Multispecific polypeptide constructs having 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 based on U.S. Provisional Application No. 62 / 656,331, filed on April 11, 2018, entitled "MULTISPECIFIC POLYPEPTIDE CONSTRUCTS HAVING CONSTRAINED CD3 BINDING AND RELATED METHODS AND USES", the content of which is hereby incorporated by reference in its entirety.

[0002] Incorporation by Reference of Sequence Listing This application is filed together with a sequence listing in electronic format. The sequence listing is provided as a file named 744952000240SeqList.TXT, created on April 10, 2019, with a size of 212 kilobytes. The information in the electronic format of the sequence listing is hereby incorporated by reference in its entirety.

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

Background Art

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

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

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

Summary of the Invention

[0007] Summary The present disclosure provides multispecific polypeptide constructs that exhibit restricted CD3 binding. In some 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; one or both of the first and second components comprise 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 of the multispecific polypeptide construct and / or carboxy-terminal to the CD3 binding region. 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 of the multispecific polypeptide construct and / or carboxy-terminal to the CD3 binding region. In a specific embodiment of the provided multispecific polypeptide construct, at least one antigen-binding domain is positioned carboxy-terminal to the CD3 binding region of the multispecific polypeptide construct.

[0008] In order from the N-terminus to the C-terminus, a multispecific polypeptide construct comprising: 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 domain that binds to CD3 (CD3ε); and a second antigen-binding domain that binds to a tumor-associated antigen (TAA) is provided herein. Also provided is a multispecific polypeptide construct comprising, in order from the N-terminus to the C-terminus: an immunoglobulin Fc region; a linker such as a non-cleavable linker; a CD3-binding domain that binds to CD3 (CD3ε); and an antigen-binding domain that binds to a tumor-associated antigen (TAA). Also provided is a multispecific polypeptide construct comprising, in order from the N-terminus to the 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 domain 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 the Fc region on the N-terminal side of the CD3-binding region reduces or prevents the ability of the CD3-binding region to bind CD3. In some embodiments, the first component (Component #1) and the second component (Component #2) of the multispecific polypeptide construct are linked, and binding to CD3 is not permitted 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 of which can bind CD3 when the multispecific construct binds an antigen recognized by the antigen-binding domain of Component #1 or Component #2. Thus, the linkage of the CD3-binding region as described to the Fc region ensures that the multispecific polypeptide construct does not bind CD3 and does not engage in other ways 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 ability of the CD3-binding region at the site of antigen expression. This may be beneficial because it may reduce or eliminate the major binding sink of peripheral T cells and allow for more favorable distribution and localization to the site of antigen expression, such as tumor cells or the tumor microenvironment.

[0011] When the antigen-binding domain binds 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 co-engagement mediates antigen-dependent activation, cytotoxicity, cytokine release, degranulation, and proliferation of T cells. In some embodiments, the multispecific polypeptide construct can interact with FcγR and mediate innate immune effector functions, such as antibody-dependent cell cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP). In some embodiments, the multispecific polypeptide construct can interact with a complement protein, namely C1q, and mediate complement-dependent cytotoxicity.

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

[0013] Thus, in the 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 on the N-terminal side of the CD3-binding region; one or both of the first and second components comprise 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 on the amino-terminal side with respect to the Fc region of the multispecific polypeptide construct and / or on the carboxy-terminal side with respect to the CD3-binding region. 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 on the amino-terminal side with respect to the Fc region of the multispecific polypeptide construct and / or on the carboxy-terminal side with respect to the CD3-binding region. In a specific embodiment of the provided multispecific polypeptide construct, at least one antigen-binding domain is positioned on the carboxy-terminal side with respect 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 a specific embodiment, the antibody or antigen-binding fragment is a double-stranded 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 a specific embodiment, the Fv is a disulfide-stabilized Fv (dsFv) containing an interchain disulfide bond between the VH chain and the VL chain.

[0015] 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 antigen-binding fragment that is an anti-CD3 antibody or an Fv antibody fragment comprising a variable heavy chain region (VH) and a variable light chain region (VL); Fc is a heterodimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide, and the VH and VL of the anti-CD3 antibody or antigen-binding fragment are linked to the opposing polypeptides of the heterodimeric Fc; the first and second components are coupled by a non-cleavable linker, and the Fc region is positioned on the N-terminal side of the CD3 binding region; the first component comprises a first antigen-binding domain, the second component comprises a second antigen-binding domain, and each of the antigen-binding domains binds to a tumor-associated antigen (TAA). Multispecific polypeptide constructs are provided herein. In some embodiments, the CD3 binding region binds to CD3 (CD3ε). In some embodiments, the first antigen-binding domain is positioned on the amino-terminal side relative to the Fc region of the multispecific construct, and the second antigen-binding domain is positioned on the carboxy-terminal side relative to the CD3 binding region of the multispecific construct. In some embodiments, the multispecific construct comprises, in order from the N-terminus to the 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] 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 antigen-binding fragment that 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); Fc is a heterodimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide, and the VH and VL of the anti-CD3 antibody or antigen-binding fragment are linked to the opposing polypeptides of the heterodimeric Fc; the first and second components are coupled by a non-cleavable linker, and the Fc region is positioned on the N-terminal side of the CD3-binding region; one or both of the first and second components comprise an antigen-binding domain that binds to a tumor-associated antigen (TAA), is provided herein. In a specific embodiment of the provided multispecific polypeptide construct, at least one antigen-binding domain is positioned on the carboxy-terminal side relative to the CD3-binding region of the multispecific polypeptide construct. In some embodiments, the CD3-binding region binds to CD3 (CD3ε).

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

[0018] In an embodiment provided herein, the multispecific construct comprises, in order from the N-terminus to the 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 an embodiment provided herein, the multispecific construct comprises, in order from the N-terminus to the 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 aspects provided herein, the multispecific construct comprises, in order from the N-terminus to the 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 domain that binds to CD3 (CD3ε).

[0021] Multispecific polypeptide constructs are provided herein that comprise, in order from the N-terminus to the 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 domain that binds to CD3 (CD3ε); and a second antigen-binding domain that binds to a tumor-associated antigen (TAA). Also provided are multispecific polypeptide constructs that comprise, in order from the N-terminus to the C-terminus, an immunoglobulin Fc region; a linker such as a non-cleavable linker; a CD3-binding domain that binds to CD3 (CD3ε); and an antigen-binding domain that binds to a tumor-associated antigen (TAA). Also provided are multispecific polypeptide constructs that comprise, in order from the N-terminus to the 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 domain that binds to CD3 (CD3ε).

[0022] 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 native cognate binding partner, an Anticalin (modified lipocalin), a Darpin, a Fynomer, a Centyrin (modified fibronectin type III domain), a cystine knot domain, an Affilin, an Affibody, or a modified CH3 domain. In some embodiments, the native cognate binding partner includes the extracellular domain of the native cognate binding partner of the TAA or a binding fragment thereof, or variants thereof that exhibit binding activity to the TAA.

[0023] In some embodiments, the antigen-binding domain comprises one or more copies of an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding domain comprises one or more copies of an antibody or an antigen-binding fragment thereof selected from the group consisting of Fab fragments, F(ab')2 fragments, Fv fragments, scFv, scAb, dAb, single-domain heavy-chain antibodies, and single-domain light-chain antibodies. In some embodiments, the antigen-binding domain comprises one or more copies of one or more single-domain antibody (sdAb) fragments, such as V H H, V NAR , modified V H domains, or modified V K domains. V H H can be generated from the heavy-chain-only antibodies of camelids. V NAR can be generated from the heavy-chain-only antibodies of cartilaginous fish. Various methods, including interface modification and selection of specific germline families, have been implemented to generate monomeric sdAbs from conventional heterodimeric V H domains and V K 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, is 1-92-LFA-3, 5T4, α4 integrin, αV integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis Y, apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9 (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132 (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), claudin 3, claudin 4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor α (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIt binds to tumor antigens selected from IIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged1, Jagged2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin 16 (MUC16, CA-125), Na / K ATPase, NGF, nicastrin, Notch receptor, Notch1, Notch2, Notch3, Notch4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine 1-phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

[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 IgG1 isotype, IgG2 isotype, IgG3 isotype, and IgG4 subclass. In some examples, the Fc region is the Fc region of human IgG1, human IgG2, human IgG3, or human IgG4, or an immunologically active fragment thereof. In some embodiments, the Fc region comprises a polypeptide comprising the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO:1. In some cases, the Fc region comprises a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO:2. In some of such embodiments, the Fc region comprises a polypeptide comprising the amino acid sequence shown in SEQ ID NO:4, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO:4. In some examples, the Fc region comprises a polypeptide comprising the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO:5. In some examples, the Fc region comprises a polypeptide comprising the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with 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 containing 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 for inducing 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 shown in SEQ ID NO: 1 or an immunologically active fragment thereof. In some cases, one or both of the Fc polypeptides of the heterodimeric Fc region optionally comprise at least one modification for inducing heterodimerization compared to the polypeptide of the homodimeric Fc region and compared to the Fc polypeptide shown in SEQ ID NO: 1 or an immunologically active fragment thereof. In some embodiments, each of the Fc polypeptides of the heterodimeric Fc independently comprises at least one amino acid modification. In some cases, the at least one modification is selected from a steric modification, a knob-into-hole modification, a charge variant for increasing the electrostatic complementarity of the polypeptide, a modification for changing the isoelectric point (pI variant), or a combination thereof.

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

[0031] In some examples, 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 combinations 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 modification T366S / L368A / Y407V / Y349C. In some embodiments, the first Fc polypeptide comprises the modification L368D / K370S, and the second Fc polypeptide comprises the modification S364K / E357Q.

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

[0034] In some embodiments, at least one of the first and second polypeptides comprises the modification 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, an 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 amino acid sequence set forth in any of SEQ ID NO: 82, 86, or 201, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NO: 83, 87, 90, 92, 202, or 205. In some embodiments, the first Fc polypeptide and the second Fc polypeptide comprise sequences selected from the group consisting of SEQ ID NO: 82 and 83; SEQ ID NO: 86 and 87; SEQ ID NO: 201 and 202; SEQ ID NO: 82 and 90; SEQ ID NO: 86 and 92; and SEQ ID NO: 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. In some embodiments, the immunoglobulin Fc region is for preventing glycosylation, for altering Fc receptor interaction, for reducing Fc receptor binding, for enhancing interaction with CD32A, for reducing complement protein C1q binding, for extending half-life, for enhancing FcRn binding, for altering antibody-dependent cell cytotoxicity (ADCC) and / or complement-dependent cell cytotoxicity (CDC), for inducing heterodimerization, for preventing dimerization, for stabilizing homodimerization at the CH3:CH3 interface, and 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 for combinations thereof.

[0039] In some embodiments, the modification within the Fc region reduces binding to Fcγ receptors but has minimal effect on binding to the neonatal Fc receptor (FcRn). In some embodiments, the mutant or modified Fc polypeptide, using the Kabat numbering system, comprises the following mutations: 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 for enhancing FcRn binding. In some examples, the modification is at a position selected from the group consisting of Met252, Ser254, Thr256, Met428, Asn434, and combinations thereof. In some 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 amino acid sequence set forth in any of SEQ ID NO: 94, 96, or 207, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NO: 98, 100, or 209. In some embodiments, the first Fc polypeptide and the second Fc polypeptide comprise sequences selected from the group consisting of SEQ ID NO: 94 and 98, respectively; SEQ ID NO: 96 and 100, respectively; and SEQ ID NO: 207 and 209, respectively.

[0042] In some embodiments, the Fc region comprises a polypeptide comprising at least one modification for enhancing FcγR binding. In some cases, the modification is a modification at Ser239 or Ile332. In some embodiments, the glycosylation of the Fc region is modified to enhance FcγR binding as compared to the unmodified Fc region. In some 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 Fcγ receptors or C1q. In 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 amino acid sequence set forth in any of SEQ ID NO: 82, 86, 94, or 96, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NO: 83, 87, 90, 92, 98, or 100. In some embodiments, the Fc region comprises a polypeptide comprising at least one amino acid modification that reduces effector function and / or reduces binding to an effector molecule selected from Fcγ receptors 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 amino acid sequence set forth in any of SEQ ID NO: 84, 88, 95, or 97, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NO: 85, 89, 91, 93, 99, or 101.

[0045] In some embodiments, the first polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NO: 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 NO: 85, 89, 91, 93, 99, 101, 204, 206, or 210. In some embodiments, the first Fc polypeptide and the second Fc polypeptide comprise the sequences consisting of SEQ ID NO: 84 and 85, respectively; SEQ ID NO: 88 and 89, respectively; SEQ ID NO: 203 and 204, respectively; SEQ ID NO: 95 and 99, respectively; SEQ ID NO: 97 and 101, respectively; SEQ ID NO: 208 and 210, respectively; SEQ ID NO: 84 and 91, respectively; SEQ ID NO: 88 and 93, respectively; and SEQ ID NO: 203 and 206, respectively, selected from the group consisting of.

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

[0048] In some embodiments, the CD3 binding region is unable or substantially unable to bind or engage CD3 unless at least one of the antigen-binding domains binds to its TAA. In some aspects, the CD3 binding region is unable or substantially unable to bind or engage 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 - 24 amino acids, 2 - 20 amino acids, 2 - 18 amino acids, 2 - 14 amino acids, 2 - 12 amino acids, 2 - 10 amino acids, 2 - 8 amino acids, 2 - 6 amino acids, 6 - 24 amino acids, 6 - 20 amino acids, 6 - 18 amino acids, 6 - 14 amino acids, 6 - 12 amino acids, 6 - 10 amino acids, 6 - 8 amino acids, 8 - 24 amino acids, 8 - 20 amino acids, 8 - 18 amino acids, 8 - 14 amino acids, 8 - 12 amino acids, 8 - 10 amino acids, 10 - 24 amino acids, 10 - 20 amino acids, 10 - 18 amino acids, 10 - 14 amino acids, 10 - 12 amino acids, 12 - 24 amino acids, 12 - 20 amino acids, 12 - 18 amino acids, 12 - 14 amino acids, 14 - 24 amino acids, 14 - 20 amino acids, 14 - 18 amino acids, 18 - 24 amino acids, 18 - 20 amino acids, or 20 - 24 amino acids, or a polypeptide of about 2 - 24 amino acids, about 2 - 20 amino acids, about 2 - 18 amino acids, about 2 - 14 amino acids, about 2 - 12 amino acids, about 2 - 10 amino acids, about 2 - 8 amino acids, about 2 - 6 amino acids, about 6 - 24 amino acids, about 6 - 20 amino acids, about 6 - 18 amino acids, about 6 - 14 amino acids, about 6 - 12 amino acids, about 6 - 10 amino acids, about 6 - 8 amino acids, about 8 - 24 amino acids, about 8 - 20 amino acids, about 8 - 18 amino acids, about 8 - 14 amino acids, about 8 - 12 amino acids, about 8 - 10 amino acids, about 10 - 24 amino acids, about 10 - 20 amino acids, about 10 - 18 amino acids, about 10 - 14 amino acids, about 10 - 12 amino acids, about 12 - 24 amino acids, about 12 - 20 amino acids, about 12 - 18 amino acids, about 12 - 14 amino acids, about 14 - 24 amino acids, about 14 - 20 amino acids, about 14 - 18 amino acids, about 18 - 24 amino acids, about 18 - 20 amino acids, or about 20 - 24 amino acids.In some embodiments, the linker is a polypeptide that is 3 amino acids in length, 4 amino acids in length, 5 amino acids in length, 6 amino acids in length, 7 amino acids in length, 8 amino acids in length, 9 amino acids in length, 10 amino acids in length, 11 amino acids in length, 12 amino acids in length, 13 amino acids in length, 14 amino acids in length, 15 amino acids in length, 16 amino acids in length, 17 amino acids in length, 18 amino acids in length, 19 amino acids in length, or 20 amino acids in length.

