Multispecific polypeptide constructs with constrained CD3 binding and methods of using same

Abstract

To provide multispecific polypeptides having constrained CD3 binding, where, the multispecific polypeptides contain cleavable linkers that, when cleaved, result in dual effector functions and are used in a variety of therapeutic, diagnostic and prophylactic applications.SOLUTION: A multispecific polypeptide construct comprises a first component comprising an immunoglobulin Fc region, and a second component comprising a CD3-binding region, where the first and second components are coupled by a 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 comprise an antigen binding domain that binds a tumor associated antigen (TAA).SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 484,217, filed April 11, 2017, entitled "MULTISPECIFIC POLYPEPTIDES HAVING DUAL EFFECTOR FUNCTION AND METHODS OF USING THE SAME," the contents of which are incorporated by reference in their entirety.

[0002] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING This application is submitted with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 744952000140SeqList.TXT, created on April 11, 2018, and having a size of 174,179 bytes. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field of Disclosure The present invention generally relates to multispecific polypeptides with constrained CD3 binding. In some embodiments, the multispecific polypeptides contain a cleavable linker that, when cleaved, provides dual effector functions. Methods for making and using these multispecific polypeptides in a variety of therapeutic, diagnostic, and prophylactic applications are also provided. [Background technology]

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

[0005] The CD3 (cluster of differentiation 3) T-cell coreceptor is a multimeric protein composed of four distinct polypeptide chains called the epsilon, gamma, delta, and zeta chains. The CD3 complex serves as the signaling module of the T-cell receptor (TCR), noncovalently associating with the antigen-binding a / b chains of the TCR.

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

[0007] Disclosure Overview The present disclosure provides multispecific polypeptide constructs that exhibit constrained CD3 binding. In some embodiments, the multispecific polypeptide constructs are composed of a first component comprising an immunoglobulin Fc region and a second component comprising a CD3-binding region, wherein the first and second components are coupled or operably linked by a linker, and the Fc region is located N-terminal to the CD3-binding region; one or both of the first and second components comprises an antigen-binding domain that binds to a tumor-associated antigen (TAA). In some embodiments, the multispecific polypeptide constructs are composed of a first component and a second component in an inactive state, wherein the first and second components are operably linked, and each of the first and second components comprises an antigen-binding domain that binds to a tumor-associated antigen (TAA), the first component comprises an Fc region, and the second component comprises a CD3-binding region, and the first and second components are coupled by a cleavable linker. In some embodiments, the CD3-binding region binds to CD3 (CD3ε).

[0008] In some embodiments, the antigen-binding domain is positioned amino-terminal to the Fc region and / or carboxy-terminal to the CD3-binding region of the multispecific polypeptide construct. In some embodiments, the first component comprises a first antigen-binding domain and the second component comprises a second antigen-binding domain, wherein each of the antigen-binding domains binds to a tumor-associated antigen (TAA). In some cases, the first antigen-binding domain is positioned at the amino-terminus of the multispecific construct and the second antigen-binding domain is positioned at the carboxy-terminus of the multispecific construct. In some embodiments, the first antigen-binding domain is positioned amino-terminal to the Fc region and / or carboxy-terminal to the CD3-binding region of the multispecific polypeptide construct.

[0009] Provided herein is a multispecific polypeptide construct comprising, from N-terminus to C-terminus, a first antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker; a CD3-binding region that binds to CD3 (CD3ε); and a second antigen-binding domain that binds to a tumor-associated antigen (TAA). Also provided herein is a multispecific polypeptide construct comprising, from N-terminus to C-terminus, an immunoglobulin Fc region; a linker; a CD3-binding region that binds to CD3 (CD3ε); and an antigen-binding domain that binds to a tumor-associated antigen (TAA). Provided herein is a multispecific polypeptide construct comprising, from N-terminus to C-terminus, an antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker; and a CD3-binding region that binds to CD3 (CD3ε).

[0010] Embodiments of the present disclosure include multispecific polypeptide constructs that bind to at least CD3 and a second antigen, such as a tumor-associated antigen (TAA). The multispecific polypeptide constructs provided herein include at least a first component comprising one or more copies of an antigen-binding domain that binds to the antigen linked to an immunoglobulin Fc region, a second component comprising at least one or more copies of a binding domain that binds to CD3 (referred to herein as an anti-CD3 binding domain or CD3 binding region, used interchangeably herein), and a linker, such as a cleavable linker, joining the first component and the second component.

[0011] Positioning the Fc region N-terminal to the CD3-binding region reduces or prevents the ability of the CD3-binding region to bind to CD3. In some embodiments, in the uncleaved / inactive state, the first component (component #1) and the second component (component #2) of the multispecific polypeptide construct are linked, disallowing binding to CD3 unless the antigen-binding domain binds to the cognate antigen. This is advantageous because it prevents systemic binding of the CD3-binding region to T cells and concentrates it at the site of antigen expression. This is beneficial because it eliminates the primary binding sink of peripheral T cells, allowing for more favorable distribution and localization to the site of antigen expression, e.g., tumor cells or the tumor microenvironment. In some cases, CD3 binding and / or engagement is amplified or increased by including a cleavable linker joining component #1 and component #2, allowing increased binding of the CD3-binding region upon cleavage of the cleavable linker, such as by proteolysis.

[0012] In the inactive, i.e., uncleaved, state, components #1 and #2 of the multispecific polypeptide construct are operably linked and do not bind or otherwise engage CD3 unless the antigen-binding domain binds to a cognate antigen. In some embodiments, the uncleaved multispecific polypeptide construct is capable of interacting with FcγR and mediating innate immune effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the uncleaved multispecific polypeptide construct is capable of interacting with complement proteins, i.e., C1q, and mediating complement-dependent cytotoxicity.

[0013] The multispecific polypeptide constructs of the present disclosure generally have multiple antigen-binding domains. In provided aspects where the multispecific polypeptide construct contains a cleavable linker, after the linker joining the first and second components is cleaved, such as by a protease, each component retains at least one antigen-binding domain. The first component (i.e., component #1) contains at least an Fc region and an antigen-binding domain. The second component (i.e., component #2) contains at least an anti-CD3 binding domain and an antigen-binding domain.

[0014] Cleavage of the cleavable linker, such as by proteolysis, physically separates components #1 and #2, each of which has therapeutic utility, albeit relying on different effector cells. Component #1 contains at least one antigen-binding domain and an Fc region. In some embodiments, component #1 can induce innate immune effector functions, such as ADCC, cytokine release, degranulation, and / or phagocytosis. Component #2 contains at least a CD3-binding region and an antigen-binding domain, the former of which can bind to CD3 (when separated from component #1). Component #2 can form an immune synapse between antigen-expressing cells and T cells. This simultaneous engagement mediates antigen-dependent T cell activation, cytotoxicity, cytokine release, degranulation, and proliferation. In the cleaved / activated state, component #2 is not operably linked to the Fc region of component #1; therefore, component #2 does not interact with FcRn and has enhanced serum clearance when localized to sites lacking antigen-expressing cells. This is advantageous because it limits systemic exposure of the activated anti-CD3 binding domain and concentrates it directly in tissues expressing the antigen, such as tumor cells or the tumor microenvironment.

[0015] In some embodiments, the multispecific polypeptide is in an inactive state, i.e., uncleaved state, and binding of the CD3 binding region to CD3 is inhibited or substantially reduced when the multispecific polypeptide construct is in the uncleaved state compared to the cleaved state. In some embodiments, the multispecific polypeptide is in an activated state, and the first and second components are not operably linked. In some embodiments, the multispecific polypeptide is in an activated state, i.e., cleaved state, and the second component binds to the epsilon chain of CD3 (CD3ε) and a tumor-associated antigen (TAA).

[0016] In some aspects, the antigen-binding domain, or each of the antigen-binding domains, independently, is selected from an antibody or antigen-binding fragment, a natural cognate binding partner, anticalin (a modified lipocalin), darpin, fynomer, centyrin (a modified fibronectin III domain), a cystine-knot domain, affilin, affibody, or a modified CH3 domain. In some embodiments, the natural cognate binding partner comprises the extracellular domain of the native cognate binding partner of the TAA or a binding fragment thereof, or a variant thereof that exhibits binding activity with the TAA.

[0017] In some aspects, the antigen binding domain, or each of the antigen binding domains independently, comprises the extracellular domain of the native cognate binding partner of the TAA or a binding fragment thereof, or a variant thereof that exhibits binding activity with the TAA.

[0018] In some embodiments, the first component comprises one or more copies of an antigen-binding domain. In some embodiments, the first component contains at least two antigen-binding domains, such as two antigen-binding domains. In some embodiments, at least two antigen-binding domains of the first component bind to the same TAA. In some cases, at least two antigen-binding domains of the first component bind to different epitopes of the same TAA. In some cases, at least two antigen-binding domains of the first component bind to different epitopes of the same TAA. In some embodiments, at least two antigen-binding domains of the first component bind to different TAAs.

