier.

31. A method for treating a T cell comprising contacting a target cell and a T cell with a multispecific polypeptide construct according to any one of claims 1 to 25 or a pharmaceutical composition according to claim 30, the target cells express a tumor-associated antigen recognized by the multispecific polypeptide construct; Ex vivo or in vitro methods of stimulating or inducing an immune response.

32. A composition comprising a multispecific polypeptide construct described in any one of claims 1 to 25 or a pharmaceutical composition described in claim 30 for use in stimulating or inducing an immune response in a subject having a disease or condition.

33. Use of a multispecific polypeptide construct described in any one of claims 1 to 25 or a pharmaceutical composition described in claim 30 for the manufacture of a medicament for stimulating or inducing an immune response in a subject or for treating a disease or condition in a subject.

34. The composition described in claim 32, which increases the immune response against tumors or cancer.

35. The composition of claim 32 or claim 34 for treating a disease or condition in a subject.

36. A composition comprising a multispecific polypeptide construct described in any one of claims 1 to 25 or a pharmaceutical composition described in claim 30 for use in treating a disease or condition in a subject.

37. A composition described in claim 35 or claim 36, wherein the disease or condition is a tumor or cancer.

38. The use described in claim 33, wherein the disease or condition is a tumor or cancer.

39. A composition described in any one of claims 32 and 34 to 37, wherein the subject is a human.

40. The use of claim 33 or claim 38, wherein the subject is a human.

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