[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 by a protease for cleavage. In some embodiments, the protease is produced by immune effector cells, by tumors, or by cells present in the tumor microenvironment. In some embodiments, the protease is produced by immune effector cells, and the immune effector cells are activated T cells, natural killer (NK) cells, or NK T cells. In some embodiments, the protease is selected from matriptase, matrix metalloprotease (MMP), granzyme B, and combinations thereof. In some embodiments, the protease is granzyme B.

[0052] In some embodiments, the linker has the amino acid sequence TIFF2025102819000002.tif4128 and combinations thereof. In some embodiments, the linker contains the amino acid sequence (GGS)n, where n is from 1 to 10. In some embodiments, the linker contains the amino acid sequence (GGGGS)n (SEQ ID NO:173), where n is from 1 to 10. In some embodiments, the linker contains (GGGGGS)n (SEQ ID NO:172), where n is from 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, GGGGGGGS (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, TIFF2025102819000003.tif4128 or comprises the same. In some embodiments, the linker is, TIFF2025102819000004.tif4128 or comprises the same. In some embodiments, the linker is, TIFF2025102819000005.tif4128 or comprises the same. In some embodiments, the linker is, TIFF2025102819000006.tif4128 or comprises the same. In some embodiments, the linker is, or comprises, GGGGGG (SEQ ID NO:192).

[0054] In some embodiments, the antigen-binding domain of the first component (which is the first antigen-binding domain in some cases) and the immunoglobulin Fc region are functionally linked via one or more additional amino acid linkers (referred to herein as an intra-component linker). The intra-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 intra-peptide linker present in the first component, i.e., linking the Fc region and the antigen-binding domain, can be of various lengths, for example, 5 amino acids long, 6 amino acids long, 7 amino acids long, 8 amino acids long, 9 amino acids long, 10 amino acids long, 11 amino acids long, 12 amino acids long, 13 amino acids long, 14 amino acids long, 15 amino acids long, 16 amino acids long, 17 amino acids long, 18 amino acids long, 19 amino acids long, 20 amino acids long. In some embodiments, these intra-component linkers are mainly composed of the amino acids glycine and serine and are referred to herein as GS linkers. In some embodiments, the GS linker comprises an amino acid sequence selected from the group consisting of TIFF2025102819000007.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 an 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 an antigen-binding fragment thereof, wherein one or both of the first and second polypeptides comprise 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 at least one antigen-binding domain that binds to a 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 a tumor-associated antigen (TAA). In a specific embodiment of the provided multispecific polypeptide construct, at least one antigen-binding domain is positioned carboxy-terminally with respect 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 that can bind or engage 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, an F(ab')2 fragment, an Fv fragment, a scFv, a scAb, a dAb, a single-domain heavy-chain antibody, and a single-domain light-chain antibody.

[0058] In some embodiments, the anti-CD3-binding domain comprises an Fv fragment that binds CD3ε (referred to herein 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 consisting of SEQ ID NOs: 32-62, 196-198, and 211 and 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 instances, 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 instances, 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 of the second component (which in some cases is the second antigen-binding domain) and the CD3-binding region are functionally linked via one or more additional amino acid linkers (referred to herein as an intramolecular linker within the component). The intramolecular 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 intramolecular linker present in the second component, i.e., linking the CD3-binding region and the antigen-binding domain, can be of various lengths, for example, 5 amino acids in length, 6 amino acids in length, 7 amino acids in length, 8 amino acids in length, 9 amino acids in length, 10 amino acids in length, 11 amino acids in length, 12 amino acids in length, 13 amino acids in length, 14 amino acids in length, 15 amino acids in length, 16 amino acids in length, 17 amino acids in length, 18 amino acids in length, 19 amino acids in length, 20 amino acids in length. In some embodiments, the intramolecular linker of the second component is mainly composed of the amino acids glycine and serine and is referred to herein as a GS linker. In some embodiments, the GS linker comprises an amino acid sequence selected from the group consisting of the amino acid sequence of TIFF2025102819000008.tif19160.

[0065] A multispecific polypeptide construct is provided herein that comprises a first component comprising a heterodimeric Fc region and a second component comprising an anti-CD3 antibody or an antigen-binding fragment comprising a heavy-chain variable region (VH) and a light-chain variable region (VL), wherein the VH and VL that constitute 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, 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 a TAA result in monovalent, divalent, trivalent, or tetravalent binding to the TAA. In some embodiments, the one or more antigen-binding domains that bind to a TAA are independently selected from sdAb, scFv, or Fab. In some embodiments, the one or more antigen-binding domains that bind to a TAA are single-chain antibody fragments containing VH and VL, such as single-chain molecules like sdAb or scFv. In some embodiments, the one or more antigen-binding domains that bind to a TAA are sdAb H such as VH or VH NAR . In some embodiments, at least one of the antigen-binding domains is a Fab containing 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 a V H H or VH NAR . In some embodiments, the antigen-binding domain that binds to the TAA is a V H H or VH NAR . In some embodiments, the antigen-binding domain that binds to the TAA is a V H H. In some embodiments, the antigen-binding domain that binds to the TAA is a V H H. In some embodiments, the antigen-binding domain that binds to the TAA is a VH NAR . 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 Fc domain to which the VH of the anti-CD3 binding domain is attached. NAR .

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

[0071] In some embodiments, at least one antigen-binding domain is positioned on the amino-terminal side with respect to the Fc region of one of the first or second polypeptides of the multispecific polypeptide construct and / or on the carboxy-terminal side with respect to the CD3-binding region. In some cases, at least one antigen-binding domain is positioned on the amino-terminal side with respect to the Fc region of the multispecific construct, and a second antigen-binding domain is positioned on the carboxy-terminal side with respect to the CD3-binding region of the multispecific construct. In a specific embodiment of the provided multispecific polypeptide construct, at least one antigen-binding domain is positioned on the carboxy-terminal side with respect 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 on the carboxy-terminal side with respect to the CD3-binding region of the multispecific construct. In some embodiments, at least one antigen-binding domain that is an sdAb is positioned on the amino-terminal side with respect 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, at least one antigen-binding domain that is a V H H is positioned on the carboxy-terminal side with respect to the CD3-binding region of the multispecific construct. In some embodiments, at least one antigen-binding domain that is a V H H is positioned on the amino-terminal side with respect to the Fc region of the multispecific construct.

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

[0073] In some embodiments, the multispecific polypeptide construct has the following structural arrangement from the N-terminus to the 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 the N-terminus to the 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 linker peptides, LP1 and LP2, are not identical to each other. In some cases, LP1 or LP2 is, independently, a peptide about 1 to 20 amino acids in length. In some examples, LP1 or LP2 is, independently, the Gly-Ser linker shown in SEQ ID NOs: 10-13, 119, 135, 147, 149, or a peptide containing it.

[0075] In some embodiments, the multispecific construct is a construct having any of the structural arrangements shown in FIG. 1. In some embodiments, the construct is a bispecific construct having the following structural arrangement from the N-terminus to the 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. Coupled to the first antigen-binding domain is a central immunoglobulin Fc polypeptide region that controls FcγR interaction and / or FcRn interaction. In some embodiments, the central immunoglobulin Fc polypeptide region is a heterodimer. The immunoglobulin Fc polypeptide region is coupled to a linker that is located on the C-terminal side relative to the end of the immunoglobulin Fc polypeptide region. The linker is attached to an anti-CD3 binding sequence that is located on the C-terminal side relative to the Fc region and, 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 the TAA. In some embodiments, the second antigen-binding domain binds to the same TAA as the first antigen-binding domain located in 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 TAA different from that of the first antigen-binding domain.

[0076] In some 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 a modified Fv antibody fragment that includes a disulfide bond between the heavy chain variable region (VH) and the light chain variable region (VL) to thereby create a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv). In some embodiments, the VH domain and the VL domain that constitute 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 aspects such as provided, the anti-CD3 dsFv does not engage CD3 unless the multispecific polypeptide construct binds to cognate antigen.

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

[0079] In any of the provided embodiments, the anti-CD3 antibody or antigen-binding fragment comprises a VH CDR1 with the amino acid sequence TYAMN (SEQ ID NO:16), an amino acid sequence TIFF2025102819000009.tif4128 for VH CD2; an amino acid sequence TIFF2025102819000010.tif4128 for VH CDR3; an amino acid sequence TIFF2025102819000011.tif4128 for VL CDR1; a VL CDR2 with the amino acid sequence GTNKRAP (SEQ ID NO:20); and a VL CDR3 with 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); an amino acid sequence comprising VH CDR3 comprising TIFF2025102819000012.tif4128; an amino acid sequence comprising VL CDR1 comprising TIFF2025102819000013.tif4128; a VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO:20); and a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO:21).

[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); an amino acid sequence comprising VH CDR3 sequence comprising at least TIFF2025102819000014.tif4128; an amino acid sequence comprising VL CDR1 sequence comprising at least TIFF2025102819000015.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 of the provided embodiments, the anti-CD3 antibody or antigen-binding fragment has a VH having the amino acid sequence of any of SEQ ID NO: 14, 32-62, 196-198, and 211, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 14, 32-62, 196-198, and 211; and a VL having the amino acid sequence of any of SEQ ID NO: 15, 63-81, 191, 199, 200, and 212, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 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 has a VH having the amino acid sequence of any of SEQ ID NO: 14, 32-43, 45-47, 48, 196, and 211, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 14, 32-43, 45-47, 48, 196, and 211; and a VL having the amino acid sequence of any of SEQ ID NO: 15, 63, 65-71, 73, 75, 77, and 199, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 15, 63, 65-71, 73, 75, 77, and 199. In some cases, the anti-CD3 dsFv contains 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 contains 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 the VL chain region of the CD3-binding domain each independently comprise at least one amino acid modification. In some embodiments, at least one amino acid modification of the VH chain region and the VL chain region of the CD3-binding domain increases the stability of the CD3-binding domain. In some embodiments, at least one amino acid modification of the VH chain region and the VL chain region of the CD3-binding domain increases the ability of the CD3-binding domain to bind to CD3. In some embodiments, at least one amino acid modification of the VH chain region and the VL chain region of the CD3-binding domain increases the stability of the CD3-binding domain by creating a disulfide linkage between the VH chain region and the VL chain region.

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

[0086] In some embodiments, the anti-CD3 dsFv comprises a VH having the amino acid sequence of any of SEQ ID NO: 44, 49-62, 197, and 198, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 44, 49-62, 197, and 198; and a VL having the amino acid sequence of any of SEQ ID NO: 64, 72, 74, 76, 78-81, 191, 200, and 212, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 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 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 construct can be used 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 each other, for example, each is 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 plan. 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 include one or more disulfide bonds.

[0091] The present disclosure also provides an isolated nucleic acid molecule or polynucleotide encoding at least a portion of the multispecific construct described herein, and / or one or more nucleic acid molecules encoding the multispecific construct described herein, such as, for example, 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, and vectors containing these isolated nucleic acid sequences.

[0092] The provided aspects include a polynucleotide encoding any of the provided multispecific polypeptide constructs. Also provided is a polynucleotide encoding any of the polypeptide chains of the provided multispecific polypeptide constructs. Further provided is a polynucleotide 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 within the sequence. In some cases, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter. In some aspects, the multispecific polypeptide construct includes a third polypeptide chain, and the polynucleotide further includes a third nucleic acid encoding the third polypeptide of the multispecific construct. In some aspects, the third nucleic acid is separated from the first and / or second polypeptides by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, and / or the third nucleic acid sequence is operably linked to the same promoter as the first and / or second nucleic acid sequences. In some examples, the nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping is selected from T2A, P2A, E2A, or F2A (encoded by the sequences shown in SEQ ID NO: 159-164, or SEQ ID NO: 165).

[0093] Vectors comprising any of the provided polynucleotides are provided herein. In some aspects, 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] A cell comprising either the provided polynucleotide or vector is provided. In some cases, the cell is recombinant or isolated. In some examples, the cell is a mammalian cell. In some examples, the cell is a HEK293 cell or a CHO cell.

[0095] The present disclosure provides a method for making a multispecific construct by culturing a cell comprising such a nucleic acid sequence under conditions that result in the expression of the multispecific construct. In some embodiments, the cell comprises such a vector.

[0096] A method for making a multispecific polypeptide construct is provided herein, comprising the steps of introducing either the provided polynucleotide or vector into a cell, and culturing the cell under conditions that result in the expression of the multispecific construct for making the multispecific polypeptide construct. A method for making a multispecific polypeptide construct is also provided, comprising culturing any of the provided cells under conditions under which the multispecific polypeptide is expressed or produced by the cell. In some cases, the cell is a mammalian cell. In some examples, the cell is a HEK293 cell or a CHO cell. In some embodiments, the method further comprises the step of isolating or purifying the multispecific polypeptide construct from the cell. In some cases, the multispecific polypeptide construct is a heterodimer.

[0097] A multispecific polypeptide construct made by any of the provided methods is provided herein.

[0098] A method for stimulating or inducing an immune response is provided herein, comprising contacting a target cell and a T cell with either the provided multispecific polypeptide construct or a pharmaceutical composition, wherein the target cell expresses a tumor-associated antigen recognized by the multispecific polypeptide construct. In some embodiments, the target cell is a tumor cell expressing a tumor-associated antigen (TAA).

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

[0100] Provided is a method of stimulating or inducing an immune response in a subject, comprising administering to the subject in need thereof, in a therapeutically effective amount, either the provided multispecific conjugate or pharmaceutical composition. In some cases, the method increases cellular immunity. In some embodiments, the method increases T cell activity. In some embodiments, the method increases cytotoxic T lymphocyte (CTL) activity. In some examples, the immune response against a tumor or cancer is increased. In some embodiments, the method treats a disease or condition in a subject.

[0101] The present disclosure also provides a method of treating, preventing, delaying the progression of, or otherwise alleviating one or more pathologies, or alleviating symptoms associated with such pathologies, by administering to a subject in need thereof the multispecific polypeptide construct of the present disclosure. Also provided herein is a method of treating a disease or condition in a subject, comprising administering to the subject in need thereof, in a therapeutically effective amount, either the provided multispecific conjugate or pharmaceutical composition. In some embodiments, the disease or condition is a 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), a farm animal, 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 modes of use can be administered at any stage of the disease. For example, such multispecific polypeptide constructs can be administered to patients suffering from 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 modes of use can be used in treatment regimens including neoadjuvant therapy.

[0105] The multispecific polypeptide constructs of the present disclosure used in any of these methods and modes of use 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 biologic agents. In some embodiments, the multispecific polypeptide construct is administered in combination with one or more additional agents such as, by way of non-limiting example, chemotherapeutic agents such as alkylating agents, antimetabolites, antimicrotubule 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 platinum-based chemotherapy such as carboplatin or cisplatin. In some embodiments, the additional agent is a kinase inhibitor, e.g., a targeted agent such as sorafenib or erlotinib. In some embodiments, the additional agent is a targeted 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 one of ordinary skill in the art. In some embodiments, the additional agent is a chemotherapeutic agent well known to one of ordinary 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 agent or an anti-tumor agent. In some embodiments, the additional agent is a vaccine, an oncolytic virus, and / or, by way of non-limiting example, a toll-like receptor (TLR) agonist and / or a DC activator such as αCD40. In some embodiments, the additional agent is a tumor-targeting antibody designed to kill tumors via ADCC or via direct conjugation to 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 Btk inhibitors 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 IDO inhibitors, αCSF1R inhibitors, αCCR4 inhibitors, TGFβ, myeloid-derived suppressor cells, or regulatory T cells. 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 combinations 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 myeloid-derived suppressor cells. In some embodiments, the inhibitor is regulatory T cells.

[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 during and / or after treatment in combination with one or more additional agents such as, for example, chemotherapeutic agents, anti-inflammatory agents, and / or immunosuppressive agents. 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, for example, 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 either singly or in multiple doses.