[0019] In some embodiments, the antigen-binding domain of the first component, in some cases the first antigen-binding domain, comprises one or more copies of an antibody or antigen-binding fragment thereof. In some embodiments, the antigen-binding domain of the first component, such as the first antigen-binding domain, comprises one or more copies of an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a Fv fragment, a scFv, a scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. In some embodiments, the first antigen-binding domain comprises one or more single-domain antibody (sdAb) fragments, such as a V H H, V NAR , Modified V H domain, or modified V K Contains one or more copies of the domain. V H H can be generated from camelid heavy chain-only antibodies. NAR can be generated from cartilaginous fish heavy chain-only antibodies. Various methods, including interface engineering and specific germline family selection, have been used to generate V-heterodimeric antibodies. H Domains and V K This has been done to generate monomeric sdAbs from the domains.

[0020] In some embodiments, the antigen binding domain of the first component, such as the first antigen binding domain, binds to an antigen, such as a tumor-associated antigen (TAA). In some embodiments, the TAA is selected from the group consisting of 1-92-LFA-3, 5T4, alpha4 integrin, alphaV integrin, alpha4beta1 integrin, alpha4beta7 integrin, AGR2, anti-Lewis Y, apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD 20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD 64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD16 6, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), claudin 3, claudin 4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EG FR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE , IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL 23, IL23R, IL27 / IL27R (wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin 16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch1, Notch2, Notch3, Notch4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine-1-phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TI Selected from the group consisting of GIT, 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.

[0021] In some embodiments, the Fc region is a homodimeric Fc region. In some embodiments, the Fc region is a heterodimeric Fc region.

[0022] In some embodiments, the immunoglobulin Fc region of the first component is an IgG isotype selected from the group consisting of an IgG1 isotype, an IgG2 isotype, an IgG3 isotype, and an IgG4 subclass. In some examples, the Fc region is an 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 set forth in SEQ ID NO:1, or a sequence of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1. In some cases, the Fc region comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, or a sequence of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2. In some such embodiments, the Fc region comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4, or a sequence of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:4. In some examples, the Fc region comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:5, or a sequence of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:5.

[0023] 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.

[0024] 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. 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 a polypeptide comprising an amino acid sequence derived from an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, comprising one or more modifications to prevent glycosylation, alter Fc receptor interaction, reduce Fc receptor binding, enhance interaction with CD32A, reduce complement protein C1q binding, extend half-life, enhance FcRn binding, alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), induce heterodimerization, prevent dimerization, stabilize homodimerization at the CH3:CH3 interface, and combinations thereof.

[0025] In some embodiments, the Fc is a heterodimeric Fc. In some cases, one or both Fc polypeptides of the heterodimeric Fc region comprise at least one modification to induce heterodimerization compared to the polypeptide of the homodimeric Fc region, optionally compared to the Fc polypeptide set forth in SEQ ID NO:1 or an immunologically active fragment thereof. In some embodiments, each of the Fc polypeptides of the heterodimeric Fc comprises at least one modification. In some cases, each of the Fc polypeptides of the heterodimeric Fc comprises a knobs-into-holes modification or a charge mutation to increase electrostatic complementarity of the polypeptides. In some embodiments, the amino acid modification is a knobs-into-holes modification.

[0026] In some embodiments, the first Fc polypeptide of the heterodimeric Fc comprises a modification selected from Thr366Ser, Leu368Ala, Tyr407Val, and a combination thereof, and the second Fc polypeptide of the heterodimeric Fc comprises the modification T366W. In some cases, the first and second Fc polypeptides further comprise a modification of a non-cysteine ​​residue to a cysteine ​​residue, wherein the modification of the first polypeptide is at one of positions Ser354 and Y349, and the modification of the second Fc polypeptide is at the other of positions Ser354 and Y349.

[0027] In some instances, the amino acid modification is a charge mutation to increase the electrostatic complementarity of the polypeptides. In some embodiments, the first and / or second Fc polypeptide comprises a modification at a complementary position that is a substitution of an amino acid with an opposite charge to the complementary amino acid of the other polypeptide. In some embodiments, the first or second polypeptide comprises a modification at a complementary position that is a substitution of an amino acid with an opposite charge to the complementary amino acid of the other polypeptide. In some embodiments, at least the first or second Fc polypeptide each comprises a modification at a complementary position that is a substitution of an amino acid with an opposite charge to the complementary amino acid of the other polypeptide. In some embodiments, the first and second Fc polypeptide each comprise a modification at a complementary position that is a substitution of an amino acid with an opposite charge to the complementary amino acid of the other polypeptide.

[0028] 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. In some embodiments, the Fc region comprises a polypeptide lacking Lys447.

[0029] In some embodiments, modifications in the Fc region reduce binding to Fcγ receptors but minimally affect binding to neonatal Fc receptors (FcRn). In some embodiments, the variant or modified Fc polypeptide comprises the following mutations, using the Kabat numbering system: Met252Tyr and Met428Leu or Met252Tyr and Met428Val (M252Y, M428L, or M252Y, M428V).

[0030] In some embodiments, the Fc region comprises a polypeptide comprising at least one modification to enhance FcRn binding. In some instances, the modification is at a position selected from the group consisting of Met252, Ser254, Thr256, Met428, Asn434, and combinations thereof. In some cases, the modification is at a position selected from the group consisting of Met252Y, Ser254T, Thr256E, Met428L, Met428V, Asn434S, and combinations thereof. In some specific embodiments, the modification is at position Met252 and position Met428. In some cases, the modification is Met252Y and Met428L. In some cases, the modification is Met252Y and Met428V.

[0031] In some embodiments, the first polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs:82, 86, 94, or 96, and the second polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs:83, 87, 90, 92, 98, or 100.

[0032] In some embodiments, the Fc region comprises a polypeptide comprising at least one amino acid modification that reduces effector function and / or reduces binding to an effector molecule selected from an Fcγ receptor or C1q. In some examples, the one or more amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235. In some aspects, a first polypeptide of the heterodimeric Fc comprises a sequence of amino acids set forth in any of SEQ ID NOs: 84, 88, 95, or 97, and a second polypeptide of the heterodimeric Fc comprises a sequence of amino acids set forth in any of SEQ ID NOs: 85, 89, 91, 93, 99, or 101.

[0033] In some embodiments, the Fc region comprises a polypeptide comprising at least one modification to enhance FcγR binding. In some cases, the modification is at Ser239 or Ile332. In some embodiments, the glycosylation of the Fc region is modified to enhance FcγR binding compared to an unmodified Fc region. In some examples, the Fc region lacks fucose or has a reduced fucose content.

[0034] 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 heavy chain variable region (VH) and a light chain variable region (VL). In some such embodiments, the CD3 binding region is monovalent.

[0035] In some embodiments, the anti-CD3 antibody or antigen-binding fragment is not a single-chain antibody, and optionally is not a single-chain variable fragment (scFv). In some embodiments, the Fc is a heterodimeric Fc, and the VH and VL comprising the anti-CD3 antibody or antigen-binding fragment are linked to opposite polypeptides of the heterodimeric Fc. In some embodiments, the CD3 binding region is incapable of, or substantially incapable of, binding to or engaging with CD3 unless at least one of the antigen-binding domains binds to its TAA. In some aspects, the CD3 binding region is incapable of, or substantially incapable of binding to or engaging with CD3 unless at least two of the antigen-binding domains bind to its TAA.

[0036] 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 a polypeptide of about 2-24 amino acids, 2-20 amino acids, 2-18 amino acids, 2-14 amino acids, 2-12 amino acids, 2-10 amino acids, 2-8 amino acids, 2-6 amino acids, 6-24 amino acids, 6-20 amino acids, 6-18 amino acids, 6-14 amino acids, 6-12 amino acids, 6-10 amino acids, 6-8 amino acids, 8-24 amino acids, 8-20 amino acids, 8-18 amino acids, 8-14 amino acids, 8-12 amino acids, 8-10 amino acids, 10-24 amino acids, 10-20 amino acids, 10-18 amino acids, 10-14 amino acids, 10-12 amino acids, 12-24 amino acids, 12-20 amino acids, 12-18 amino acids, 12-14 amino acids, 14-24 amino acids, 14-20 amino acids, 14-18 amino acids, 18-24 amino acids, 18-20 amino acids, or 20-24 amino acids. In some embodiments, the linker is a polypeptide that is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some cases, the linker is a cleavable linker.

[0037] In some embodiments, the first antigen-binding domain and the immunoglobulin Fc polypeptide are operably linked via an amino acid linker. In some embodiments, these intracomponent linkers are primarily composed of the amino acids glycine and serine, and are referred to herein as GS linkers. The GS linkers of the fusion proteins of the present disclosure can be of various lengths, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0038] In some embodiments, the GS linker is TIFF0007798942000001.tif18146.

[0039] In some embodiments, the second component also comprises one or more copies of an anti-CD3 binding domain. In some embodiments, the anti-CD3 binding domain comprises one or more copies of an antibody or antigen-binding fragment thereof. In some embodiments, the anti-CD3 binding domain comprises one or more copies of an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a Fv fragment, a scFv, a scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. In some embodiments, the 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 comprises an amino acid sequence selected from the group 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 to 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 to 62 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 63 to 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.

[0040] In some embodiments, the anti-CD3ε Fv antibody fragment is a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv).

[0041] In some embodiments, the second component also comprises one or more copies of an antigen-binding domain. In certain embodiments, the second component contains at least two antigen-binding domains, such as two antigen-binding domains. In some embodiments, at least two antigen-binding domains of the second component bind to the same TAA. In some cases, at least two antigen-binding domains of the second component bind to different epitopes of the same TAA. In some cases, at least two antigen-binding domains of the second component bind to different epitopes of the same TAA. In some embodiments, at least two antigen-binding domains of the second component bind to different TAAs.

[0042] 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 second antigen-binding domain of the second component binds to the same antigen as the first antigen-binding domain of the first component. In some embodiments, the second antigen-binding domain of the second component binds to a different epitope of the same antigen as the first antigen-binding domain of the first component. In some embodiments, the second antigen-binding domain of the second component binds to the same epitope of the antigen as the first antigen-binding domain of the first component.

[0043] In some embodiments, the antigen-binding domain of the second component, such as the second antigen-binding domain, comprises one or more copies of an antibody or antigen-binding fragment thereof. In some embodiments, the second antigen-binding domain comprises one or more copies of an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a Fv fragment, a scFv, a scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. In some embodiments, the second antigen-binding domain comprises one or more single-domain antibody (sdAb) fragments, such as a V H H, V NAR , Modified V H domain, or modified V KContains one or more copies of the domain. V H H can be generated from camelid heavy chain-only antibodies. NAR can be generated from cartilaginous fish heavy chain-only antibodies. Various methods, including interface engineering and specific germline family selection, have been used to generate V-heterodimeric antibodies. H Domains and V K This has been done to generate monomeric sdAbs from the domains.

[0044] In some embodiments, the antigen binding domain of the second component, such as the second antigen binding domain, binds to an antigen, such as a tumor-associated antigen (TAA). In some embodiments, the TAA is selected from the group consisting of 1-92-LFA-3, 5T4, alpha4 integrin, alphaV integrin, alpha4beta1 integrin, alpha4beta7 integrin, AGR2, anti-Lewis Y, apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD 20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD 64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD16 6, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), claudin 3, claudin 4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EG FR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL2 1, IL23, IL23R, IL27 / IL27R(wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin 16(MUC16, CA-125), Na / K ATPase, NGF, nicastrin, Notch receptor, Notch1, Notch2, Notch3, Notch4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine-1-phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TI M-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.

[0045] In some embodiments, the antigen-binding domain of the second component, such as the second antigen-binding domain, and the anti-CD3 binding domain are operably linked via an amino acid linker. In some embodiments, these intracomponent linkers are primarily composed of the amino acids glycine and serine, and are referred to herein as GS linkers. The GS linkers of the fusion proteins of the present disclosure can be of various lengths, for example, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids.

[0046] In some embodiments, the GS linker is TIFF0007798942000002.tif18145.

[0047] Provided herein are multispecific polypeptide constructs comprising a first component comprising a heterodimeric Fc region and a second component comprising an anti-CD3 antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprising the anti-CD3 antibody or antigen-binding fragment are linked to opposite polypeptides of the heterodimeric Fc; the first and second components are coupled by a cleavable linker, and the heterodimeric Fc region is positioned N-terminal to the anti-CD3 antibody; and one or both of the first and second components comprises an antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0048] In some embodiments, binding of the CD3 binding region to CD3 is substantially reduced when the multispecific polypeptide construct is in an uncleaved state compared to a cleaved state, hi some embodiments, in the cleaved state the first and second components are unlinked.

[0049] In some embodiments, the cleavable linker is a polypeptide. In some embodiments, the cleavable linker is a polypeptide that is a substrate for a protease. In some embodiments, the protease is produced by immune effector cells, by tumors, or by cells present in the tumor microenvironment. In some embodiments, the protease is produced by tumors in the vicinity of cells expressing CD3ε and / or by tumors co-localized with cells expressing CD3ε in tissues, and when the multispecific polypeptide construct is exposed to the protease, the protease cleaves the cleavable linker in the multispecific polypeptide construct. In some embodiments, the protease is produced by tumors in the vicinity of cells expressing one or more tumor-associated antigens (TAA) and / or by tumors co-localized with cells expressing the target TAA in tissues, and when the multispecific polypeptide construct is exposed to the protease, the protease cleaves the cleavable linker in the multispecific polypeptide construct. In some embodiments, the protease is produced by immune effector cells. In some embodiments, the protease is produced by an immune effector cell in the vicinity of the cell expressing the TAA. In some instances, the protease is produced by an immune effector cell, and the immune effector cell is an activated T cell, a natural killer (NK) cell, or an NK T cell. In some embodiments, the protease cleaves a cleavable linker within the multispecific polypeptide construct when the multispecific polypeptide construct is exposed to the protease. In some embodiments, the protease is produced by an immune effector cell in the vicinity of the cell expressing the TAA, and the protease cleaves a cleavable linker within the multispecific polypeptide construct when the multispecific polypeptide construct is exposed to the protease.

[0050] In some embodiments, the cleavable linker is a polypeptide up to 50 amino acids in length. In some embodiments, the cleavable linker is a polypeptide up to 25 amino acids in length. In some embodiments, the cleavable linker is a polypeptide up to 15 amino acids in length.

[0051] In some embodiments, the cleavable linker is a substrate for a protease selected from the proteases described herein. In some embodiments, the cleavable linker is a substrate for a protease selected from the group consisting of uPA, legumain, matriptase (also referred to herein as MT-SP1 or MTSP1), ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-9, MMP-12, MMP-13, MMP-14, and combinations thereof. In some embodiments, the cleavable linker is a substrate for a protease selected from the group consisting of uPA, legumain, and matriptase. In some embodiments, the protease is selected from matriptase, matrix metalloproteinase (MMP), granzyme B, and combinations thereof.

[0052] In some embodiments, the protease is granzyme B. In some examples, the cleavable linker comprises an amino acid sequence of the general formula P4 P3 P2 P1↓P1' (SEQ ID NO: 150) [P4 is amino acid I, L, Y, M, F, V, or A; P3 is amino acid A, G, S, V, E, D, Q, N, or Y; P2 is amino acid H, P, A, V, G, S, or T; P1 is amino acid D or E; and P1' is amino acid I, L, Y, M, F, V, T, S, G, or A]. In some embodiments, the cleavable linker comprises an amino acid sequence of the general formula P4 P3 P2 P1↓P1' (SEQ ID NO: 151) [P4 is amino acid I or L; P3 is amino acid E; P2 is amino acid P or A; P1 is amino acid D; and P1' is amino acid I, V, T, S, or G]. In some embodiments, the cleavable linker comprises the amino acid sequence IEPDI (SEQ ID NO:136), LEPDG (SEQ ID NO:152), LEADT (SEQ ID NO:137), IEPDG (SEQ ID NO:138), IEPDV (SEQ ID NO:139), IEPDS (SEQ ID NO:140), IEPDT (SEQ ID NO:141), or LEADG (SEQ ID NO:153). In some cases, the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:22, 105-112, 136-141, 148, and 150-153.

[0053] In some embodiments, the protease is matriptase. In some cases, the cleavable linker comprises the sequence P1QAR↓(A / V) (SEQ ID NO:154) [wherein P1 is any amino acid]; or the cleavable linker comprises the sequence RQAR(A / V) (SEQ ID NO:155). In some examples, the cleavable linker comprises the sequence RQARV (SEQ ID NO:156). In some cases, the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:23, 154-156.

[0054] In some embodiments, the protease is an MMP. In some examples, the MMP is MMP-2. In some embodiments, the cleavable linker comprises the general formula P3 P2 P1↓P1′ (SEQ ID NO:157) [P3 is P, V, or A; P2 is Q or D; P1 is A or N; P1′ is L, I, or M]. In some cases, the cleavable linker comprises the general formula P3 P2 P1↓P1′ (SEQ ID NO:158) [P; P2 is Q or D; P1 is A or N; P1′ is L or I]. In some embodiments, the cleavable linker comprises the sequence PAGL (SEQ ID NO:24). In some embodiments, the cleavable linker is a substrate for a matrix metalloprotease (MMP).