[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 the treatment regimen.

[0113] In addition to the foregoing elements, the multispecific polypeptide construct may contain additional elements, such as amino acid sequences on the N-terminal side or C-terminal side of the multispecific polypeptide construct. For example, the multispecific polypeptide construct may include a targeting moiety that facilitates delivery to cells or tissues 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, together with the multispecific polypeptide constructs of the present disclosure, a conjugated agent, a linker, and any of the other components described herein.

[0115] The present disclosure also relates to immunoconjugates comprising a multispecific polypeptide construct conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin derived from bacteria, fungi, plants, or animals, or fragments thereof), or a radioisotope (i.e., a radioconjugate). Suitable cytotoxic agents for use in targeting diseased T cells in T cell-derived lymphomas and the like include, for example, dolastatin and its derivatives (e.g., auristatin E, AFP, MMAD, MMAF, MMAE). In some embodiments, the agent is 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 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, for example, cleavable or non-cleavable, or the two or more linkers are different, for example, at least one is cleavable and at least one is non-cleavable.

[0117] Multispecific polypeptide constructs and their conjugates are useful in methods of 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 injury, ischemia, etc. For example, indications include leukemias including T cell acute lymphoblastic leukemia (T-ALL), lymphocytic disorders including multiple myeloma, and solid tumors including breast cancer including lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, and triple negative breast cancer. For example, indications include bone disorders or metastases of cancers regardless of primary tumor origin; non-limiting examples include breast cancer including ER / PR+ breast cancer, Her2+ breast cancer, triple negative breast cancer; colorectal cancer; endometrial cancer; gastric cancer; glioblastoma; head and neck cancer such as esophageal cancer; lung cancer such as non-small cell lung cancer as a non-limiting example; multiple myeloma; ovarian cancer; pancreatic cancer; prostate cancer; sarcoma such as osteosarcoma; kidney cancer such as renal cell cancer as a non-limiting example; and / or skin cancer such as squamous cell carcinoma, basal cell carcinoma, or melanoma as a non-limiting example. 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] There is provided a pharmaceutical composition comprising any of the multispecific polypeptide constructs provided herein and a pharmaceutically acceptable carrier. In some cases, the pharmaceutical composition is sterile. The pharmaceutical compositions according to the present disclosure may comprise the multispecific polypeptide constructs and carriers of the present disclosure. These pharmaceutical compositions may be included, for example, in kits such as diagnostic kits.