[0055] In some embodiments, the multispecific polypeptide construct comprises at least (i) a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and a VH domain of an anti-CD3 antibody or antigen-binding fragment; and (ii) a second polypeptide comprising a second Fc polypeptide of a heterodimeric Fc region, a linker, and a VL domain of an anti-CD3 antibody or antigen-binding fragment, wherein one or both of the first and second polypeptides comprises at least one antigen-binding domain that binds to a tumor-associated antigen (TAA). In some cases, only one of the first or second polypeptides comprises at least one antigen-binding domain that binds to a TAA.

[0056] In some provided embodiments, the antigen-binding domain provides monovalent, bivalent, trivalent, or tetravalent binding to the TAA. In some embodiments, the one or more antigen-binding domains that bind to the TAA are independently selected from an sdAb, an scFv, or a Fab. In some embodiments, the one or more antigen-binding domains that bind to the TAA are single-chain molecules such as single-chain antibody fragments, e.g., sdAb or scFv, containing a VH and a VL. In some embodiments, at least one of the antigen-binding domains is a Fab containing a first chain comprising a VH-CH1(Fd) and a second chain comprising a VL-CL.

[0057] In some embodiments, at least one antigen-binding domain is positioned amino terminal to the Fc region and / or carboxy terminal to the CD3 binding region of one of the first or second polypeptides of the multispecific polypeptide construct. In some cases, at least one antigen-binding domain is positioned amino terminal to the Fc region of the multispecific construct and the second antigen-binding domain is positioned carboxy terminal to the CD3 binding region of the multispecific construct.

[0058] In some embodiments, at least one of the antigen-binding fragments is a Fab. In some embodiments, the multispecific polypeptide construct comprises (i) a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and a VH domain of an anti-CD3 antibody or antigen-binding fragment; (ii) a second polypeptide comprising a second Fc polypeptide of a heterodimeric Fc region, a linker, and a VL domain of an anti-CD3 antibody or antigen-binding fragment; and (iii) a third polypeptide comprising a VH-CH1(Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen, wherein the first and / or second polypeptide further comprises the other of the VH-CH1(Fd) or VL-CL of the Fab antibody fragment. In some cases, only one of the first or second polypeptide comprises the other of the VH-CH1(Fd) or VL-CL of the Fab antibody fragment. In some embodiments, both the first or second polypeptide comprise the other of the VH-CH1(Fd) or VL-CL of the Fab antibody fragment. In some cases, the other of the VH-CH1(Fd) or VL-CL of the Fab antibody fragment is positioned amino terminal to the Fc region and / or carboxy terminal to the CD3 binding region of one of the first or second polypeptides of the multispecific polypeptide construct. In some embodiments, the other of the VH-CH1(Fd) or VL-CL of the Fab antibody fragment is positioned amino terminal to the Fc region of the first polypeptide or the second polypeptide and carboxy terminal to the CD3 binding region of the other of the first or second polypeptide.

[0059] In some examples, 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, CD3, CD6 , CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD 51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD13 7, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), claudin 3, claudin 4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL2 1, IL23, IL23R, IL27 / IL27R(wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin 16(MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch1, Notch2, Notch3, Notch4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine-1-phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, 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.

[0060] 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.

[0061] In some embodiments, the multispecific polypeptide construct comprises a first connecting peptide (LP1) between the first antigen-binding domain and the immunoglobulin Fc polypeptide region (Fc region). In some embodiments, the multispecific polypeptide construct comprises a second connecting peptide (LP2) between the anti-CD3 binding domain (CD3 binding region) and the second antigen-binding domain. In some embodiments, the multispecific polypeptide construct comprises a first connecting peptide (LP1) between the first antigen-binding domain and the immunoglobulin Fc polypeptide region (Fc region) and a second connecting peptide (LP2) between the anti-CD3 binding domain (CD3 binding region) and the second antigen-binding domain.

[0062] In some embodiments, the uncleaved multispecific polypeptide construct has the following structural arrangement, from N-terminus to C-terminus: first antigen-binding domain - LP1 - immunoglobulin Fc polypeptide region (Fc region) - linker (such as a cleavable linker) - anti-CD3 binding domain - LP2 - second antigen-binding domain. In some embodiments, the uncleaved multispecific polypeptide construct has the following structural arrangement, from N-terminus to C-terminus: second antigen-binding domain - LP2 - anti-CD3 binding domain (CD3 binding region) - linker (such as a cleavable linker) - immunoglobulin Fc polypeptide region - LP1 - first antigen-binding domain. In some examples, the linker is a cleavable linker. In some embodiments, the two connecting peptides are not identical to each other. In some cases, LP1 or LP2 are independently peptides of about 1 to 20 amino acids in length. In some examples, LP1 or LP2 independently comprise a peptide that is or includes a Gly-Ser linker set forth in SEQ ID NOs: 10-13, 119, 135, 147, 149.

[0063] In some embodiments, the multispecific construct is a construct having any of the structural arrangements shown in FIG. 1. In some embodiments, the multispecific construct is a bispecific construct having the structural arrangement shown in FIG. 2. In some embodiments, the bispecific construct has the following structural arrangement, from N- to C-terminus: The N-terminal side 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. A central immunoglobulin Fc polypeptide region that controls FcγR and / or FcRn interactions is coupled to the first antigen-binding domain. In some embodiments, the central immunoglobulin Fc polypeptide region is heterodimeric. The immunoglobulin Fc polypeptide region is coupled to a cleavable linker containing one or more proteolytic cleavage sites located C-terminal to the end of the immunoglobulin Fc polypeptide region. In some embodiments, the one or more proteolytic cleavage sites are substrates for matriptase, matrix metalloproteinase (MMP), or granzyme B. The cleavable linker is attached to the anti-CD3 binding sequence located C-terminal to the Fc region, and in some cases, to the distal end of the second moiety.

[0064] In some embodiments, the anti-CD3 antibody or antigen-binding fragment is an Fv antibody fragment. In some embodiments, the Fv antibody fragment comprises a disulfide-stabilized anti-CD3-binding Fv fragment (dsFv). In some embodiments, the anti-CD3-binding sequence is an Fv antibody fragment engineered to include a disulfide bond between the heavy chain variable region (VH) and the light chain variable region (VL), thereby creating a disulfide-stabilized anti-CD3-binding Fv fragment (dsFv). In some embodiments, the VH and VL domains comprising the anti-CD3 Fv are operably linked to opposite members of a heterodimeric Fc region. In these embodiments, the anti-CD3 Fv binds to CD3 monovalently. When the cleavable linker is intact, i.e., uncleaved or inactive, the anti-CD3 dsFv does not engage CD3. The C-terminus of the bispecific construct comprises a second antigen-binding domain that binds to a TAA. In some embodiments, the second antigen-binding domain binds to the same TAA as the first antigen-binding domain located on the first component. In some embodiments, the second antigen-binding domain binds to a second epitope of the TAA that does not compete with the first epitope of the TAA. In some embodiments, the second antigen-binding domain binds to a different TAA than the first antigen-binding domain.

[0065] In some embodiments, each of the first and second antigen-binding domains of the bispecific construct comprises one or more copies of an antibody or antigen-binding fragment thereof. In some embodiments, each of the first and second antigen-binding domains of the bispecific construct comprises one or more copies of an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, an F(ab')2 fragment, an Fv fragment, an scFv, an scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. In some embodiments, the antigen-binding domain, or each of the antigen-binding domains, independently, is an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, an F(ab')2 fragment, an Fv fragment, an scFv, an scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. In some embodiments, each of the first and second antigen-binding domains of the bispecific construct comprises one or more single-domain antibody (sdAb) fragments, e.g., V H H, V NAR , Modified V H domain, or modified V K Contains one or more copies of the domain. V H H can be generated from natural camelid heavy chain-only antibodies, genetically modified rodents producing heavy chain-only antibodies, or from naive / synthetic camelid or humanized camelid single domain antibody libraries. NAR can be generated from cartilaginous fish heavy chain-only antibodies. Various methods, including interface engineering and specific germline family selection, have been used to generate V-heterodimeric antibodies. H Domains and V K This has been done to generate monomeric sdAbs from the domains.

[0066] In some embodiments, the antibody or antigen-binding fragment is an sdAb. In some cases, the sdAb is a human or humanized sdAb. In some aspects, the sdAb is a VHH, VNAR, modified VH domain, or modified VK domain. In some examples, the antibody or antigen-binding fragment thereof is an scFv. In some cases, the antibody or antigen-binding fragment thereof is a Fab.

[0067] In any of the provided embodiments, the anti-CD3 antibody or antigen-binding fragment comprises a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO:16), an amino acid sequence VH CD2 containing TIFF0007798942000003.tif4128; amino acid sequence VH CDR3 containing TIFF0007798942000004.tif4128; amino acid sequence TIFF0007798942000005.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).

[0068] In some embodiments, the anti-CD3 dsFv comprises a VH having the amino acid sequence of any of SEQ ID NOs: 14, 44, and 32-62, or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 14, 44, and 32-62; and a VL having the amino acid sequence of any of SEQ ID NOs: 15, 72, and 63-81, or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 14, 44, and 32-62. In some cases, the anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO: 14 and the amino acid sequence of SEQ ID NO: 15. In 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.

[0069] In some embodiments, the immunoglobulin Fc region of the first component is an IgG subtype selected from the group consisting of an IgG1 isotype, an IgG2 isotype, an IgG3 isotype, and an IgG4 subclass. 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.

[0070] 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. 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 a polypeptide comprising an amino acid sequence derived from an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, comprising one or more modifications to prevent glycosylation, alter Fc receptor interaction, reduce Fc receptor binding, enhance interaction with CD32A, reduce complement protein C1q binding, extend half-life, enhance FcRn binding, alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), induce heterodimerization, prevent dimerization, stabilize homodimerization at the CH3:CH3 interface, and combinations thereof. In some embodiments, modifications within the Fc region reduce binding to the Fc receptor gamma receptor but have minimal effect on binding to the neonatal Fc receptor (FcRn). In some embodiments, the variant or modified Fc polypeptide comprises the following mutations, using the Kabat numbering system: Met252Tyr and Met428Leu or Met252Tyr and Met428Val (M252Y, M428L or M252Y, M428V).

[0071] In some embodiments, the first antigen-binding domain and the immunoglobulin Fc polypeptide are operably linked via an amino acid linker. In some embodiments, these intracomponent linkers are mainly composed of the amino acids glycine and serine, and are referred to herein as GS linkers. The GS linker of the fusion protein of the present disclosure can be of various lengths, for example, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids.

[0072] In some embodiments, the GS linker is TIFF0007798942000006.tif18145.

[0073] In some embodiments, the anti-CD3ε dsFv antibody fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32 to 81. In some embodiments, the anti-CD3ε dsFv 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 to 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 to 62 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 63 to 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.

[0074] In some embodiments, the second antigen-binding domain and the anti-CD3 binding domain are operably linked via an amino acid linker. In some embodiments, these intracomponent linkers are mainly composed of the amino acids glycine and serine, and are referred to herein as GS linkers. The GS linker of the fusion protein of the present disclosure can be of various lengths, for example, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids.

[0075] In some embodiments, the GS linker is TIFF0007798942000007.tif18146.

[0076] In some embodiments, the cleavable linker is a polypeptide. In some embodiments, the cleavable linker is a polypeptide that is a substrate for a protease. In some embodiments, the protease is produced by a tumor in the vicinity of cells expressing CD3ε and / or produced by a tumor co-localized with cells expressing CD3ε in a tissue, wherein when the multispecific polypeptide construct is exposed to the protease, the protease cleaves the cleavable linker in the multispecific polypeptide construct. In some embodiments, the protease is produced by a tumor in the vicinity of cells expressing one or more tumor-associated antigens (TAA) and / or produced by a tumor co-localized with cells expressing a target TAA in a tissue, wherein when the multispecific polypeptide construct is exposed to the protease, the protease cleaves the cleavable linker in the multispecific polypeptide construct.

[0077] In some embodiments, the cleavable linker is a polypeptide up to 50 amino acids in length. In some embodiments, the cleavable linker is a polypeptide up to 25 amino acids in length. In some embodiments, the cleavable linker is a polypeptide up to 15 amino acids in length. In some embodiments, the cleavable linker is a substrate for a protease selected from the proteases described herein. In some embodiments, the cleavable linker is a substrate for a protease selected from the group consisting of uPA, legumain, matriptase (also referred to herein as MT-SP1 or MTSP1), ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-9, MMP-12, MMP-13, MMP-14, and combinations thereof. In some embodiments, the cleavable linker is a substrate for a protease selected from the group consisting of uPA, legumain, and matriptase. In some embodiments, the cleavable linker is a substrate for a matrix metalloprotease (MMP).

[0078] 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 cleavable linker. In some embodiments, the linker is a non-cleavable linker.

[0079] In some embodiments, the multispecific constructs described herein are used in conjunction with one or more additional agents or combinations of additional agents. Suitable additional agents include, for example, current pharmaceutical and / or surgical treatments for the intended application, such as cancer. For example, the multispecific constructs can be used in conjunction with additional chemotherapy or anti-tumor agents.

[0080] In some embodiments, the multispecific construct and the additional agent are formulated into a single therapeutic composition, and the multispecific construct and the additional agent are administered simultaneously. In some embodiments, the multispecific construct and the additional agent are separate from one another, e.g., each formulated into a separate therapeutic composition, and the multispecific construct and the additional agent are administered simultaneously, or the multispecific construct and the additional agent are administered at different times during the treatment regimen. For example, the multispecific construct is administered before the administration of the additional agent, the multispecific construct is administered after the administration of the additional agent, or the multispecific construct and the additional agent are administered alternately. As described herein, the multispecific construct and the additional agent are administered once or multiple times.

[0081] In some embodiments, the multispecific construct naturally contains one or more disulfide bonds. In some embodiments, the multispecific construct may be modified to contain one or more disulfide bonds.

[0082] The present disclosure also provides isolated nucleic acid molecules or polynucleotides encoding at least a portion of the multispecific constructs described herein, and / or one or more nucleic acid molecules encoding the multispecific constructs described herein, such as, for example, at least a first nucleic acid encoding at least a portion of a first component of the multispecific construct and a second nucleic acid encoding at least a portion of a second component of the multispecific construct, as well as vectors comprising these isolated nucleic acid sequences.

[0083] The provided embodiments include polynucleotides encoding any of the provided multispecific polypeptide constructs. Polynucleotides encoding any of the polypeptide chains of the provided multispecific polypeptide constructs are also provided. Further provided are polynucleotides comprising a first nucleic acid sequence encoding a first polypeptide of any of the provided multispecific constructs and a second nucleic acid sequence encoding a second polypeptide of the multispecific construct, wherein the first and second nucleic acid sequences are separated by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping. In some cases, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter. In some embodiments, the multispecific polypeptide construct comprises a third polypeptide chain, and the polynucleotide further comprises a third nucleic acid encoding the third polypeptide of the multispecific construct. In some embodiments, the third nucleic acid is separated from the first and / or second polypeptide by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, and / or the third nucleic acid sequence is operably linked to the same promoter as the first and / or second nucleic acid sequence. In some examples, the nucleic acid encoding the self-cleaving peptide or the peptide that causes ribosome skipping is selected from T2A, P2A, E2A, or F2A (encoded by the sequences set forth in SEQ ID NOs:159-164, or SEQ ID NO:165).

[0084] Provided herein is a vector comprising any of the provided polynucleotides. In some embodiments, the vector is an expression vector. In some examples, the vector is a viral vector or a eukaryotic vector, and optionally, the eukaryotic vector is a mammalian vector.

[0085] Cells containing any of the provided polynucleotides or vectors are provided. In some cases, the cells are recombinant or isolated. In some examples, the cells are mammalian cells. In some examples, the cells are HEK293 cells or CHO cells.

[0086] The present disclosure provides methods of making multispecific constructs by culturing cells containing such nucleic acid sequences under conditions that result in expression of the multispecific construct. In some embodiments, the cells contain such vectors.

[0087] Provided herein are methods for producing a multispecific polypeptide construct, comprising introducing any of the provided polynucleotides or vectors into cells and culturing the cells under conditions resulting in expression of the multispecific construct to produce the multispecific polypeptide construct. Also provided are methods for producing a multispecific polypeptide construct, comprising culturing any of the provided cells under conditions in which the multispecific polypeptide is expressed or produced by the cells. In some cases, the cells are mammalian cells. In some examples, the cells are HEK293 cells or CHO cells. In some embodiments, the method further comprises isolating or purifying the multispecific polypeptide construct from the cells. In some cases, the multispecific polypeptide construct is a heterodimer.

[0088] Provided herein are multispecific polypeptide constructs produced by any of the provided methods.

[0089] Provided herein are methods for stimulating or inducing an immune response, comprising contacting target cells and T cells with any of the provided multispecific polypeptide constructs or pharmaceutical compositions, wherein the target cells express a tumor-associated antigen recognized by the multispecific polypeptide construct. In some embodiments, the target cells are tumor cells that express a tumor-associated antigen (TAA).

[0090] In some embodiments, the multispecific polypeptide construct comprises a cleavable linker that functions as a substrate for a protease, and the induction or stimulation of an immune response is increased in the presence of the protease. In some cases, the protease is produced by immune effector cells, by the tumor, or by cells present in the tumor microenvironment.

[0091] In some embodiments, the protease is produced by an immune effector cell, and the immune effector cell is an activated T cell, a natural killer (NK) cell, or an NK T cell. In some cases, the immune effector cell is in the vicinity of a cell expressing an antigen. In some embodiments, the protease is produced by a tumor in the vicinity of a cell expressing a TAA in a tissue and / or is produced by a tumor co-localized with a TAA in a tissue, and when the multispecific polypeptide construct is exposed to the protease, the protease cleaves the cleavable linker in the multispecific polypeptide construct. In some examples, the protease is selected from matriptase, matrix metalloproteinase (MMP), granzyme B, and combinations thereof. In some cases, the protease is granzyme B.