[0119] Those skilled in the art will recognize that the antibodies of the present disclosure have diverse uses. For example, the proteins of the present disclosure are used as therapeutic agents for various disorders. The antibodies of the present disclosure are also used as reagents or diagnostic tools within diagnostic kits, or these antibodies may be used in competitive assays to generate therapeutic reagents. [Invention 1001] A multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3-binding region, wherein the CD3-binding region is an antigen-binding fragment that is an anti-CD3 antibody or an Fv antibody fragment comprising a variable heavy chain region (VH) and a variable light chain region (VL); Fc is a heterodimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide, and the VH and VL of the anti-CD3 antibody or antigen-binding fragment are linked to the opposing polypeptides of the heterodimeric Fc; the first and second components are coupled by a 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), a multispecific polypeptide construct. [Invention 1002] The multispecific polypeptide construct of Invention 1001, wherein the CD3-binding region binds to CD3 (CD3ε). [Invention 1003] The multi - specific polypeptide construct of the present invention 1001 or 1002, wherein the first antigen - binding domain is positioned on the amino - terminal side with respect to the Fc region of the multi - specific construct, and the second antigen - binding domain is positioned on the carboxyl - terminal side with respect to the CD3 - binding region of the multi - specific construct. [The present invention 1004] In order from the N - terminus to the 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) The multi - specific polypeptide construct according to any one of the present inventions 1001 to 1003, comprising: [The present invention 1005] A multi - specific polypeptide construct comprising a first component containing an immunoglobulin Fc region and a second component containing a CD3 - binding region, wherein the CD3 - binding region is an antigen - binding fragment that is an anti - CD3 antibody or an Fv antibody fragment (dsFv) stabilized by a disulfide bond containing a variable heavy chain (VH) and a variable light chain (VL); Fc is a heterodimeric Fc containing a first Fc polypeptide and a second Fc polypeptide, and the VH and VL of the anti - CD3 antibody or antigen - binding fragment are linked to the opposing polypeptides of the heterodimeric Fc; the first and second components are coupled by a non - cleavable linker, the Fc region is positioned on the N - terminal side of the CD3 - binding region; and one or both of the first and second components contain an antigen - binding domain that binds to a tumor - associated antigen (TAA), The multi - specific polypeptide construct. [The present invention 1006] A multi - specific polypeptide construct comprising a first component containing an immunoglobulin Fc region and a second component containing a CD3 - binding region, The CD3-binding region is an antigen-binding fragment that is an anti-CD3 antibody or an Fv antibody fragment containing a variable heavy chain (VH) and a variable light chain (VL); The Fc is a heterodimeric Fc containing a first Fc polypeptide and a second Fc polypeptide, and the VH and VL of the anti-CD3 antibody or antigen-binding fragment are linked to the opposing polypeptides of the heterodimeric Fc; The first and second components are coupled by a non-cleavable linker, and the Fc region is positioned on the N-terminal side of the CD3-binding region; and One or both of the first and second components contain an antigen-binding domain that binds to a tumor-associated antigen (TAA), and the antigen-binding domain is a single-chain antibody fragment, Multispecific polypeptide construct. [Invention 1007] The multispecific polypeptide construct of Invention 1006, wherein the single-chain antibody fragment is a single-domain antibody or a single-chain variable fragment (scFv). [Invention 1008] The multispecific polypeptide construct according to any one of Inventions 1005 to 1007, wherein the CD3-binding region binds to CD3 (CD3ε). [Invention 1009] In order from the N-terminus to the 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) The multispecific polypeptide construct according to any one of Inventions 1005 to 1008, comprising [Invention 1010] In order from the N-terminus to the 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) A multispecific polypeptide construct according to any one of the present inventions 1005 to 1008, comprising [Present Invention 1011] In order from the N-terminus to the 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) A multispecific polypeptide construct according to any one of the present inventions 1005 to 1008, comprising [Present Invention 1012] A multispecific polypeptide construct according to any one of the present inventions 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 for inducing heterodimerization as compared to the Fc region of human IgG1, human IgG2, or human IgG4. [Present Invention 1013] A multispecific polypeptide construct according to any one of the present inventions 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 for inducing heterodimerization as compared to the Fc region of human IgG1 and optionally as compared to the Fc polypeptide shown in SEQ ID NO:1 or an immunologically active fragment thereof. [Present Invention 1014] A multispecific polypeptide construct according to present invention 1012 or present invention 1013, wherein each of the Fc polypeptides of the heterodimeric Fc independently comprises at least one amino acid modification. [Present Invention 1015] A multispecific polypeptide construct according to any one of the present inventions 1012 to 1014, wherein the at least one modification is selected from a steric modification, a knob-into-hole modification, a charge variant for increasing the electrostatic complementarity of the polypeptide, a modification for changing the isoelectric point (pI variant), or a combination thereof. [Present Invention 1016] A multispecific polypeptide construct according to any one of inventions 1001 to 1015, wherein the first Fc polypeptide of the heterodimeric Fc comprises a modification selected from Thr366Ser, Leu368Ala, Tyr407Val, and combinations thereof, and the second Fc polypeptide of the heterodimeric Fc comprises the modification T366W. [Invention 1017] A multispecific polypeptide construct according to invention 1016, wherein the first and second Fc polypeptides further comprise a modification of a non-cysteine residue to a cysteine residue, the modification of the first polypeptide 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. [Invention 1018] A multispecific polypeptide construct according to any one of inventions 1001 to 1017, wherein the first Fc polypeptide comprises the modification T366W / S354C, and the second Fc polypeptide comprises the modifications T366S / L368A / Y407V / Y349C. [Invention 1019] A multispecific polypeptide construct according to any one of inventions 1001 to 1015, wherein the first Fc polypeptide comprises the modification L368D / K370S, and the second Fc polypeptide comprises the modification S364K / E357Q. [Invention 1020] A multispecific polypeptide construct according to any one of inventions 1001 to 1015, wherein at least one of the first and second polypeptides comprises the modifications Q295E / N384D / Q418E / N421D. [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 the modification is His35Arg, A multispecific polypeptide construct according to any one of inventions 1001 to 1020. [Invention 1022] A multispecific polypeptide construct according to any one of inventions 1001 to 1021, wherein one or both of the first and second Fc polypeptides comprise a polypeptide lacking Lys447. [Invention 1023] A multispecific polypeptide construct according to any one of inventions 1001 to 1022, wherein the first polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any of SEQ ID NO: 82, 86, or 201, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any of SEQ ID NO: 83, 87, 90, 92, 202, or 205. [Invention 1024] A multispecific polypeptide construct according to any one of inventions 1001 to 1023, wherein the first Fc polypeptide and the second Fc polypeptide comprise sequences selected from the group consisting of SEQ ID NO: 82 and 83 respectively; SEQ ID NO: 86 and 87 respectively; SEQ ID NO: 201 and 202 respectively; SEQ ID NO: 82 and 90 respectively; SEQ ID NO: 86 and 92 respectively; and SEQ ID NO: 201 and 205 respectively. [Invention 1025] A multispecific polypeptide construct according to any one of inventions 1001 to 1022, comprising a polypeptide in which the Fc region comprises at least one modification for enhancing FcRn binding. [Invention 1026] The multispecific fusion polypeptide construct of invention 1025, wherein the modification is at a position selected from the group consisting of Met252Y, Ser254T, Thr256E, Met428L, Met428V, Asn434S, and combinations thereof. [Invention 1027] The multispecific fusion polypeptide construct of invention 1025 or invention 1026, wherein the modification is Met252Y and Met428L, or Met252Y and Met428V. [Invention 1028] The first polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any of SEQ ID NO: 94, 96, or 207, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any of SEQ ID NO: 98, 100, or 209, a multispecific polypeptide construct of any of the present inventions 1001 - 1022 and 1025 - 1027. [Present Invention 1029] A multispecific polypeptide construct of any of the present inventions 1001 - 1028, wherein the first Fc polypeptide and the second Fc polypeptide each comprise a sequence selected from the group consisting of SEQ ID NO: 94 and 98; SEQ ID NO: 96 and 100 respectively; and SEQ ID NO: 207 and 209 respectively. [Present Invention 1030] 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 Fcγ receptor or C1q. A multispecific polypeptide construct of any of the present inventions 1001 - 1022 and 1025 - 1027, comprising the same. [Present Invention 1031] A multispecific polypeptide construct of Present Invention 1030, wherein one or more of the amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235. [Present Invention 1032] The first polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any of SEQ ID NO: 84, 88, 95, 97, 203, or 208, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any of SEQ ID NO: 85, 89, 91, 93, 99, 101, 204, 206, or 210, a multispecific polypeptide construct of any of the present inventions 1001 - 1022, 1025 - 1027, 1030, and 1031. [Present Invention 1033] The multispecific polypeptide constructs of any of Inventions 1001-1022, 1025-1027, and 1030-1032 of the present invention, wherein the first Fc polypeptide and the second Fc polypeptide each comprise a sequence selected from the group consisting of SEQ ID NO:84 and 85; SEQ ID NO:88 and 89; SEQ ID NO:203 and 204; SEQ ID NO:95 and 99; SEQ ID NO:97 and 101; SEQ ID NO:208 and 210; SEQ ID NO:84 and 91; SEQ ID NO:88 and 93; and SEQ ID NO:203 and 206 respectively. [Invention 1034] The multispecific polypeptide constructs of any of Inventions 1001-1033 of the present invention, wherein the CD3 binding region cannot or substantially cannot bind or engage CD3 unless at least one of the antigen binding domains binds to the TAA. [Invention 1035] The multispecific polypeptide constructs of any of Inventions 1001-1033 of the present invention, wherein the CD3 binding region cannot or substantially cannot bind or engage CD3 unless at least two of the antigen binding domains bind to the TAA. [Invention 1036] The multispecific polypeptide constructs of any of Inventions 1001-1035 of the present invention, wherein the linker is a polypeptide linker. [Invention 1037] The multispecific polypeptide construct of Invention 1036, wherein the linker is a polypeptide having a maximum length of 25 amino acids. [Invention 1038] The linker is a polypeptide of 2 to 24 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to 24 amino acids, 6 to 20 amino acids, 6 to 18 amino acids, 6 to 14 amino acids, 6 to 12 amino acids, 6 to 10 amino acids, 6 to 8 amino acids, 8 to 24 amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14 amino acids, 8 to 12 amino acids, 8 to 10 amino acids, 10 to 24 amino acids, 10 to 20 amino acids, 10 to 18 amino acids, 10 to 14 amino acids, 10 to 12 amino acids, 12 to 24 amino acids, 12 to 20 amino acids, 12 to 18 amino acids, 12 to 14 amino acids, 14 to 24 amino acids, 14 to 20 amino acids, 14 to 18 amino acids, 18 to 24 amino acids, 18 to 20 amino acids, or 20 to 24 amino acids, or 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, 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, the multispecific polypeptide construct of the present invention 1036 or the present invention 1037. [The present invention 1039] The linker is a polypeptide of 3 amino acids in length, 4 amino acids in length, 5 amino acids in length, 6 amino acids in length, 7 amino acids in length, 8 amino acids in length, 9 amino acids in length, 10 amino acids in length, 11 amino acids in length, 12 amino acids in length, 13 amino acids in length, 14 amino acids in length, 15 amino acids in length, 16 amino acids in length, 17 amino acids in length, 18 amino acids in length, 19 amino acids in length, or 20 amino acids in length, the multispecific polypeptide construct of any one of the present inventions 1036 to 1038. [The present invention 1040] A multi - specific polypeptide construct according to any one of the present inventions 1036 - 1039, wherein the linker is a polypeptide having a length of 3 to 18 amino acids. [The present invention 1041] A multi - specific polypeptide construct according to any one of the present inventions 1036 - 1040, wherein the linker is a polypeptide having a length of 12 to 18 amino acids. [The present invention 1042] A multi - specific polypeptide construct according to any one of the present inventions 1036 - 1040, wherein the linker is a polypeptide having a length of 15 to 18 amino acids. [The present invention 1043] The non - cleavable linker is A multi - specific polypeptide construct according to any one of the present inventions 1001 - 1042, including TIFF2025102819000016.tif4128 and combinations thereof. [The present invention 1044] A multi - specific polypeptide construct according to any one of the present inventions 1001 - 1043, wherein the non - cleavable linker contains (GGS)n and n is 1 - 10. [The present invention 1045] A multi - specific polypeptide construct according to any one of the present inventions 1001 - 1044, wherein the non - cleavable linker contains (GGGGS)n (SEQ ID NO: 173) and n is 1 - 10. [The present invention 1046] A multi - specific polypeptide construct according to any one of the present inventions 1001 - 1044, wherein the non - cleavable linker contains (GGGGGS)n (SEQ ID NO: 172) and n is 1 - 4. [The present invention 1047] A multi - specific polypeptide construct according to any one of the present inventions 1001 - 1044, wherein the non - cleavable linker is GGS or contains it. [The present invention 1048] A multi - specific polypeptide construct according to any one of the present inventions 1001 - 1043 and 1045, wherein the non - cleavable linker is GGGGS (SEQ ID NO: 149) or contains it. [The present invention 1049] A multispecific polypeptide construct according to any one of inventions 1001 to 1043 and 1046, wherein the non-cleavable linker is GGGGG S (SEQ ID NO: 135) or comprises the same. [Invention 1050] A multispecific polypeptide construct according to any one of inventions 1001 to 1044, wherein the non-cleavable linker is (GGS)2 (SEQ ID NO: 10) or comprises the same. [Invention 1051] A multispecific polypeptide construct according to any one of inventions 1001 to 1044, wherein the non-cleavable linker is GGSGGSGGS (SEQ ID NO: 11) or comprises the same. [Invention 1052] A multispecific polypeptide construct according to any one of inventions 1001 to 1044, wherein the non-cleavable linker is GGSGGSGGSGGS (SEQ ID NO: 12) or comprises the same. [Invention 1053] The non-cleavable linker is TIFF2025102819000017.tif4128 or a multispecific polypeptide construct according to any one of inventions 1001 to 1044 that includes the same. [Invention 1054] The non-cleavable linker is TIFF2025102819000018.tif4128 or a multispecific polypeptide construct according to any one of inventions 1001 to 1043 and 1046 that includes the same. [Invention 1055] The non-cleavable linker is TIFF2025102819000019.tif4128 or a multispecific polypeptide construct according to any one of inventions 1001 to 1049 that includes the same. [Invention 1056] The non-cleavable linker is TIFF2025102819000020.tif4128 or a multispecific polypeptide construct according to any one of inventions 1001 to 1043 and 1045 that includes the same. [The present 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 an 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 an antigen-binding fragment thereof comprising at least, wherein one or both of the first and second polypeptides comprise at least one antigen-binding domain that binds to a tumor-associated antigen (TAA), A multispecific polypeptide construct according to any one of the present inventions 1001 to 1056. [The present invention 1058] A multispecific polypeptide construct according to any one of the present inventions 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). [The present invention 1059] A multispecific polypeptide construct according to the present invention 1058, 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] A multispecific polypeptide construct according to any one of the present inventions 1001 to 1004, 1009, and 1012 to 1059, showing a divalent, trivalent, or tetravalent binding to a TAA. [The present invention 1061] A multispecific polypeptide construct according to any one of the present inventions 1005 to 1008 and 1010 to 1059, showing a monovalent, divalent, trivalent, or tetravalent binding to a TAA. [The present invention 1062] A multispecific polypeptide construct according to any one of the present inventions 1001 to 1061, wherein only one of the first and second polypeptides comprises at least one antigen-binding domain that binds to a TAA. [The present invention 1063] At least one antigen-binding domain is positioned on the amino-terminal side with respect to the Fc region of one of the first or second polypeptides of the multispecific polypeptide construct, and / or is positioned on the carboxy-terminal side with respect to the CD3-binding region of one of the first or second polypeptides of the multispecific polypeptide construct, any multispecific polypeptide construct of the present invention from 1001 to 1062. [The present invention 1064] At least one antigen-binding domain is positioned at the amino terminus with respect to the Fc region of the multispecific construct, and a second antigen-binding domain is positioned at the carboxy terminus with respect to the CD3-binding region of the multispecific construct, any multispecific polypeptide construct of the present invention from 1001 to 1062. [The present invention 1065] The antigen-binding domain, or each of the antigen-binding domains, independently comprises an extracellular domain of a native cognate binding partner of a TAA or a binding fragment thereof, or a variant thereof that exhibits binding activity to a TAA, any multispecific polypeptide construct of the present invention from 1001 to 1064. [The present invention 1066] The antigen-binding domain, or each of the antigen-binding domains, independently is an antibody or an antigen-binding fragment thereof selected from the group consisting of a Fab fragment, an F(ab')2 fragment, an Fv fragment, a scFv, a scAb, a dAb, a single-domain heavy-chain antibody, and a single-domain light-chain antibody, any multispecific polypeptide construct of the present invention from 1001 to 1005 and 1008 to 1065. [The present invention 1067] The antibody or an antigen-binding fragment thereof is an Fv, a scFv, a Fab, or a single-domain antibody (sdAb), the multispecific polypeptide construct of the present invention 1066. [The present invention 1068] The antibody or an antigen-binding fragment thereof is an sdAb, any multispecific polypeptide construct of the present invention from 1001 to 1067. [The present invention 1069] The sdAb is a human sdAb or a humanized sdAb, the multispecific polypeptide construct of the present invention 1068. [Invention 1070] The multispecific polypeptide construct of Invention 1068 or Invention 1069, wherein the sdAb is a VHH, VNAR, modified VH domain, or modified VK domain. [Invention 1071] The multispecific polypeptide construct of any one of Inventions 1001 to 1067, wherein the antibody or antigen-binding fragment thereof is a scFv. [Invention 1072] The multispecific polypeptide construct of any one of Inventions 1001 to 1005 and 1008 to 1067, wherein the antibody or antigen-binding fragment thereof is a Fab. [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 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 comprising wherein the first and / or second polypeptide further comprises the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment. The multispecific polypeptide construct of Invention 1072. [Invention 1074] The multispecific polypeptide construct of Invention 1073, wherein only one of the first and second polypeptides comprises the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment. [Invention 1075] The multispecific polypeptide construct of Invention 1073, wherein both the first and second polypeptides comprise the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment. [Invention 1076] The other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment is positioned on the amino-terminal side with respect to the Fc region of one of the first and second polypeptides of the multispecific polypeptide construct and / or on the carboxy-terminal side with respect to the CD3-binding region of one of the first and second polypeptides of the multispecific polypeptide construct, the multispecific polypeptide construct of the present invention 1074 or the present invention 1075. [The present invention 1077] The other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment is positioned on the amino-terminal side with respect to the Fc region of the first or second polypeptide and on the carboxy-terminal side with respect to the CD3-binding region of the other of the first or second polypeptides, the multispecific polypeptide construct of any one of the present inventions 1074 to 1076. [The present invention 1078] The antigen-binding domain, or each of the antigen-binding domains independently, is 1-92-LFA-3, 5T4, α4 integrin, αV integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis Y, apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9 (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132 (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), claudin 3, claudin 4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor α (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPA multispecific polypeptide construct according to any one of the present inventions 1001 to 1077 that binds to a tumor antigen selected from IIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged1, Jagged2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin 16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch1, Notch2, Notch3, Notch4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine 1-phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3. [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. Any of the multispecific polypeptide constructs of the present invention from 1001 to 1078. [The present invention 1080] The multispecific polypeptide construct of the present invention 1079, wherein the first antigen-binding domain and the second antigen-binding domain bind to different epitopes of the same TAA. [The present invention 1081] The multispecific polypeptide construct of the present invention 1079, wherein the first antigen-binding domain and the second antigen-binding domain bind to the same epitope of the same TAA. [The present invention 1082] 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 different TAAs. Any of the multispecific polypeptide constructs of the present invention from 1001 to 1078. [The present invention 1083] The anti-CD3 antibody or an 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); The amino acid sequence VH CDR3 comprising TIFF2025102819000021.tif4128; The amino acid sequence VL CDR1 comprising TIFF2025102819000022.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) comprises; or The anti-CD3 antibody or antigen-binding fragment, 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 A VH CDR3 sequence comprising at least TIFF2025102819000023.tif4128; Amino acid sequence A VL CDR1 sequence comprising at least TIFF2025102819000024.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) Comprising Any of the multispecific polypeptide constructs of the present invention 1001 to 1082. [The present invention 1084] The anti-CD3 Fv is Any amino acid sequence of SEQ ID NO:14, 32 - 43, 45 - 47, 48, 196, and 211, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO:14, 32 - 43, 45 - 47, 48, 196, and 211, for VH; and Any amino acid sequence of SEQ ID NO:15, 63, 65 - 71, 73, 75, 77, and 199, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO:15, 63, 65 - 71, 73, 75, 77, and 199, for VL Comprising any of the multispecific polypeptide constructs of the present invention 1001 - 1004 and 1006 - 1083. [The present invention 1085] The anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO:14 and the amino acid sequence of SEQ ID NO:15, any of the multispecific polypeptide constructs of the present invention 1001 - 1004 and 1006 - 1084. [Invention 1086] A multispecific polypeptide construct of any of Inventions 1001 - 1004 and 1006 - 1084, wherein the anti - CD3 Fv comprises the amino acid sequences of SEQ ID NO:196 and SEQ ID NO:199. [Invention 1087] A multispecific polypeptide construct of any of Inventions 1001 - 1004 and 1006 - 1083, wherein the Fv antibody fragment comprises a disulfide - stabilized anti - CD3 - binding Fv fragment (dsFv). [Invention 1088] A multispecific polypeptide construct of Invention 1005 or Invention 1087, wherein the disulfide - stabilized anti - CD3 - binding Fv fragment (dsFv) comprises a VH chain containing the mutation G44C and a VL chain containing the mutation G100C according to Kabat numbering. [Invention 1089] The anti - CD3 Fv is an amino acid sequence of any of SEQ ID NO:44 and 49 - 62, 197, and 198, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO:44 and 49 - 62, 197, and 198 for VH; and an amino acid sequence of any of SEQ ID NO:64, 72, 74, 76, 78 - 81, 191, 200, and 212, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO:64, 72, 74, 76, 78 - 81, 191, 200, and 212 for VL and is included in a multispecific polypeptide construct of any of Inventions 1005, 1087, and 1088. [Invention 1090] A multispecific polypeptide construct of any of Inventions 1005 and 1087 - 1089, wherein the anti - CD3 dsFv comprises the amino acid sequences of SEQ ID NO:44 and SEQ ID NO:72. [Invention 1091] A multispecific polypeptide construct according to any one of Inventions 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 comprises the amino acid sequence of SEQ ID NO: 197 and the amino acid sequence of SEQ ID NO: 200. [Invention 1092] A multispecific polypeptide construct according to any one of Inventions 1001 to 1091, conjugated to a drug. [Invention 1093] The multispecific polypeptide construct of Invention 1092, wherein the drug is a therapeutic agent, an anti-tumor agent, a toxin or a fragment thereof, a detectable moiety, or a diagnostic agent. [Invention 1094] The multispecific polypeptide construct of Invention 1093, wherein the drug is conjugated to the multispecific polypeptide construct via a linker. [Invention 1095] A polynucleotide encoding a multispecific polypeptide construct according to any one of Inventions 1001 to 1094. [Invention 1096] A polynucleotide encoding any polypeptide chain of a multispecific polypeptide construct according to any one of Inventions 1001 to 1094. [Invention 1097] A polynucleotide comprising a first nucleic acid sequence encoding a first polypeptide of a multispecific polypeptide construct according to any one of Inventions 1001 to 1094 and a second nucleic acid sequence encoding a second polypeptide of the multispecific polypeptide construct, wherein the first and second nucleic acid sequences are separated by an internal ribosome entry site (IRES) within the sequence, or by a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping. Polynucleotide. [Invention 1098] The polynucleotide of the present invention 1097, wherein the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter. [The present invention 1099] The polynucleotide of the present invention 1097 or the present invention 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) within the sequence, or by a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, and / or the third nucleic acid sequence is operably linked to the same promoter as the first and / or second nucleic acid sequence. [The present invention 1101] The polynucleotide of any one of the present inventions 1097 to 1100, wherein the nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping is selected from T2A, P2A, E2A, or F2A. [The present invention 1102] A vector comprising the polynucleotide of any one of the present inventions 1095 to 1101. [The present invention 1103] The vector of the present invention 1102, which is an expression vector. [The present invention 1104] Which is a viral vector or a eukaryotic vector, Optionally, the eukaryotic vector is a mammalian vector, The vector of the present invention 1102 or 1103. [The present invention 1105] A cell comprising one or more of the polynucleotides of any one of the present inventions 1095 to 1101, or one or more of the vectors of any one of the present inventions 1102 to 1104. [The present invention 1106] The cell of the present invention 1105, which is recombinant or isolated. [The present invention 1107] A cell of the invention 1105 or the invention 1106, which is a mammalian cell. [The invention 1108] A cell of the invention 1107, which is a HEK293 cell or a CHO cell. [The invention 1109] A step of introducing into a cell one or more polynucleotides of any one or more of the inventions 1095 to 1101 or one or more vectors of any one or more of the inventions 1102 to 1104, and A step of culturing the cell under conditions under which a multispecific polypeptide construct is produced A method for producing a multispecific polypeptide construct, comprising the above steps. [The invention 1110] A step of culturing a cell of any one of the inventions 1105 to 1108 under conditions under which a multispecific polypeptide is produced by the cell A method for producing a multispecific polypeptide construct, comprising the above step. [The invention 1111] The method of the invention 1109 or the invention 1110, further comprising a step of isolating or purifying the multispecific polypeptide construct from the cell. [The invention 1112] The method of any one of the inventions 1109 to 1111, wherein the multispecific polypeptide construct is a heterodimer. [The invention 1113] A multispecific polypeptide construct produced by the method of any one of the inventions 1109 to 1112. [The invention 1114] A pharmaceutical composition comprising any multispecific polypeptide construct of the inventions 1001 to 1094 or the invention 1113 and a pharmaceutically acceptable carrier. [The invention 1115] The pharmaceutical composition of the invention 1114, which is sterile. [The invention 1116] A step of contacting a target cell and a T cell with any multispecific polypeptide construct of the inventions 1001 to 1094 or the invention 1113 or the pharmaceutical composition of the invention 1114 or the invention 1115, comprising A method for stimulating or inducing an immune response, wherein the target cell expresses a tumor-associated antigen recognized by a multispecific polypeptide construct. [Invention 1117] The method of Invention 1116, wherein the target cell is a tumor cell expressing a tumor-associated antigen (TAA). [Invention 1118] The method of Invention 1116 or Invention 1117, wherein the contacting step is performed ex vivo or in vitro. [Invention 1119] The method according to any one of Inventions 1116 to 1118, wherein the contacting step is performed in vivo in a subject. [Invention 1120] Administering to a subject in need thereof, in a therapeutically effective amount, a multispecific polypeptide construct according to any one of Inventions 1001 to 1094 or Invention 1113, or a pharmaceutical composition according to Invention 1114 or Invention 1115 A method for stimulating or inducing an immune response in a subject, comprising [Invention 1121] The method according to any one of Inventions 1116 to 1120, which increases cellular immunity. [Invention 1122] The method according to any one of Inventions 1116 to 1121, which increases T cell activity. [Invention 1123] The method according to any one of Inventions 1116 to 1122, which increases cytotoxic T lymphocyte (CTL) activity. [Invention 1124] The method according to any one of Inventions 1116 to 1123, in which the immune response against a tumor or cancer is increased. [Invention 1125] The method according to any one of Inventions 1116 to 1124, for treating a disease or condition in a subject. [Invention 1126] ​Administering to a subject in need thereof, in a therapeutically effective amount, a multispecific conjugate of any of the present invention 1001-1094 or the present invention 1113, or a pharmaceutical composition of the present invention 1114 or the present invention 1115 A method of treating a disease or condition in a subject, comprising: [The present invention 1127] The method of the present invention 1125 or the present invention 1126, wherein the disease or condition is a tumor or cancer. [The present invention 1128] Any method of the present invention 1119-1127, wherein the subject is human. BRIEF DESCRIPTION OF THE DRAWINGS

[0120]

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Mode for Carrying Out the Invention

[0121] Detailed Description The present disclosure provides a chimeric T cell engaging fusion protein in the form of a multispecific polypeptide construct that binds at least to CD3 and a second antigen. The multispecific polypeptide constructs provided herein include a first component comprising one or more copies of an antigen-binding domain that binds to an 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 interchangeably as an anti-CD3 binding domain or a CD3 binding region), and at least a linker such as a polypeptide linker that joins the first component and the second component. In 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 granzyme B, MMP, or matriptase.

[0122] The provided multispecific polypeptide constructs include an arrangement in which a first component containing an Fc region is on the N-terminal side of a second component containing a CD3 binding region. In such embodiments, the first and second components are joined via a linker on the C-terminal side of the end of the Fc region. In some embodiments, the antigen-binding domain is positioned in the amino-terminal (N-terminal) region of the multispecific polypeptide construct. In some embodiments, the antigen-binding domain is positioned in the carboxy-terminal (C-terminal) region of the multispecific polypeptide construct. In some embodiments, the antigen-binding domain is positioned in both the N-terminal and C-terminal regions of the multispecific polypeptide construct. The various arrangements of the multispecific polypeptide constructs provided herein are shown in Figure 1.