[0092] In some embodiments, the contacting is performed ex vivo or in vitro. In some embodiments, the contacting is performed in vivo in a subject.

[0093] Methods are provided for stimulating or inducing an immune response in a subject, comprising administering a therapeutically effective amount of any of the provided multispecific conjugates or pharmaceutical compositions to a subject in need thereof. In some cases, the methods increase cellular immunity. In some embodiments, the methods increase T cell activity. In some embodiments, the methods increase cytolytic T cell (CTL) activity. In some examples, the immune response against tumors or cancer is increased. In some embodiments, the methods treat a disease or condition in the subject.

[0094] The present disclosure also provides methods for treating, preventing, slowing the progression of, or otherwise ameliorating the symptoms of, one or more pathologies, or alleviating the symptoms associated with such pathologies, by administering a multispecific polypeptide construct of the present disclosure to a subject in whom such treatment or prevention is desired. Provided herein are methods for treating a disease or condition in a subject, comprising administering a therapeutically effective amount of any of the provided multispecific conjugates or pharmaceutical compositions to a subject in need thereof. In some embodiments, the disease or condition is tumor or cancer.

[0095] In some embodiments of any of the provided methods, the subject, such as the subject being treated, is, for example, a human or other mammal. In some embodiments of any of the provided methods, the subject is a human. In some embodiments, the subject is a non-human mammal, such as a non-human primate, a pet (e.g., a cat, dog, horse), livestock, a laboratory animal, or a zoo animal. In some embodiments, the subject is a rodent.

[0096] The multispecific polypeptide constructs of the present disclosure used in any of these methods and embodiments of use can be administered at any stage of the disease. For example, such multispecific polypeptide constructs can be administered to patients with cancer at any stage from early to metastatic. The terms subject and patient are used interchangeably herein.

[0097] The multispecific polypeptide constructs of the present disclosure used in any of these methods and embodiments of use may be used in treatment regimens including neoadjuvant therapy.

[0098] The multispecific polypeptide constructs of the present disclosure used in any of these methods and use embodiments may be administered alone or in combination with one or more additional agents, including small molecule inhibitors, other antibody-based therapies, polypeptide- or peptide-based therapies, nucleic acid-based therapies, and / or other biologics. In some embodiments, the multispecific polypeptide constructs are administered in combination with one or more additional agents, such as chemotherapeutic agents, including, but not limited to, alkylating agents, antimetabolites, anti-microtubule agents, topoisomerase inhibitors, cytotoxic antibiotics, and other nucleic acid damaging agents. In some embodiments, the additional agent is a taxane, such as paclitaxel (e.g., Abraxane®). In some embodiments, the additional agent is an antimetabolite, such as gemcitabine. In some embodiments, the additional agent is an alkylating agent, such as a platinum-based chemotherapy, such as carboplatin or cisplatin. In some embodiments, the additional agent is a targeted agent, such as a kinase inhibitor, e.g., sorafenib or erlotinib. In some embodiments, the additional agent is a 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 those of skill in the art. In some embodiments, the additional agent is a chemotherapeutic agent well known to those of skill in the art. In some embodiments, the multispecific polypeptide construct and the additional agent are formulated into a single composition. In some embodiments, the multispecific polypeptide construct and the additional agent are administered as two or more separate compositions. In some embodiments, the multispecific polypeptide construct and the additional agent are administered simultaneously.In some embodiments, the multispecific polypeptide construct and the additional agent are administered sequentially.

[0099] 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.

[0100] In some embodiments, the additional agent is a checkpoint inhibitor, a kinase inhibitor, an agent targeting an inhibitor of the tumor microenvironment, and / or a T cell or NK agonist. In some embodiments, the additional agent is radiation therapy, alone or in combination with another additional agent, such as a chemotherapeutic or anti-tumor agent. In some embodiments, the additional agent is a vaccine, an oncovirus, and / or a DC activator, such as, by way of non-limiting example, a Toll-like receptor (TLR) agonist and / or αCD40. In some embodiments, the additional agent is a tumor-targeted antibody designed to kill tumors via ADCC or via direct conjugation with a toxin (e.g., an antibody-drug conjugate (ADC)).

[0101] In some embodiments, the checkpoint inhibitor is an inhibitor of a target selected from the group consisting of CTLA-4, LAG-3, PD-1, PDL1, TIGIT, TIM-3, B7H3, B7H4, and Vista. In some embodiments, the kinase inhibitor is selected from the group consisting of B-RAFi, MEKi, and a Btk inhibitor such as ibrutinib. In some embodiments, the kinase inhibitor is crizotinib. In some embodiments, the tumor microenvironment inhibitor is selected from the group consisting of an IDO inhibitor, an αCSF1R inhibitor, an αCCR4 inhibitor, TGFβ, a myeloid-derived suppressor cell, or a T regulatory cell. In some embodiments, the agonist is selected from the group consisting of OX40, GITR, CD137, CD28, ICOS, CD27, and HVEM. In some embodiments, the checkpoint inhibitor is an antibody that binds to a target selected from CTLA-4, PD-1, and / or PD-L1. In some embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody, an anti-PD-1 antibody, and an anti-PD-L1 antibody, and / or a combination thereof. In some embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody, such as, for example, Yervoy™. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody, such as, for example, Opdivo™ and / or Keytruda™.

[0102] In some embodiments, the inhibitor is a CTLA-4 inhibitor. In some embodiments, the inhibitor is a LAG-3 inhibitor. In some embodiments, the inhibitor is a PD-1 inhibitor. In some embodiments, the inhibitor is a PDL1 inhibitor. In some embodiments, the inhibitor is a TIGIT inhibitor. In some embodiments, the inhibitor is a TIM-3 inhibitor. In some embodiments, the inhibitor is a B7H3 inhibitor. In some embodiments, the inhibitor is a B7H4 inhibitor. In some embodiments, the inhibitor is a Vista inhibitor. In some embodiments, the inhibitor is a B-RAFi inhibitor. In some embodiments, the inhibitor is a MEKi inhibitor. In some embodiments, the inhibitor is a Btk inhibitor. In some embodiments, the inhibitor is ibrutinib. In some embodiments, the inhibitor is crizotinib. In some embodiments, the inhibitor is an IDO inhibitor. In some embodiments, the inhibitor is an αCSF1R inhibitor. In some embodiments, the inhibitor is an αCCR4 inhibitor. In some embodiments, the inhibitor is TGFβ. In some embodiments, the inhibitor is a myeloid-derived suppressor cell. In some embodiments, the inhibitor is a T regulatory cell.

[0103] 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.

[0104] In some embodiments, the multispecific polypeptide construct is administered in combination with one or more additional agents, such as, for example, chemotherapeutic agents, anti-inflammatory agents, and / or immunosuppressants, during and / or after treatment. In some embodiments, the multispecific polypeptide construct and the additional agent are formulated into a single therapeutic composition, and the multispecific polypeptide construct and the additional agent are administered simultaneously. Alternatively, the multispecific polypeptide construct and the additional agent are separate from each other, e.g., each is formulated into a separate therapeutic composition, and the multispecific polypeptide construct and the additional agent are administered simultaneously, or the multispecific polypeptide construct and the additional agent are administered at different times during the treatment regimen. For example, the multispecific polypeptide construct is administered before the administration of the additional agent, the multispecific polypeptide construct is administered after the administration of the additional agent, or the multispecific polypeptide construct and the additional agent are administered alternately. As described herein, the multispecific polypeptide construct and the additional agent are administered once or multiple times.

[0105] In some embodiments, the multispecific polypeptide construct and the additional agent are administered simultaneously. For example, the multispecific polypeptide construct and the additional agent may be formulated into a single composition or may be administered as two or more separate compositions. In some embodiments, the multispecific polypeptide construct and the additional agent are administered sequentially, or the multispecific polypeptide construct and the additional agent are administered at different times during a treatment regimen.

[0106] In addition to the above elements, the multispecific polypeptide construct may contain additional elements, such as an amino acid sequence at the N- or C-terminus of the multispecific polypeptide construct. For example, the multispecific polypeptide construct may include a targeting moiety that facilitates delivery to a cell or tissue of interest. The multispecific polypeptide construct may be conjugated to an agent such as a therapeutic agent, a detectable moiety, or a diagnostic agent. Examples of agents are disclosed herein.

[0107] The multispecific polypeptide construct may also include a multispecific polypeptide construct of the present disclosure along with a conjugated agent, linker, and any of the other components described herein.