[0123] The provided multispecific polypeptide construct exhibits restricted T cell engagement activity because it binds substantially to CD3 only after the antigen binds via the antigen-binding domain. This is illustrated in the examples and figures provided herein, which demonstrate that the restricted CD3-engaging protein can bind efficiently to TAA-positive cells but binds little to no T cells. This unique property allows the restricted CD3-engaging protein to distribute to sites where TAAs are present without binding to peripheral T cells. This format provides significant benefits in that it allows for preferential distribution to sites where antigens recognized by the antigen-binding domain are present by not allowing or excluding constitutive CD3 binding, avoiding peripheral T cell binding, and is different from other CD3-engaging multispecific constructs. Additionally, 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 restricted T cell engagement activity of the provided multispecific polypeptide constructs is, in some aspects, by positioning the Fc region on the N-terminal side of 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 ability. 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 bind to CD3 by the CD3-binding region 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 the 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 of the tumor microenvironment. In some aspects, the provided multispecific polypeptide constructs can be used to increase the T cell activity against a tumor or cancer, for example, an immune response such as cytotoxic activity. In some embodiments, the provided multispecific polypeptide constructs can be used to treat a tumor or cancer in a subject.

[0126] In some embodiments, the CD3 binding regions of the multispecific polypeptide constructs of the present disclosure are restricted by the presence of the Fc region or otherwise blocked and / or inhibited, such that these constructs ensure that binding of T cells via CD3 in peripheral blood does not occur. Accordingly, the multispecific polypeptide constructs of the present disclosure offer a number of 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 provided multispecific polypeptide constructs of the present disclosure enable controlled in vivo distribution to a desired site in a subject, such as, for example, the site of tumor-associated antigen (TAA) expression. Sites of TAA expression include, for example, tumors and the surrounding tumor microenvironment.

[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. For example, refer to FIG. 1. In some embodiments, one or more antigen-binding domains bind to the same antigen. In some embodiments, the multispecific polypeptide constructs include multiple antigen-binding domains that bind to different epitopes of the same antigen. In some embodiments, the multispecific polypeptide constructs include multiple antigen-binding domains that bind to one or more different antigens. In some embodiments, the multispecific polypeptide constructs include multiple antigen-binding domains that bind to different epitopes of the same antigen and also include additional antigen-binding domains that bind to one or more different antigens. In some aspects, the provided multispecific polypeptide constructs are bispecific polypeptide constructs capable of binding to CD3 and another antigen, such as a TAA, via the binding of the antigen-binding domains of the multispecific polypeptide constructs. In some examples, the provided multispecific polypeptide constructs are bispecific polypeptide constructs that provide 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 includes 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 the ability to bind to CD3 and then activate T cells: (1) an "inactive" state in which CD3 binding is restricted and T cell interaction is removed, which occurs when none or all of the binding of the antigen-binding domains is present. (2) An "active" state in which the CD3-binding region can bind to CD3 and T cell interaction is enabled, which occurs upon antigen binding by any or all of the antigen-binding domains.

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

[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 cell cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP). In some embodiments, the Fc region of the multispecific polypeptide construct can interact with a complement protein, namely 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 enable the simultaneous occurrence of cytotoxicity mediated by T cells and NK cells. In some cases, such activity can occur in a multispecific polypeptide construct containing a first antigen-binding domain, such as a first anti-TAA antigen-binding domain, and a second antigen-binding domain, such as a second anti-TAA antigen-binding domain, that can target different and / or non-competing epitopes of a given TAA.

[0133] The CD3-binding constructs can be used with any TAA-binding domain and are intended to enable better therapeutic exposure in tumors or the tumor microenvironment by avoiding interaction with peripheral T cells and mediating potent TAA-dependent T cell cytotoxicity. In some embodiments, the second portion or component contains a CD3-binding region that is monovalent with respect to CD3 such that T cell activation does not occur unless TAA is present.

[0134] In some aspects, the multispecific polypeptide constructs of the present disclosure offer a number of advantages compared to current bispecific therapeutics. The multispecific polypeptide constructs of the present disclosure are smaller than conventional therapeutic antibodies, e.g., 125 kDa versus 150 kDa, which will allow for better penetration to targets, e.g., tumors. In some aspects, the size of the entire multispecific polypeptide construct provides for a long half-life of the construct. In some aspects, binding to CD3 by the CD3-binding region is dependent on TAA binding before CD3 engagement occurs, such that the multispecific polypeptide constructs of the present disclosure exhibit reduced systemic toxicity or toxicity in regions outside of the tumor and / or tumor microenvironment.

[0135] All publications and patent documents cited herein are hereby incorporated by reference as if each such publication or document was specifically and individually indicated to be incorporated by reference herein. The citation of publications and patent documents is not intended as an admission that anything is relevant prior art, nor does it constitute an admission as to the content or date thereof. Although the invention has been described in the specification, those skilled in the art will recognize that the invention can be practiced in various embodiments, and that the above description and the following examples are for purposes of illustration and not limitation of the appended claims.

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

[0137] As used in accordance with the present disclosure, the following terms are to be understood to have the following meanings unless otherwise indicated.

[0138] As used herein, the term "antibody" refers to an immunoglobulin molecule and the antigen-binding portion of an immunoglobulin (Ig) molecule, i.e., a molecule that contains an antigen-binding site that specifically binds (immunoreacts) with an antigen. "Specifically binds" or "immunoreacts" or "immunologically specifically binds" means that an antibody reacts with one or more antigenic determinants of a desired antigen and does not react with or has a much lower affinity (K d >10 -6 ) for binding. 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 the heavy and / or light chains of an immunoglobulin 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 chain (VH) sequence and variable light chain (VL) sequence derived from an antibody that binds to an antigen, and generally, for an antibody containing VH and VL, six CDRs ("CDR1", "CDR2", and "CDR3" for each of the heavy and light chains), or for an antibody containing a single variable domain, three CDRs may be included. Antigen-binding fragments include single-domain antibodies, e.g., those containing only VH or only VL, e.g., V H H, V NAR , modified V H domains, or modified V K domains.

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

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

[0141] The terms "antigen-binding site" or "binding portion" refer to the part of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by the amino acid residues of 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 inserted between more conserved adjacent 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 an immunoglobulin. 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 so as to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and each of the three hypervariable regions of the heavy and light chains is called a "complementarity-determining region" or "CDR". The amino acid assignments to each domain follow the definitions of 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 a specific portion of an antigen that is targeted by an antibody, an 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 that can specifically bind to an immunoglobulin or a T cell receptor. Epitope determinants generally consist of surface groups having the chemical activity of a molecule such as an amino acid or a sugar side chain, and generally have specific three-dimensional structural features and specific charge features. For example, an antibody can be produced against a peptide at the N-terminus, central, or C-terminus of a polypeptide. Further, an antibody can be produced against a linear or discontinuous epitope of a polypeptide. An antibody is said to specifically bind to an antigen when the dissociation constant is ≤1 μM, for example, in some embodiments, ≤100 nM, and in some embodiments, ≤10 nM, and shows no binding to other proteins, even if closely related or different.

[0143] As used herein, the terms "specific binding", "immunological binding", and "immunological binding properties" refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction is represented by the dissociation constant (K d ) of the interaction, and a 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 rates depend on the concentrations of the complex partners, the affinity of the interaction, and the geometric parameters that equally affect the rates in both directions. Thus, both the "association rate constant" (K on ) and the "dissociation rate constant" (K off ) can be determined by calculation of the concentrations as well as the actual rates of association and dissociation (see Nature 361:186-87 (1993)). K off / Kon The ratio allows for the cancellation of all parameters independent of affinity and is equal to the dissociation constant K d (see generally Davies et al. (1990) Annual Rev Biochem 59, parameter 73). The antibodies of the present disclosure have a binding constant (K d ) that 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 and are said to specifically bind to EGFR when measured by an assay such as a radioligand binding assay or a similar assay known to those of skill in the art.

[0144] As used herein, the term "isolated polynucleotide" means a polynucleotide derived from genomic, cDNA, or synthetic, or combinations thereof, and due to its origin, the "isolated polynucleotide" is (1) not related to all or part of the polynucleotide in which the "isolated polynucleotide" is found in nature, (2) functionally linked to a polynucleotide to which it is not naturally linked, or (3) does not naturally exist as part of a larger sequence. The polynucleotides according to the present disclosure include nucleic acid molecules encoding the heavy chain immunoglobulin molecules shown herein and nucleic acid molecules encoding the light chain immunoglobulin molecules shown herein.

[0145] As used herein, the term "isolated protein" means a protein derived from cDNA, recombinant RNA, or synthetic, or combinations thereof, and due to its origin or derivation, the "isolated protein" is (1) not related to a protein found in nature, (2) does not contain other proteins from the same origin, e.g., does not contain mouse proteins, (3) is expressed by cells from different species, or (4) does not exist in nature.

[0146] The term "polypeptide" is used herein as a general term to refer to a native protein, fragment, or analog of a polypeptide sequence. Thus, fragments and analogs of native proteins are a type of polypeptides. The polypeptides according to the present disclosure include the heavy chain immunoglobulin molecules shown herein, and the light chain immunoglobulin molecules shown herein, and antibody molecules formed by a combination including heavy chain immunoglobulin molecules together with light chain immunoglobulin molecules such as κ light chain immunoglobulin molecules, and vice versa, and also include fragments and analogs thereof.

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

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

[0149] As used herein, the term "control sequence" refers to a polynucleotide sequence necessary to achieve expression and processing of a ligated coding sequence. The nature of such control sequences varies depending on the host organism. In prokaryotes, such control sequences generally include a promoter, a ribosome binding site, and a transcription termination sequence. In eukaryotes, such control sequences generally include a promoter and a transcription termination sequence. The term "control sequence" 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. As used herein, the term "polynucleotide" means a nucleotide, ribonucleotide or deoxynucleotide of at least 10 bases in length, or a modified form of any type of nucleotide. The term includes single-stranded and double-stranded forms of DNA.

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

[0151] The term "naturally occurring nucleotide" as referred to herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" as referred to herein includes nucleotides having modified or substituted sugar groups and the like. The term "oligonucleotide linkage" as referred to herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroserellorate, phosphorodiselenoate, phosphorothioanilate, phosphororaniladate, phosphoramidate, 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. Anti Cancer 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. Patent 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)). Non-natural amino acids such as stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), α,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other non-conventional amino acids can also be suitable components for the polypeptides of the present disclosure. Examples of non-conventional 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, in accordance with standard usage and convention, the left direction is the amino-terminal direction and the right direction is the carboxy-terminal direction.

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

[0154] The term "substantial identity" as applied to polypeptides means that two peptide sequences share at least 80 percent sequence identity when optimally aligned by a program such as GAP or BESTFIT using a default gap weight, for example, in some embodiments, at least 90 percent sequence identity, in some embodiments, at least 95 percent sequence identity, and in some embodiments, at least 99 percent sequence identity.

[0155] In some embodiments, the non-identical residue positions 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 to be encompassed by the present disclosure as long as the variation in the amino acid sequence maintains at least 75%, for example, in some embodiments, at least 80%, 90%, 95%, and in some embodiments, 99%. Specifically, conservative amino acid substitutions are contemplated. A conservative substitution is one that occurs within a family of amino acids that have related side chains. Genetically encoded amino acids are generally classified into the following families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include (i) serine and threonine, which are aliphatic hydroxy families; (ii) asparagine and glutamine, which are amide-containing families; (iii) alanine, valine, leucine, and isoleucine, which are aliphatic families; and (iv) phenylalanine, tryptophan, and tyrosine, which are aromatic families. For example, particularly when the substitution does not involve amino acids within the framework region, it is reasonable to expect that isolated substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitutions of structurally related amino acids will not have a significant effect on the binding or properties of the resulting molecule. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. The assays are described in detail herein.Antibodies or fragments or analogs of immunoglobulin molecules can be readily prepared by those skilled in the art. In some embodiments, the amino and carboxy termini of the fragment or analog are located near the boundaries of the functional domains. The structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains present in other proteins having 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 in accordance with the present disclosure.

[0157] In some embodiments, the amino acid substitutions are those that (1) decrease the susceptibility of such analogs to proteolysis, (2) decrease the susceptibility of such analogs to oxidation, (3) alter the binding affinity for forming protein complexes, (4) alter the binding affinity, and (4) confer or modify other physicochemical or functional properties. The analogs can include various muteins of sequences other than the naturally occurring peptide sequences. For example, one or more amino acid substitutions (e.g., conservative amino acid substitutions) can be made in a naturally occurring sequence, e.g., in a portion of the polypeptide outside of the intermolecular contact-forming domain. Conservative amino acid substitutions should not substantially change the structural characteristics of the parental sequence (e.g., the substituted amino acid should not tend to disrupt a helix present in the parental sequence or disrupt other types of secondary structure that characterize the parental sequence). Examples of secondary and tertiary structures recognized in the art 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, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991).

[0158] As used herein, the term "polypeptide fragment" refers to a polypeptide having amino-terminal and / or carboxy-terminal deletions and / or one or more in-sequence deletions, where the remaining amino acid sequence is identical to the corresponding position in a naturally occurring sequence, e.g., 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. As used herein, the term "analog" refers to a polypeptide composed of at least 25 amino acid segments that have substantial identity to a portion of a deduced amino acid sequence and have specific binding to EGFR under appropriate binding conditions. Typically, polypeptide analogs contain conservative amino acid substitutions (or additions or deletions) relative to a 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 more, and often can be the same length as the full-length naturally occurring polypeptide.

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

[0160] As used herein, the term "label" or "labeled" refers to the incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid into a polypeptide, or by attachment of a biotinyl moiety detectable by a labeled avidin (e.g., streptavidin containing a fluorescent marker or enzyme activity detectable by optical or calorimetric methods). In certain situations, the label or marker may also be therapeutic. A variety of methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include radioisotopes or radionuclides (e.g.,3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., fluorophores, rhodamines, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), but are not limited thereto. In some embodiments, the label is attached by spacer arms of various lengths to reduce possible steric hindrance. The term "pharmaceutical agent or drug" as used herein refers to a chemical compound or composition that can induce 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. It can 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 mammalian subjects, often humans. Pharmaceutical compositions typically contain 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] As used herein, the terms "treating," "treatment," or "therapy" of a disease or disorder, alone or in combination with another compound described herein, mean the slowing, halting, or reversal of the progression of a disease or disorder as evidenced by a decrease, cessation, or elimination of clinical or diagnostic symptoms by administration of a pharmaceutical composition of the disclosure. "Treating," "treatment," or "therapy" also means a decrease in the severity of symptoms or a decrease in the recurrence rate in acute or chronic diseases or disorders. As used herein, with respect to cancer, the terms "treatment" of cancer, or "inhibiting," "inhibition," or "inhibit" are not limited, but include at least one of a statistically significant decrease in the rate of tumor growth, a halt in tumor growth, or a decrease in the size, amount, metabolic activity, or volume of a tumor, or a statistically significant increase in progression-free survival (PFS) or overall survival (OS), as measured by standard criteria such as Response Evaluation Criteria for Solid Tumors (RECIST). "Preventing," "prevention," or "prevention" of a disease or disorder means administration of a pharmaceutical composition, alone or in combination with another compound, for preventing the occurrence or onset of a disease or disorder, or some or all of the symptoms of a disease or disorder, or for reducing the likelihood of onset of a disease or disorder.

[0164] The term "effective amount" or "therapeutically effective amount" refers to the amount and / or concentration of a composition that, when administered to a patient, alone (i.e., as monotherapy) or in combination with an additional therapeutic agent, gives a statistically significant decrease in disease progression, for example, by alleviating or eliminating the symptoms and / or causes of a disease. An effective amount can be an amount that reduces, lessens, or alleviates at least one symptom or biological response or effect associated with a disease or disorder, prevents the progression of a disease or disorder, or improves the physical function of a patient.

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

[0166] Generally, a substantially pure composition will constitute more than about 80 percent of all polymeric species present in the composition, for example, in some embodiments, more than about 85%, 90%, 95%, and 99%. In some embodiments, the species of matter is purified to a substantially homogeneous state in which the composition consists essentially of a single polymeric species (such that 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 according to their 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 the ordinary error range for each value readily known to one of ordinary skill in the art. Reference to a value or parameter herein with "about" includes (describes) aspects regarding that value or parameter itself. For example, a description that refers to "about X" includes a description of "X".

[0170] II. Multispecific polypeptide constructs A multispecific polypeptide construct containing a first component containing an immunoglobulin Fc region and a second component containing a CD3 binding region is provided herein, wherein the first and second components are coupled by a linker and the Fc region is positioned on the N-terminal side of the CD3 binding region; one or both of the first and second components contain 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 specifically recognized by a protease for cleavage.

[0171] In some embodiments, the multispecific polypeptide construct contains, in order from the N-terminus to the C-terminus, an immunoglobulin Fc region; a linker; a CD3 binding region that binds to CD3 (CD3ε); and an antigen-binding domain that binds to a tumor-associated antigen (TAA). In some embodiments, the multispecific polypeptide construct contains, in order from the N-terminus to the 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, in order from the N-terminus to the 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 construct of the present disclosure will be described in more detail below.

[0173] 1. Anti-CD3 binding domain: The multispecific polypeptide constructs of the present disclosure include one or more copies of an anti-CD3 binding domain. The anti-CD3 binding 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 activities of CD3 include, for example, T cell activation and other signaling through the interaction between CD3 and the antigen-binding subunits of the T cell receptor (TCR). For example, the anti-CD3 binding domain of the present disclosure fully or partially activates T cells through engagement of CD3ε on T cells by, for example, partially or fully regulating CD3-mediated T cell activation, such as 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, for example, 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 includes one or more copies of an antibody or its antigen-binding fragment.

[0175] In some embodiments, the anti-CD3ε binding domain includes 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 is a VH CDR1 sequence that includes at least the amino acid sequence TYAMN (SEQ ID NO:16); an amino acid sequence TIFF2025102819000025.tif4128 that includes at least a VH CD2 sequence; and an amino acid sequence Selected from VH CDR3 sequences comprising at least TIFF2025102819000026.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 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); and an amino acid sequence Selected from VH CDR3 sequences comprising at least TIFF2025102819000027.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 an amino acid sequence Selected from VL CDR1 sequences comprising at least TIFF2025102819000028.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).

[0177] In some embodiments, the anti-CD3ε binding domain is a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO:16); an amino acid sequence Selected from VH CDR2 sequences comprising at least TIFF2025102819000029.tif4128; an amino acid sequence Selected from VH CDR3 sequences comprising at least TIFF2025102819000030.tif4128; an amino acid sequence A VL CDR1 sequence comprising at least TIFF2025102819000031.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); an amino acid sequence A VH CDR3 sequence comprising at least TIFF2025102819000032.tif4128; an amino acid sequence A VL CDR1 sequence comprising at least TIFF2025102819000033.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); an amino acid sequence A VH CDR3 sequence comprising at least TIFF2025102819000034.tif4128; an amino acid sequence A VL CDR1 sequence comprising at least TIFF2025102819000035.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 VH CDR1 sequence having a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence TYAMN (SEQ ID NO:16); an amino acid sequence 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 the sequence TIFF2025102819000036.tif4128; and an amino acid sequence selected from 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 the sequence TIFF2025102819000037.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 having a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence GFTFNTYAMN (SEQ ID NO:211); a VH CDR2 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence RIRSKYNNYATY (SEQ ID NO:212); and an amino acid sequence selected from 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 the sequence TIFF2025102819000038.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 an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the sequence TIFF2025102819000039.tif4128; a VL CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the 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 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); an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the VH CD2 sequence TIFF2025102819000040.tif4128; an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the VH CDR3 sequence TIFF2025102819000041.tif4128, an amino acid sequence A VL CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to TIFF2025102819000042.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-CD3ε binding domain has a VH CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence GFTFNTYAMN (SEQ ID NO:211); a VH CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence RIRSKYNNYATY (SEQ ID NO:212); an amino acid sequence A VH CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to TIFF2025102819000043.tif4128, an amino acid sequence A VL CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to TIFF2025102819000044.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-CD3ε binding domain has a VH CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence GFTFNTYAMN (SEQ ID NO: 211); a VH CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence RIRSKYNNYATY (SEQ ID NO: 212); an amino acid sequence TIFF2025102819000045.tif4128 and a VH CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence TIFF2025102819000046.tif4128 and a VL CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence GTNKRAP (SEQ ID NO: 230); 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 has a VH CDR1 sequence that at least includes the amino acid sequence GFTFSTYAMN (SEQ ID NO: 227); a VH CDR2 sequence that at least includes the amino acid sequence RIRSKYNNYATY (SEQ ID NO: 228); an amino acid sequence TIFF2025102819000047.tif4128 and a VH CDR3 sequence that at least includes the amino acid sequence A VL CDR1 sequence comprising at least TIFF2025102819000048.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-CD3ε binding domain has 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 GFTFSTYAMN (SEQ ID NO:227); a VH CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence RIRSKYNNYATY (SEQ ID NO:228); an amino acid sequence A VH CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to TIFF2025102819000049.tif4128, an amino acid sequence A VL CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to TIFF2025102819000050.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 that at least includes 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 an antigen-binding fragment thereof selected from the group consisting of a Fab fragment, an F(ab')2 fragment, an Fv fragment, a scFv, a scAb, a dAb, a single-domain heavy-chain antibody, and a single-domain light-chain antibody. In some embodiments, the anti-CD3 binding domain comprises an Fv antibody fragment that binds to CD3ε (referred to herein as an anti-CD3ε Fv fragment). In some embodiments, the anti-CD3ε Fv antibody fragment is a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv). In some embodiments, the anti-CD3 binding domain is monovalent for CD3 binding.

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

[0191] In some embodiments, the CD3 binding region is an Fv antibody fragment containing a variable heavy chain (also referred to as Hv or VH) and a variable light chain (also referred to as Lv or VL) such as any of those described. In aspects of such embodiments, the immunoglobulin Fc region is a heterodimeric Fc region containing two different Fc polypeptides capable of heterodimeric association between both polypeptides of the Fc heterodimer such as any of those described in Section II.2. In such embodiments, the variable heavy chain (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, the heavy chain variable region amino acid sequence being selected from the group consisting of SEQ ID NOs: 32-81. In some embodiments, the anti-CD3ε binding domain comprises a combination of a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62 and a light chain variable region amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81.

[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 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 32-81, 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 that comprises 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 consisting 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 combination of a heavy chain variable amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 32-62, 196-198, and 211 and a light chain variable amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 selected from the group consisting 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 consisting 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 consisting of SEQ ID NOs: 63-81, 191, 199, 200, and 212.

[0196] In some embodiments, the anti-CD3ε Fv antibody fragment comprises 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 thereto, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 63, 65-71, 73, 75, 77, and 199 and an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical thereto. 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: 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 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:15. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:14, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: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 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 196. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 199. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 196 and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 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, the Fv is V H -V LA disulfide-stabilized Fv (dsFv) in which the heterodimer is stabilized by an interchain disulfide bond. In some embodiments, the interchain disulfide bond is modified by a positional mutation at a framework position of the VH chain and / or the VL chain. In some embodiments, the disulfide-stabilized anti-CD3 Fv comprises an anti-CD3 VH containing a mutation to Cys at position 44 and an anti-CD3 VL containing a mutation to Cys at position 100, according to Kabat numbering. For example, in some embodiments, each according to Kabat numbering, the VH chain contains the mutation G44C and the VL chain contains the mutation G100C. In some embodiments, the disulfide-stabilized anti-CD3 Fv comprises an anti-CD3 VH containing a mutation to Cys at position 105 and an anti-CD3 VL containing a mutation to Cys at position 43, according to 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 NO: 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 NO: 64, 72, 74, 76, 78-81, 191, 200, and 212. In some of such embodiments, the anti-CD3 Fv is a dsFv having a VH chain containing the mutation G44C and a VL chain containing the mutation G100C, each according to Kabat numbering. 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 NO: 44, 49-62, 197, and 198, and an amino acid sequence selected from the group consisting of SEQ ID NO: 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 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 44, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 72. In some of such embodiments, the anti-CD3 Fv is a dsFv having a VH chain containing the mutation G44C and a VL chain containing the mutation G100C, each by Kabat numbering. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 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 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 198. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 200. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 198 and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 200. In some of such embodiments, the anti-CD3 Fv is a dsFv having a VH chain containing the mutation G44C and a VL chain containing the mutation G100C, each by Kabat numbering. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 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 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 197. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 200. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 197 and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 200. In some of such embodiments, the anti-CD3 Fv is a dsFv having a VH chain containing the mutation G44C and a VL chain containing the mutation G100C, each by Kabat numbering. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 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 polypeptide: 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 a human Fc. In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6. In some embodiments, the immunoglobulin Fc region contains an Fc chain that is an immunologically active fragment of any of SEQ ID NOs: 1-6. In some embodiments, the immunoglobulin Fc region contains an Fc polypeptide chain that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences of SEQ ID NOs: 1-6, or an immunologically active fragment thereof.

[0205] In some embodiments, the multispecific polypeptide construct is a dimer formed by polypeptides each containing an Fc. In some specific embodiments, the same or substantially the same polypeptide will dimerize to create a homodimer. In some embodiments, the dimer is a homodimer in which the two 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 for promoting 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 cell-mediated cytotoxicity (ADCC) in addition to the antigen-binding ability, which is the major function of immunoglobulins. Further, the FcRn sequence present in the Fc region plays a role in controlling the IgG level in serum by increasing the in vivo half-life through conjugation with the in vivo FcRn receptor. In some embodiments, in the Fc for use with the provided multispecific polypeptide construct, such functions may be altered, for example, they may be decreased or enhanced.

[0207] In some embodiments, the Fc region of the provided multispecific polypeptide construct exhibits one or more effector functions. In some cases, the Fc region can provide Fc-mediated effector functions such as, for example, ADCC (e.g., release of granzyme B by NK cells), ADCP, and / or CDC. Thus, in some embodiments where the multispecific polypeptide construct contains a cleavable linker, cleavage of the linker can produce two components each having 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 a target-specific effector function. In certain embodiments provided herein, the multispecific polypeptide construct contains a non-cleavable linker and, in some aspects, the Fc may not exhibit an independent 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 of the Fc polypeptide for altering, e.g., reducing, effector functions are known and include the following. In some embodiments, unless otherwise specified with respect to a particular SEQ ID NO, references to amino acid substitutions in the Fc region are by EU numbering (also called Kabat numbering) by Kabat. EU numbering is known and is based on the EU index reported in the most recent IMGT Scientific Chart (IMGT®, the international ImMunoGeneTics information system®, http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created: 17 May 2001, last updated: 10 Jan 2013)) and Kabat, E. A. et al. Sequences of Proteins of Immunological interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0209] In some embodiments, provided multispecific polypeptide constructs containing an Fc region that exhibits reduced effector function may be desirable candidates for applications where restricted CD3 binding is desired but certain effector functions (such as CDC and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays may be performed to confirm a decrease / depletion of CDC activity and / or ADCC activity. For example, to ensure that a multispecific polypeptide construct and / or its cleaved components lack FcγR binding (and thus are likely to lack ADCC activity) but retain FcRn binding ability, an Fc receptor (FcR) binding assay may be performed. The main cells that mediate ADCC, NK cells, express only FcγRIII, and monocytes express FcγRI, FcγRII, and FcγRIII. Non-limiting examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assay methods may be utilized (see, e.g., ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96™ non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the multispecific polypeptide construct or its cleaved components are unable to bind C1q and thus lack CDC activity. See, for example, the C1q binding ELISA and C3c binding ELISA of WO 2006 / 029879 and WO 2005 / 100402. A CDC assay can be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, for example, Petkova, S. B. et al., Int'l. Immunol. 18 (12): 1759-1769 (2006)).

[0210] In some embodiments, the immunoglobulin Fc region or an immunologically active fragment thereof is of the IgG isotype. For example, the immunoglobulin Fc region of the fusion protein is of the human IgG1 isotype having the following amino acid sequence. TIFF2025102819000051.tif34138

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

[0212] In some embodiments, an IgG1 Fc polypeptide or variant thereof, such as any of the following, can be made in the G1 m1 allotype or the 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 for example. In some cases, the Fc polypeptide may contain the amino acid substitutions E356D and M358L to reconstruct the 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, as shown in SEQ ID NO:194 and 195 by EU numbering for example. In some cases, the Fc polypeptide may contain the amino acid substitutions D356E and L358M to reconstruct the residues of allotype G1 m3. In some embodiments, the human IgG1 Fc region is modified to alter antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). For example, amino acid modifications described in Natsume et al., 2008 Cancer Res,68(10): 3863-72;Idusogie et al., 2001 J Immunol, 166 (4):2571-5;Moore et al., 2010 mAbs, 2 (2): 181-189;Lazar et al., 2006 PNAS, 103 (11): 4005-4010, Shields et al., 2001 JBC, 276 (9): 6591-6604;Stavenhagen et al., 2007 Cancer Res,67(18): 8882-8890;Stavenhagen et al.,2008 Advan. Enzyme Regul., 48: 152-164;Alegre et al, 1992 J Immunol, 148: 3461-3468; and reviewed in Kaneko and Niwa,2011 Biodrugs,25(1):1-11.The content of each of them is hereby incorporated by reference in its entirety.

[0213] In some embodiments, an Fc region, such as a human IgG1 Fc region, is modified to enhance ADCC activity or CDC activity. Examples of mutations that enhance ADCC include modifications at Ser239 and Ile332, such as Ser239Asp and Ile332Glu (S239D, I332E). Examples of mutations that enhance CDC include modifications at Lys326 and Glu333. In some embodiments, the Fc region is modified at one or both of these positions using the Kabat numbering system (e.g., Lys326Ala and / or Glu333Ala (K326A and E333A)).

[0214] In some embodiments, the human IgG1 Fc region fusion protein of the present disclosure lacks fucose attached to the N-linked glycan chain at N297 or has reduced fucose. There are numerous ways to prevent fucosylation, including production in FUT8-deficient cell lines; addition of inhibitors, such as castanospermine, to the mammalian cell culture medium; and metabolic engineering of the production cell line, but are not limited thereto. In some embodiments, the human IgG1 Fc region is modified at amino acid Asn297 (boxed, Kabat numbering) to prevent glycosylation of the fusion protein (e.g., Asn297Ala (N297A) or Asn297Asp (N297D)).

[0215] In some embodiments, the Fc region is modified to provide reduced Fc-mediated effector function, for example, through reduced binding to Fc receptors, such as binding to FcγR rather than normal 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 interaction (e.g., Leu235Glu (L235E) or Leu235Ala (L235A)). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (within the box of SEQ ID NO:1, Kabat numbering) to alter Fc receptor interaction (e.g., Leu234Ala (L234A)). In some embodiments, the Fc region of the fusion protein is altered at both amino acids 234 and 235 (e.g., Leu234Ala and Leu235Ala (L234A / L235A) or Leu234Val and Leu235Ala (L234V / L235A)). In a preferred embodiment, the modification within the Fc region reduces binding to the Fc receptor γ receptor but has a 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 binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E respectively) (Kabat numbering, Dall'Acqua et al 2006, J. Biol Chem Vol. 281(33)23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al 2010 Nature Biotech, Vol. 28(2)157-159) (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest). In some embodiments, the mutant or modified Fc polypeptide 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 the amino acid at one or more of the following positions to reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (L235). In these embodiments, the Fc deletion of these three amino acids reduces complement protein C1q binding. TIFF2025102819000052.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 can include E233P, L234V, and / or L235A.