[0108] The present disclosure also relates to immunoconjugates comprising multispecific polypeptide constructs conjugated to a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin, or fragment thereof, derived from bacteria, fungi, plants, or animals), or a radioisotope (i.e., a radioconjugate). Suitable cytotoxic agents for use in targeting diseased T cells, such as in T cell-derived lymphomas, include, for example, dolastatin and its derivatives (e.g., auristatin E, AFP, MMAD, MMAF, MMAE). In some embodiments, the agent is a dolastatin. In some embodiments, the agent is an auristatin or a derivative thereof. In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine.

[0109] In some embodiments, the linker between the multispecific polypeptide construct and the cytotoxic agent is cleavable. In some embodiments, the linker is non-cleavable. In some embodiments, there are two or more linkers. The two or more linkers are all the same, e.g., cleavable or non-cleavable, or the two or more linkers are different, e.g., at least one is cleavable and at least one is non-cleavable.

[0110] The multispecific polypeptide constructs and conjugates thereof are useful in methods for treating a variety of disorders and / or diseases. Non-limiting examples of diseases include all types of cancer (breast cancer, lung cancer, colorectal cancer, prostate cancer, melanoma, head and neck cancer, pancreatic cancer, etc.), rheumatoid arthritis, Crohn's disease, SLE, cardiovascular disorders, ischemia, etc. For example, indications may include leukemias, including T-cell acute lymphoblastic leukemia (T-ALL), lymphoblastic diseases, including multiple myeloma, and solid tumors, including lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, and breast cancer, including triple-negative breast cancer. For example, indications include bone disease or metastasis of cancer regardless of primary tumor origin; breast cancer, including, but not limited to, ER / PR+ breast cancer, Her2+ breast cancer, and triple-negative breast cancer; colorectal cancer; endometrial cancer; gastric cancer; glioblastoma; head and neck cancer, such as esophageal cancer; lung cancer, including, but not limited to, non-small cell lung cancer; multiple myeloma; ovarian cancer; pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; kidney cancer, including, but not limited to, renal cell carcinoma; and / or skin cancer, including, but not limited to, squamous cell carcinoma, basal cell carcinoma, or melanoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is cutaneous squamous cell carcinoma. In some embodiments, the cancer is esophageal squamous cell carcinoma. In some embodiments, the cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is lung squamous cell carcinoma.

[0111] Pharmaceutical compositions are provided that include any of the multispecific polypeptide constructs provided herein and a pharmaceutically acceptable carrier. In some cases, the pharmaceutical composition is sterile. Pharmaceutical compositions according to the present disclosure may include a multispecific polypeptide construct of the present disclosure and a carrier. These pharmaceutical compositions may be included in a kit, such as a diagnostic kit.