[0219] In some embodiments, the Fc region of the fusion protein is modified at Gly236 (within the box of SEQ ID NO: 1) to reduce Fc receptor binding. For example, Gly236 is deleted from the fusion protein. In some embodiments, the human IgG1 Fc region is modified at amino acid Gly236 to enhance interaction with CD32A (e.g., Gly236Ala (G236A)).

[0220] In a specific embodiment, for example, mutations in the Fc region to reduce Fc effector function via reduced binding of the Fc receptor to FcγR include any of the mutations G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G, or E233P / L234V / L235A / G236del.

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

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

[0223] In some embodiments, the immunoglobulin Fc region of the fusion protein or its immunologically active fragment is of the human IgG2 isotype and has the following amino acid sequence. TIFF2025102819000053.tif33138

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

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

[0226] In some embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG3 isotype and has the following amino acid sequence. TIFF2025102819000054.tif34138

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

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

[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. TIFF2025102819000055.tif34138

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

[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. TIFF2025102819000056.tif33138

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

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

[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. A variety of methods for promoting heterodimerization of complementary Fc polypeptides are known. 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 numbers WO 1998 / 050431, WO2009 / 089004, WO2011143545, WO2014 / 067011, WO2012 / 058768, WO2018027025; Published US patent application numbers US20140363426, US20150307628, US20180016354, US20150239991; and US Patents 5731168, 7183076, 9701759, 9605084, and 9650446. Methods for promoting heterodimerization of Fc chains include, for example, mutagenesis of the Fc region by including a set of "knob-into-hole" mutations or by including mutations to achieve electrostatic steering of the Fc that are favorable for attractive interactions between different polypeptide chains.For example, in some embodiments, the Fc polypeptide of the heterodimer has mutations to alter the charge polarity at the Fc dimer interface such that electrostatically matched co-expression of the Fc chains supports favorable attractive interactions, thereby promoting the formation of the desired Fc heterodimer, and unfavorable repulsive charge interactions suppress the formation of unwanted Fc homodimers (Guneskaran et al., (2010) JBC, 285:19637-19646). When co-expressed in cells, the chains can associate with each other, but due to charge repulsion, the chains do not substantially self-associate. Other strategies for generating heterodimeric Fc include mixing of CH3 domain segments of human IgG and IgA to create complementary CH3 heterodimers, called SEED Fc.

[0236] Methods and variants of heterodimerization also include those described in the published international PCT application WO2014 / 145806, including "knobs and holes" mutations (also called "skew" variants), mutations related to "electrostatic steering" or "charge pairs", and pI variants. Heterodimeric variants also include any of those described in the published US application numbers US2012 / 0149876 or US2018 / 011883.

[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 of the polypeptides of the Fc fusion are shown in Table 1.

[0238] (Table 1) Pairs of amino acids of heterodimeric Fc TIFF2025102819000057.tif51145

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

[0240] In some embodiments, the first Fc polypeptide modified to contain bulge (hole) amino acids protrudes from the interface of the first Fc polypeptide and thus has at least one side chain positioned in a compensatory cavity (hole) at the adjacent interface of the second polypeptide, including substitution of the native or first amino acid with an amino acid. The substituted amino acid most often has a larger side chain volume than the first amino acid residue. Methods for determining and / or evaluating the properties of amino acid residues to identify those that are ideal substituted amino acids for creating a bulge are known to those of skill in the art. In some embodiments, the substituted residue for bulge formation is a naturally occurring amino acid residue and includes, for example, arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). In some examples, the first residue identified for substitution is an amino acid residue with a small side chain, such as, for example, alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0241] In some embodiments, the second Fc polypeptide modified to contain a cavity (hole) is recessed from the interface of the second polypeptide and thus has at least one side chain of an amino acid capable of accepting a corresponding protrusion from the interface of the first polypeptide, including substitution of the native or first amino acid to the amino acid. Most often, the substituted amino acid has a side chain volume smaller than the first amino acid residue. Methods for determining and / or evaluating the properties of amino acid residues are known to those skilled in the art to identify those that are ideal substitution residues for cavity formation. Generally, substitution residues for cavity formation are naturally occurring amino acids, including, for example, alanine (A), serine (S), threonine (T), and valine (V). In some examples, the first amino acid identified for substitution is an amino acid with a large side chain, such as tyrosine, arginine, phenylalanine, or tryptophan.

[0242] The CH3 interface of human IgG1 contains, for example, 16 residues of each domain located in four antiparallel β-strands buried 1090 Å2 from each surface (see, for example, Deisenhofer et al. (1981) Biochemistry, 20:2361-2370; Miller et al., (1990) J Mol. Biol., 216, 965-973; Ridgway et al., (1996) Prot. Engin., 9:617-621; U.S. Patent No. 5,731,168). Modifications of the CH3 domain to create a protrusion or cavity 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 examples, modifications of the CH3 domain to create a protrusion or cavity typically target residues located in two central antiparallel β-strands. The goal is to minimize the risk that the created protrusion is accepted not by the compensatory cavity of the partner CH3 domain but by protruding into the surrounding solvent.

[0243] For example, in some embodiments, the heterodimeric Fc can preferentially pair with a second CH3 domain having amino acid modifications (T366S / L368A / Y407V) at positions Thr366, Leu368, and Tyr407 to smaller amino acids, such as Ser, Ala, Val, respectively, when exchanged with a bulkier amino acid, such as Try (T366W). Heterodimerization via CH3 modification can be further stabilized by the introduction of disulfide bonds, for example, by 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 a specific embodiment, the multispecific polypeptide construct contains a first and a second Fc that can mediate Fc heterodimerization, the first Fc polypeptide contains the mutations T366W and S354C, and the second Fc polypeptide contains the mutations T366S, L368A, Y407V, and Y349C. In some embodiments, the first Fc polypeptide is selected from Fc polypeptides comprising the sequences shown in SEQ ID NO:201 or 207, and the second Fc polypeptide is selected from Fc polypeptides comprising the sequences shown in SEQ ID NO:202, 205, or 209. In some embodiments, the first Fc polypeptide has or comprises the amino acid sequence shown in any of SEQ ID NO:82, 86, 94, or 96, and the second Fc polypeptide has or comprises the amino acid sequence shown in any of SEQ ID NO:83, 87, 90, 92, 98, or 100.

[0245] In some embodiments, the Fc polypeptide exhibits characteristics that provide Fc-mediated effector functions. In specific examples, the first Fc polypeptide is or comprises the sequence shown 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 shown in SEQ ID NO: 82, and the second Fc polypeptide is or comprises the sequence shown in SEQ ID NO: 83 or 90. In some embodiments, the first Fc polypeptide is or comprises the sequence shown in SEQ ID NO: 86, and the second Fc polypeptide is or comprises the sequence shown in SEQ ID NO: 87 or 92. The first and second Fc polypeptides can be formatted in any polypeptide chain of the construct.

[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 functions include any of those described. In some embodiments, the modification can be a deletion of one or more positions Glu233 (E233), Leu234 (L234), or Leu235 (L235), such as a deletion of Glu233 (E233), Leu234 (L234), and Leu235 (L235). In some embodiments, the first Fc polypeptide is selected from Fc polypeptides comprising the sequences shown in SEQ ID NO: 203 or 208, and the second Fc polypeptide is selected from Fc polypeptides comprising the sequences shown in SEQ ID NO: 204, 206, or 210. In some embodiments, the first Fc polypeptide is or comprises the amino acid sequence shown in any of SEQ ID NO: 84, 88, 95, or 97, and the second Fc polypeptide is or comprises the amino acid sequence shown in any of SEQ ID NO: 85, 89, 91, 93, 99, or 101.

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

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

[0249] Additional examples of variants that can facilitate heterodimer promotion are S364K / E357Q and L368D / K370S; L368D / K370S and S364K; L368E / K370S and S364K; T411T / E360E / Q362E and D401K; L368D / K370S and S364K / E357L, K370S and S364K / E357Q and T366S / L368A / Y407V and T366W, or 366S / L368A / Y407V / Y349C and T366W / S354C (each pair represents a mutation in the first Fc polypeptide and the second Fc polypeptide), any combination or pair of conformational variants (e.g., sc variants) of the first Fc polypeptide and the second Fc polypeptide. In a specific embodiment, the provided construct contains first and second Fc polypeptides containing the mutation pairs L368D / K370S and S364K and E357Q.

[0250] An additional mechanism that can be used in heterodimer generation is what is sometimes referred to as "electrostatic steering" as described in Gunasekaran et al., J. Biol. Chem. 285 (25): 19637 (2010). This is sometimes referred to herein as a "charge pair". In this aspect, electrostatics is used to bias the formation towards heterodimerization. As will be understood by those skilled in the art, these also have an effect on the pI and thus on purification, and thus in some cases can also be considered pI variants. However, since they are generated to force heterodimerization and not used as purification tools, they are classified as "conformational variants". In one aspect, the first Fc polypeptide may contain the mutations D221E / P228E / L368E, and the second Fc polypeptide may contain the mutations D221R / P228R / K409R. In another aspect, the first Fc polypeptide may contain the mutations C220E / P228E / 368E, and the second Fc polypeptide may contain the mutations C220R / E224R / P228R / K409R.

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

[0252] In some embodiments, combinations of steric heterodimerization variants (e.g., knob and hole) with pI variants or charge pair variants can be used.

[0253] In a specific embodiment, the provided construct contains: (a) a first Fc polypeptide comprising the skew variant S364K / E357Q; and (b) a second Fc polypeptide containing the skew variants L368D / K370S and the pI variant N208D / Q295E / N384D / Q418E / N421D. In some embodiments, one or both of the first and second polypeptides can contain additional mutations, such as the exemplary mutations E233P / L234V / L235A / G236del / S267K, to reduce Fc effector activity. Examples of such first and second Fc polypeptides that can mediate Fc heterodimerization include the sequences shown in SEQ ID NO:194 and 195. The first and second Fc polypeptides can be formatted in either polypeptide chain of the construct.

[0254] The resulting multispecific polypeptide construct can be purified by suitable methods, such as affinity chromatography on a protein A column or a protein G column. When two nucleic acid molecules encoding different polypeptides are transformed into cells, homodimer and heterodimer formation will occur. The conditions for expression can be adjusted so that heterodimer formation is favored over homodimer formation.

[0255] Techniques for the recovery of a heterodimer from its homodimer based on differential affinity for an affinity reagent of the heterodimer are known. In some aspects, such techniques include the design of a heterodimer such that one of the Fc polypeptide chains does not bind to the affinity reagent protein A. In some cases, one of the polypeptide chains may contain one or more amino acid substitutions to abolish or reduce the affinity for the protein A reagent in one of the polypeptides of the Fc heterodimer. See, for example, WO2017134440, WO2010151792, Jendeberg et al., (1997) J. Immunol. Methods, 201(1): 25-34. In some of these embodiments, the Fc region can be modified at the protein A binding site of one member of the heterodimer to prevent protein A binding and thereby enable more efficient purification of the heterodimeric fusion protein. 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 may contain a modification (pA+ / pG-) such that it can bind to protein A and cannot bind to protein G. Exemplary pA+ / pG- amino acid modifications include, for human IgG1, containing serine at position 428, serine at position 434, and optionally containing histidine at position 436, or Fc containing these residues at the corresponding positions in human IgG 2, 3, or 4. In some aspects, such amino acid modifications at positions 428 and 434, and optionally at position 436, of a given IgG Fc polypeptide reduce or prevent binding of protein G and enhance purification of the protein.

[0256] In some embodiments, any such modification that confers differential affinity for the affinity reagent can be combined with one or more of the other amino acid modifications described above. For example, the I253R modification can be combined with either the T366S / L368A / Y407V modification or the T366W modification. The T366S / L368A / Y407V modified Fc can form homodimers because there is no steric hindrance at the dimerization interface that exists in the case of the T336W modified Fc. Thus, in some embodiments, the I253R modification is combined with the T366S / L368A / Y407V modified Fc such that purification of the potentially formed homodimeric Fc is not permitted. Similar modifications can be utilized by combining T366S / L368A / Y407V and H453R.

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

[0258] In some embodiments, one Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence shown 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 shown 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 shown 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 shown 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 proteins of the present disclosure are monomeric. For example, modification to a charged residue at residue Thr366, e.g., 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 modified at one or more of the following positions to reduce Fc receptor binding: Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325), or Ala327 (A327). For example, Leu234Ala (L234A), Leu235Ala (L235A), Asp265Asn (D265N), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Asn325Glu (N325E), or Ala327Ser (A327S). In a preferred embodiment, the modification within the Fc region reduces binding to the Fc receptor γ receptor but has a 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 may contain a modified IgG1 hinge having the sequence EPKSSDKTHTCPPC (SEQ ID NO:7), where Cys220, which forms a disulfide with the C-terminal cysteine of the light chain, is mutated to serine (e.g., Cys220Ser (C220S)). In other embodiments, the fusion protein contains a shortened hinge having the sequence DKTHTCPPC (SEQ ID NO:8).

[0262] In some embodiments, the fusion protein has a modified hinge derived from IgG4 that is modified (e.g., Ser228Pro (S228P)) to prevent or reduce chain exchange and has the sequence ESKYGPPCPPC (SEQ ID NO:9). In some embodiments, the fusion protein contains a linker polypeptide. In other embodiments, the fusion protein contains a linker and a hinge polypeptide.

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

[0264] In some embodiments, the linker is positioned at the terminus of the C-terminal region of the Fc region such that the Fc region is on the N-terminal side of the CD3 binding region. Since the provided multispecific polypeptide construct is a multimer such as a dimer, the provided construct includes a linker that joins the first Fc polypeptide of the first polypeptide and the first domain (e.g., VH) of the CD3 binding region with the second Fc polypeptide of the second polypeptide and the second domain (e.g., VL) of the CD3 binding region. Typically, the linkers present in the first and second polypeptides of the multispecific polypeptide construct are the same. Thus, in some embodiments, each domain of the CD3 binding domain is linked via a linker such as the same linker to the opposing polypeptide of the Fc such as a heterodimeric Fc.

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

[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, for example, 2 - 25 amino acid residues, 5 - 20 amino acid residues, 5 - 15 amino acid residues, 8 - 12 amino acids in length. In some embodiments, the linker is about 2 - 24 amino acids, 2 - 20 amino acids, 2 - 18 amino acids, 2 - 14 amino acids, 2 - 12 amino acids, 2 - 10 amino acids, 2 - 8 amino acids, 2 - 6 amino acids, 6 - 24 amino acids, 6 - 20 amino acids, 6 - 18 amino acids, 6 - 14 amino acids, 6 - 12 amino acids, 6 - 10 amino acids, 6 - 8 amino acids, 8 - 24 amino acids, 8 - 20 amino acids, 8 - 18 amino acids, 8 - 14 amino acids, 8 - 12 amino acids, 8 - 10 amino acids, 10 - 24 amino acids, 10 - 20 amino acids, 10 - 18 amino acids, 10 - 14 amino acids, 10 - 12 amino acids, 12 - 24 amino acids, 12 - 20 amino acids, 12 - 18 amino acids, 12 - 14 amino acids, 14 - 24 amino acids, 14 - 20 amino acids, 14 - 18 amino acids, 18 - 24 amino acids, 18 - 20 amino acids, or 20 - 24 amino acids. In some embodiments, the linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0268] In certain scenarios, the longer the linker length, the greater the CD3 binding when the multispecific polypeptide conjugate binds to its antigen, for example, a TAA. Thus, in some scenarios, the linker is longer than 12 amino acids, for example, longer than 13, 14, 15, 16, 17, or 18 amino acids. In some embodiments, the linker is 12 - 40 amino acids in length, 12 - 30 amino acids, 12 - 24 amino acids, 12 - 18 amino acids, 12 - 15 amino acids, 15 - 40 amino acids, 15 - 30 amino acids, 15 - 24 amino acids, 15 - 18 amino acids, 18 - 40 amino acids, 18 - 30 amino acids, 18 - 24 amino acids, 24 - 40 amino acids, 24 - 30 amino acids, or 30 - 40 amino acids.

[0269] The linker may be naturally occurring, synthetic, or a combination of both. Particularly suitable linker polypeptides mainly contain amino acid residues selected from glycine (Gly), serine (Ser), alanine (Ala), and threonine (Thr). For example, the linker may contain at least 75% (calculated based on the total number of residues present in the peptide linker), for example, at least 80%, at least 85%, or at least 90% of amino acid residues selected from Gly, Ser, Ala, and Thr. The linker may consist only of residues of Gly, Ser, Ala, and / or Thr. In some embodiments, the linker contains 1 - 25 glycine residues, 5 - 20 glycine residues, 5 - 15 glycine residues, or 8 - 12 glycine residues. In some scenarios, suitable peptide linkers typically contain at least 50% glycine residues, for example, at least 75% glycine residues. In some embodiments, the peptide linker contains only glycine residues. In some embodiments, the peptide linker contains only glycine and serine residues.

[0270] In some embodiments, these linkers are mainly composed 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 to 10, for example, 1 to 5, for example, 1 to 3), for example, GGS(GGS)n (SEQ ID NO:171) (n is 0 to 10). In a specific embodiment, the linker contains the sequence (GGGGGS)n (SEQ ID NO:173) (n is 1 to 10 or n is 1 to 5, for example, 1 to 3). In a further embodiment, the linker contains (GGGGGS)n (SEQ ID NO:172) (n is 1 to 4, for example, 1 to 3). The linker may include any of the above combinations, for example, repeats of 2, 3, 4, or 5 GS linkers, GGS linkers, GGGGS linkers, and / or GGGGGGS linkers 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 (one-letter amino acid code): GGS, GGGGS (SEQ ID NO:149), or GGGGGGS (SEQ ID NO:135). In some embodiments, the GS linker contains the amino acid sequence of TIFF2025102819000058.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 above examples, serine may be substituted with alanine (for example, (Gly4Ala) or (Gly3Ala)).

[0272] In some embodiments, the linker has 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 x, y, and z are each integers in the range of 1 to 5) and includes a peptide linker. In some embodiments, Xaa is independently selected from the group consisting of Ser, Ala, and Thr. In a specific variation, each of x, y, and z is equal to 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) (each Xaa is selected as described above).

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

[0274] In some cases, it may be desirable to provide a certain degree of immobility to the peptide linker. 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 consists of only proline residues.

[0275] In some embodiments, the peptide linker contains at least one cysteine residue, e.g., one cysteine residue. For example, in some embodiments, the linker contains at least one cysteine residue as well as amino acid residues selected from the group consisting of Gly, Ser, Ala, and Thr. In some such embodiments, the linker contains 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 (where n and m are each an integer from 1 to 12, e.g., 3 to 9, 4 to 8, or 4 to 7) and includes a peptide linker having. In a specific 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 a specific embodiment, the structured linker has the sequence (AP)n or (EAAAK)n (SEQ ID NO:178) (n is from 2 to 20, preferably from 4 to 10), e.g., but not limited to, AS-(AP)n-GT (SEQ ID NO:179) or AS-(EAAAK)n-GT (SEQ ID NO:180) (n is from 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 has the sequence TIFF2025102819000059.tif19159 (n is from 2 to 20). In some embodiments, the linker TIFF2025102819000060.tif11159. In some embodiments, such linkers are more resistant to protein cleavage due to their structure and can thus provide an advantage when injected in vivo.

[0277] In some embodiments, the linker is not a cleavable linker (is used interchangeably with a non-cleavable linker). In some embodiments, the linker is not cleavable by a protease. In some embodiments, a linker that is not a cleavable linker or not cleavable by a protease is 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 preferably present within a cleavable peptide sequence or protease recognition site. In a specific embodiment, the non-cleavable linker is not a target substrate of the protease and thus is not preferentially or specifically cleaved by the protease as compared to a linker that contains the 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 particular protease that is cleaved by a protease and recognized by the active site of the protease. Typically, for example, a cleavage sequence for a serine protease is composed of P1-P4 amino acids and P1'-P4' amino acids within the substrate, and cleavage occurs after the P1 position. Typically, a cleavage sequence for a serine protease is 6 residues in length 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 dissociable P1-P1' binding sequence recognized by the protease.

[0279] In some situations, non-cleavable linkers, or linkers that do not contain a substrate recognition site specifically recognized for cleavage by a protease, are those in which cleavage by the protease is substantially less than the cleavage of the protease's target substrate. Typically, a protease exhibits specificity or preference for the cleavage of a particular target substrate as compared to other non-target substrates. The degree of such specificity can be determined based on the rate constant of cleavage of a sequence, such as a linker sequence, which is a measure of the protease's preference for that substrate and the efficiency of the enzyme. Any method for determining 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 (k cat / K m ) can be determined for a particular protease with respect to a particular linker. In some embodiments, non-cleavable linkers, or linkers that do not contain a substrate recognition site specifically recognized for cleavage by a protease, even if cleaved, are linkers that are cleaved by the protease at a rate of less than 1×10 4 M -1 S -1 , less than 5×10 3 M -1 S, less than 1×10 3 M -1 S, or less than 1×10 2 M -1 S, or less.

[0280] In some embodiments, the linker in the multispecific constructs provided herein does not contain a substrate recognition site for proteases including, for example, matrix metalloproteases (MMPs), cysteine proteases, serine proteases, and plasmin activators. In specific embodiments, the linker does not contain a substrate recognition site 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; ADAMDEC1; 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, e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P; cysteine proteases, e.g., Cruzipain; legumain; Otubain 2; KLK, e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, or KLK14; metalloproteases, e.g., meprin; neprilysin; PSMA; BMP-1; MMP, 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., FVIIa, 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 (TTSP), e.g., 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 amino acids that are substrates of 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) (where P4 is the amino acid I, L, Y, M, F, V, or A; P3 is the amino acid A, G, S, V, E, D, Q, N, or Y; P2 is the amino acid H, P, A, V, G, S, or T; P1 is the amino acid D or E; and P1' is the 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) (where P4 is the amino acid I or L; P3 is the amino acid E; P2 is the amino acid P or A; P1 is the amino acid D; and P1' is the amino acid I, V, T, S, or G).

[0283] In some embodiments, the linker does not contain the amino acid sequence TIFF2025102819000061.tif4145. In some embodiments, the linker does not contain the amino acid sequence TIFF2025102819000062.tif26155.

[0284] In some embodiments, the linker does not contain amino acids that are substrates of matriptase. In some embodiments, the linker does not contain the sequence P1QAR↓(A / V)(SEQ ID NO:154) (where 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 amino acids that are substrates of 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) (where P3 is P, V, or A; P2 is Q or D; P1 is A or N; and 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) (where P3 is P; P2 is Q or D; P1 is A or N; and 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 not a linker that contains the amino acid sequence shown as TIFF2025102819000063.tif99159

[0287] 4. Antigen-binding domain: The multispecific polypeptide constructs of the present disclosure include at least one antigen-binding domain, for example, including 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 native cognate binding partner, an anticalin (modified lipocalin), a darpin, a fibronomer, a centyrin (modified fibronectin type III domain), a cystine knot domain, an affilin, an affibody, or a modified CH3 domain. In some embodiments, the native cognate binding partner includes an extracellular domain or a binding fragment thereof of the native cognate binding partner of the TAA, or variants thereof that exhibit binding activity with the TAA.

[0288] In some embodiments, the TAA is a counter-structure that is predominantly present on the tumor cells of a mammalian subject and is generally not found on the normal cells of the mammalian subject. Tumor-specific antigens need not be exclusive to tumor cells and can be targeted by an antitumor therapeutic agent such as the provided multispecific polypeptide construct, and the proportion of cells having a particular mammalian tumor-associated antigen is high enough, or the level of tumor-associated antigen on the surface of the tumor is high enough, to provide prevention or treatment of the effects of mammalian tumors. In some embodiments, in a random statistical sample of cells derived from a mammalian subject having a tumor, at least 50% of the cells exhibiting the TAA are cancer cells. In other embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the cells exhibiting the TAA are cancer cells.

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

[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 on the amino-terminal side relative to the Fc region of the multispecific polypeptide construct and / or on the carboxy-terminal side relative to the CD3-binding region. In some embodiments, the multispecific polypeptide construct contains only one scFv or sdAb that binds to a TAA, which may be positioned on either the amino-terminal side relative to the Fc region and / or on the carboxy-terminal side relative to the CD3-binding region. In some embodiments, the multispecific polypeptide construct contains two scFvs or sdAbs that bind to a TAA, positioned on the amino-terminal side relative to the Fc region and / or on the carboxy-terminal side relative to the CD3-binding region. In some embodiments, the multispecific polypeptide construct contains three scFvs or sdAbs, two of which are positioned on the amino-terminal side relative to the Fc region or on the carboxy-terminal side relative 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 comprises 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 the heterodimeric Fc region, a linker, a VL domain of an anti-CD3 antibody or antigen-binding fragment (e.g., Fv), and optionally the same or a different scFv or sdAb that binds to a tumor-associated antigen, and is formed from or comprises two polypeptides. The scFv or sdAb that binds to the TAA may be positioned on the amino-terminal side relative to the Fc polypeptide of the heterodimeric Fc and / or on the carboxy-terminal side relative to the VH 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 contains 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 antigen-binding domain and the second antigen-binding domain, independently contains a plurality of chains. 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 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 VH-CH1 (Fd) and VL-CL is positioned on the amino-terminal side with respect to the Fc region of the multispecific polypeptide construct and / or on the carboxy-terminal side with respect to the CD3-binding region. In some embodiments, the multispecific polypeptide construct contains only one Fab antibody containing VH-CH1 (Fd) and VL-CL that binds to a TAA, which may be positioned either on the amino-terminal side with respect to the Fc region or on the carboxy-terminal side with respect to the CD3-binding region. In some embodiments, the multispecific polypeptide construct contains two Fab antibody fragments each containing VH-CH1 (Fd) and VL-CL that bind to a TAA, one positioned on the amino-terminal side with respect to the Fc region and the other positioned on the carboxy-terminal side with respect to the CD3-binding region.

[0294] In some embodiments, the multispecific polypeptide construct is formed from, or comprises, more than three 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 the heterodimeric Fc region and 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 a 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, an extracellular domain of a native cognate binding partner of a TAA or a binding fragment thereof, or a variant thereof that exhibits binding activity to a TAA.

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

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

[0298] In some embodiments, the TAA is 1-92-LFA-3, 5T4, α4 integrin, αV integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis Y, apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), claudin 3, claudin 4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor α (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPSelected from IIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R (wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged1, Jagged2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin 16 (MUC16, CA-125), Na / K ATPase, NGF, nicastrin, Notch receptor, Notch1, Notch2, Notch3, Notch4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine-1-phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

[0299] In some embodiments, at least one antigen-binding domain, or each antigen-binding domain independently, binds to folate receptor alpha (FRα), a tumor-associated antigen (TAA). For example, the antigen-binding domain contains the 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 the binding domain as an sdAb that binds to cMET. Exemplary cMET-binding sdAbs are 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 tumor-associated antigen (TAA) B7H3. For example, the antigen-binding domain contains the binding domain as an scFv that binds to B7H3. Exemplary B7H3-binding scFvs are shown in SEQ ID NO: 124. In some embodiments, the antigen-binding domain is an sdAb such as a VHH. Exemplary B7H3-binding sdAbs are shown in any of SEQ ID NOs: 214-218. In some embodiments, the antigen-binding domain is or contains a Fab antibody fragment comprising VH-CH1 (Fd) or LC. Exemplary B7H3 Fd is shown in SEQ ID NO: 127, and exemplary B7H3 LC is shown 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 shown in SEQ ID NO:189 and a VL shown in SEQ ID NO:190, or sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, 98%, or 99%, or about 85%, 90%, 95%, 96%, 97%, 98%, 98%, or 99% sequence identity to 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 shown in SEQ ID NO: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, binds to each of the tumor-associated antigens (TAAs) DLL3. For example, the antigen-binding domain contains a binding domain as an scFv that binds to DLL3. Exemplary DLL3-binding scFvs are shown in SEQ ID NO:126 and 188 (U.S. Publication No. US2017 / 0037130). In some embodiments, the antigen-binding domain is a sdAb such as a VHH. Exemplary DLL3-binding sdAbs are shown in either VH shown 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 an LC that bind to DLL3. An exemplary DLL3 Fd is shown in SEQ ID NO:133 and an exemplary DLL3 LC is shown in SEQ ID NO:134 (U.S. Patent No. 8,044,178).

[0304] In some embodiments, at least one antigen-binding domain, or each antigen-binding domain independently, binds to 5T4, a tumor-associated antigen (TAA). An exemplary 5T4 Fd is shown in SEQ ID NO:129, and an exemplary 5T4 LC is shown in SEQ ID NO:130. In some embodiments, the antibody-binding domain comprises the VH-CH1 (Fd) or VL-CL shown 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 gpNMB, a tumor-associated antigen (TAA). 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 shown in SEQ ID NO:131, and an exemplary gpNMB LC is shown in SEQ ID NO:132.

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

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

[0308] In some embodiments, LP1 or LP2 is independently a peptide about 1 to 20 amino acids in length. In some embodiments, LP1 or LP2 is independently the Gly-Ser linker shown in SEQ ID NOs: 10-13, 119, 135, 147, 149, or GGS, or a peptide comprising it.

[0309] III. Pharmaceutical Compositions Compositions of any of the provided multispecific polypeptide constructs are provided herein. It will be understood that administration of a therapeutic entity according to the present disclosure is administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved uptake, delivery, resistance, etc. Numerous suitable formulations can be found in the formulary known to all pharmacists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975)), in particular, chapter 87 by Blaug, Seymour therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing vesicles (such as Lipofectin™) (cationic or anionic), DNA conjugates, anhydrous absorbent 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 can be suitable in the treatments and therapies according to the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible with the route of administration and is acceptable.For additional information on formulations, excipients, and carriers well known to pharmacists, see 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 the references cited therein.

[0310] In some embodiments, the multispecific polypeptide constructs, conjugated multispecific polypeptide constructs, and their compositions, as well as their derivatives, fragments, analogs, and homologs, collectively referred to herein as therapeutic agents, can be incorporated into a pharmaceutically suitable composition for administration. 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 include a multispecific polypeptide construct or its conjugate and a pharmaceutically acceptable carrier. When the multispecific polypeptide construct includes 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 that retains the ability to bind to the target protein sequence of the antibody can be designed. 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" is intended to include any solvent, dispersion medium, coating, antibacterial, antifungal, isotonic agent, absorption delaying agent, and the like that is compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the art, 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. Non-aqueous media such as liposomes and fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent is contemplated to be used in the composition so long as it is not incompatible with the active compound.

[0313] Formulations used for in vivo administration must be sterile. This 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 routes of administration include parenteral administration, such as 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: sterile diluents such as water for injection, aqueous saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium sulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetic acid, citric acid, or phosphoric acid, and agents for adjusting the osmotic pressure such as sodium chloride or dextrose. The pH may be adjusted with an acid or base 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] Suitable pharmaceutical compositions for injectable use include sterile aqueous solutions (water-soluble) or dispersions, or sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, sui...

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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