[0112] Those skilled in the art will recognize that the antibodies of the present disclosure have a variety of uses. For example, the proteins of the present disclosure are used as therapeutic agents for a variety of disorders. The antibodies of the present disclosure can also be used as reagents or diagnostic tools in diagnostic kits, or they can be used in competitive assays to generate therapeutic reagents. [The present invention 1001] 1. A multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, the first and second components are coupled by a linker, and the Fc region is positioned N-terminal to the CD3 binding region; One or both of the first and second components comprises an antigen-binding domain that binds to a tumor-associated antigen (TAA); Multispecific polypeptide constructs. [The present invention 1002] 1001. A multispecific polypeptide construct of the present invention, wherein the CD3 binding region binds to CD3 (CD3ε). [The present invention 1003] The multispecific construct of invention 1001 or invention 1002, wherein the antigen binding domain is positioned amino-terminal to the Fc region and / or carboxy-terminal to the CD3 binding region of the multispecific polypeptide construct. [The present invention 1004] 4. The multispecific polypeptide construct of any of claims 1001 to 1003, wherein the first component comprises a first antigen-binding domain and the second component comprises a second antigen-binding domain, each of the antigen-binding domains binding to a tumor-associated antigen (TAA). [The present invention 1005] 1004. The multispecific polypeptide construct of the invention, wherein the first antigen-binding domain is positioned amino-terminal to the Fc region of the multispecific construct and the second antigen-binding domain is positioned carboxy-terminal to the CD3-binding region of the multispecific construct. [The present invention 1006] From the N-terminus to the C-terminus, a first antigen-binding domain that binds to a tumor-associated antigen (TAA); immunoglobulin Fc region; Linker; a CD3-binding region that binds to CD3 (CD3ε); and a second antigen-binding domain that binds to a tumor-associated antigen (TAA) 1. A multispecific polypeptide construct comprising: [The present invention 1007] The multispecific polypeptide construct of any of claims 1001 to 1006, wherein the Fc region is the Fc region of human IgG1, human IgG2, human IgG3, or human IgG4, or an immunologically active fragment thereof. [The present invention 1008] The Fc region a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, or a sequence of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:1; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, or a sequence of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:2; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4, or a sequence of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:4; or A polypeptide comprising the amino acid sequence set forth in SEQ ID NO:5, or a sequence of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:5. The multispecific polypeptide construct of any of claims 1001 to 1007, comprising: [The present invention 1009] The multispecific polypeptide construct of any of claims 1001 to 1008, wherein the Fc region is a heterodimeric Fc region. [The present invention 1010] 1009. The multispecific polypeptide construct of the present invention, wherein one or both Fc polypeptides of the heterodimeric Fc region comprise at least one modification to promote heterodimerization compared to the polypeptide of the homodimeric Fc region, and optionally compared to the Fc polypeptide set forth in SEQ ID NO:1 or an immunologically active fragment thereof. [The present invention 1011] 10. The multispecific polypeptide construct of the present invention 1010, wherein each of the Fc polypeptides of the heterodimeric Fc comprises a knob-into-hole modification or a charge mutation to increase the electrostatic complementarity of the polypeptides. [The present invention 1012] 10. The multispecific fusion polypeptide of claim 10, wherein the first Fc polypeptide of said heterodimeric Fc comprises a modification selected from Thr366Ser, Leu368Ala, Tyr407Val, and a combination thereof, and the second Fc polypeptide of said heterodimeric Fc comprises the modification T366W, and optionally, 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. [The present invention 1013] 1011. The multispecific polypeptide construct of the invention, wherein the amino acid modifications are charge mutations to increase electrostatic complementarity of the polypeptide. [The present invention 1014] 1014. The multispecific polypeptide construct of any of claims 1001 to 1013, wherein the Fc region comprises a polypeptide comprising the modification Ile253Arg or His435Arg. [The present invention 1015] 10. The multispecific polypeptide construct of any of claims 1001 to 1014, wherein the Fc region comprises a polypeptide comprising at least one modification to enhance FcRn binding. [The present invention 1016] 1015. The multispecific fusion polypeptide of the invention, wherein the modification is at a position selected from the group consisting of Met252Y, Ser254T, Thr256E, Met428L, Met428V, Asn434S, and combinations thereof. [The present invention 1017] 1009-1016. The multispecific polypeptide construct of any of claims 1009-1016, wherein the first polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NOs: 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 NOs: 83, 87, 90, 92, 98, or 100. [The present invention 1018] The Fc region Polypeptides comprising at least one amino acid modification that reduces effector function and / or reduces binding to an effector molecule selected from an Fcγ receptor or C1q The multispecific polypeptide construct of any of claims 1001 to 1017, comprising: [The present invention 1019] 1018. The multispecific polypeptide construct of the invention, wherein the one or more amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235. [The present invention 1020] The multispecific polypeptide construct of any of claims 1009 to 1016, 1018, and 1019, wherein the first polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any of SEQ ID NOs: 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 NOs: 85, 89, 91, 93, 99, or 101. [The present invention 1021] The multispecific polypeptide construct of any of claims 1001 to 1020, wherein the CD3 binding region is an anti-CD3 antibody or antigen-binding fragment. [The present invention 1022] 1021. The multispecific polypeptide construct of the invention, wherein the anti-CD3 antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL). [The present invention 1023] The multispecific polypeptide construct of any of claims 1001 to 1022, wherein the CD3 binding region is monovalent. [The present invention 1024] 1024. The multispecific polypeptide construct of any of claims 1021 to 1023, wherein the anti-CD3 antibody or antigen-binding fragment is not a single chain antibody, and optionally is not a single chain variable fragment (scFv). [The present invention 1025] The multispecific polypeptide construct of any of claims 1022 to 1024, wherein the Fc is a heterodimeric Fc, and the VH and VL constituting the anti-CD3 antibody or antigen-binding fragment are linked to opposite polypeptides of the heterodimeric Fc. [The present invention 1026] 6. The multispecific polypeptide construct of any of claims 1001 to 1025, wherein the CD3 binding region is incapable or substantially incapable of binding to or engaging with CD3 unless at least one of the antigen binding domains binds to its TAA, and optionally unless at least two of the antigen binding domains bind to its TAA. [The present invention 1027] 1027. The multispecific polypeptide construct of any of claims 1001 to 1026, wherein the linker is a polypeptide linker. [The present invention 1028] The multispecific polypeptide construct of any of claims 1001 to 1027, wherein the linker is a cleavable linker. [The present invention 1029] 1. A multispecific polypeptide construct comprising a first component comprising a heterodimeric Fc region and a second component comprising an anti-CD3 antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL constituting the anti-CD3 antibody or antigen-binding fragment are linked to opposing polypeptides of a heterodimeric Fc; the first and second components are coupled by a cleavable linker, and the heterodimeric Fc region is positioned N-terminal to the anti-CD3 antibody; and One or both of the first and second components comprises an antigen-binding domain that binds to a tumor-associated antigen (TAA); Multispecific polypeptide constructs. [The present invention 1030] The multispecific polypeptide construct of invention 1028 or invention 1029, wherein the binding of the CD3 binding region to CD3 is substantially reduced when the multispecific polypeptide construct is in an uncleaved state compared to when the multispecific polypeptide construct is in a cleaved state. [The present invention 1031] 1030. The multispecific polypeptide construct of any of claims 1028 to 1030, wherein the cleavable linker is a polypeptide that functions as a substrate for a protease. [The present invention 1032] The multispecific polypeptide construct of the present invention 1031, wherein the protease is produced by immune effector cells, by the tumor, or by cells present in the tumor microenvironment. [The present invention 1033] The multispecific polypeptide construct of invention 1031 or invention 1032, wherein the protease is selected from matriptase, matrix metalloproteinase (MMP), granzyme B, and combinations thereof. [The present invention 1034] The multispecific polypeptide construct of the present invention 1033, wherein the protease is Granzyme B. [This invention 1035] The cleavable linker is an amino acid sequence of the general formula P4 P3 P2 P1↓P1′ (SEQ ID NO:150), where P4 is an amino acid I, L, Y, M, F, V, or A; P3 is an amino acid A, G, S, V, E, D, Q, N, or Y; P2 is an amino acid H, P, A, V, G, S, or T; P1 is an amino acid D or E; and P1′ is an amino acid I, L, Y, M, F, V, T, S, G, or A, Optionally, an amino acid sequence of the general formula P4 P3 P2 P1↓P1' (SEQ ID NO:151), where P4 is amino acid I or L; P3 is amino acid E; P2 is amino acid P or A; P1 is amino acid D; and P1' is amino acid I, V, T, S, or G. The multispecific polypeptide construct of any of claims 1028 to 1034, comprising: [The present invention 1036] 1036. The multispecific polypeptide construct of any of claims 1028 to 1035, wherein the cleavable linker comprises the amino acid sequence IEPDI (SEQ ID NO: 136), LEPDG (SEQ ID NO: 152), LEADT (SEQ ID NO: 137), IEPDG (SEQ ID NO: 138), IEPDV (SEQ ID NO: 139), IEPDS (SEQ ID NO: 140), IEPDT (SEQ ID NO: 141), or LEADG (SEQ ID NO: 153). [This invention 1037] 1036. The multispecific polypeptide construct of any of claims 1028 to 1036, wherein the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 105-112, 136-141, 148, 150-153. [The present invention 1038] The multispecific polypeptide construct of the present invention 1033, wherein the protease is matriptase. [This invention 1039] The cleavable linker is The sequence P4QAR↓(A / V) (SEQ ID NO:154) (wherein P4 is any amino acid), Optionally, the sequence RQAR(A / V) (SEQ ID NO: 155) or the sequence RQARV (SEQ ID NO: 156) The multispecific polypeptide construct of any of claims 1028 to 1038, comprising: [The present invention 1040] 1039. The multispecific polypeptide construct of any of claims 1028 to 1039, wherein the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 154 to 156. [The present invention 1041] The multispecific polypeptide construct of the present invention 1033, wherein the protease is an MMP, optionally MMP-2. [The present invention 1042] The cleavable linker is The general formula P3 P2 P1↓P1' (SEQ ID NO:157) (P3 is P, V, or A; P2 is Q or D; P1 is A or N; P1' is L, I, or M), Optionally, a compound of the general formula P3 P2 P1↓P1′ (SEQ ID NO:158) (P; P2 is Q or D; P1 is A or N; P1′ is L or I), Optionally, the sequence PAGL (SEQ ID NO: 24) A multispecific polypeptide construct according to any one of claims 1028 to 1041, comprising: [This invention 1043] 1028-1042. The multispecific polypeptide construct of any of claims 1028-1042, wherein the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-31, 104-114, 117-118, 136-144, 148, 150-158. [This invention 1044] (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. At least one or both of the first and second polypeptides comprises at least one antigen-binding domain that binds to a tumor-associated antigen (TAA); A multispecific polypeptide construct according to any one of claims 1025 to 1043 of the present invention. [This invention 1045] The multispecific polypeptide construct of any of claims 1001 to 1044, wherein one or more antigen-binding domains that bind to the TAA result in monovalent, divalent, trivalent, or tetravalent binding to the TAA. [The present invention 1046] at least one antigen-binding domain is positioned amino-terminal to the Fc region and / or carboxy-terminal to the CD3-binding region of one of the first or second polypeptides of the multispecific polypeptide construct; or at least one antigen-binding domain is positioned amino-terminal to the Fc region of the multispecific construct and a second antigen-binding domain is positioned carboxy-terminal to the CD3-binding region of the multispecific construct; The multispecific polypeptide construct of the present invention 1044 or 1045. [This invention 1047] The multispecific polypeptide construct of any of claims 1001 to 1046, wherein the antigen-binding domain, or each of the antigen-binding domains independently, is an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a Fv fragment, a scFv, a scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. [This invention 1048] The antibody or antigen-binding fragment thereof may be an Fv, scFv, Fab, single domain antibody (sdAb), V NAR , or V H 1047. A multispecific polypeptide construct of the present invention, wherein said polypeptide is H. [This invention 1049] the antibody or antigen-binding fragment is a Fab and 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 VH-CH1(Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen; Including, the first and / or second polypeptide further comprises the other of the VH-CH1(Fd) or VL-CL of the Fab antibody fragment; The multispecific polypeptide construct of the present invention 1047 or 1048. [The present invention 1050] only one of the first or second polypeptides comprises the other of the VH-CH1(Fd) or VL-CL of the Fab antibody fragment; or Both the first and second polypeptides comprise the other of the VH-CH1(Fd) or VL-CL of a Fab antibody fragment; 1049. A multispecific polypeptide construct of the present invention. [This invention 1051] the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment is positioned at the amino end of the Fc region and / or the carboxy end of the CD3 binding region of one of the first or second polypeptides of the multispecific polypeptide construct; or the other of the VH-CH1(Fd) or VL-CL of the Fab antibody fragment is located at the amino end of the Fc region of the first polypeptide or the second polypeptide and at the carboxy end of the CD3 binding region of the other of the first or the second polypeptide; A multispecific polypeptide construct of the present invention 1049 or 1050. [This invention 1052] The antigen-binding domain, or each antigen-binding domain independently, may be: 1-92-LFA-3, 5T4, α4 integrin, αV integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis Y, apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11 a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52 , CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD13 8, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), claudin 3, claudin 4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB , EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE , IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL 23, IL23R, IL27 / IL27R (wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin 16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch1, Notch2, Notch3, Notch4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine-1-phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, 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. [This invention 1053] comprising at least a first antigen-binding domain and a second antigen-binding domain; the first antigen-binding domain and the second antigen-binding domain bind to the same TAA; A multispecific polypeptide construct according to any one of claims 1001 to 1052. [This invention 1054] comprising at least a first antigen-binding domain and a second antigen-binding domain; the first antigen-binding domain and the second antigen-binding domain bind to different TAAs; A multispecific polypeptide construct according to any one of claims 1001 to 1053 of the present invention. [This invention 1055] a first connecting peptide (LP1) between the first antigen-binding domain and the Fc region and a second connecting peptide (LP2) between the CD3-binding region and the second antigen-binding domain; having the following structural arrangement from N-terminus to C-terminus: first antigen-binding domain - LP1 - Fc region - linker - CD3-binding region - LP2 - second antigen-binding domain, A multispecific polypeptide construct according to any one of claims 1006 to 1054. [This invention 1056] 1056. The multispecific polypeptide construct of any of claims 1021 to 1055, wherein the anti-CD3 antibody or antigen-binding fragment is an Fv antibody fragment. [This invention 1057] 1056. A multispecific polypeptide construct of the invention, wherein the Fv antibody fragment comprises a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv). [This invention 1058] The anti-CD3 antibody or antigen-binding fragment is VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO:16); Amino acid sequence VH CD2 containing TIFF0007798942000008.tif4128; Amino acid sequence VH CDR3 containing TIFF0007798942000009.tif4128; Amino acid sequence VL CDR1 containing TIFF0007798942000010.tif4128; VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO:20); and VL CDR3 containing the amino acid sequence ALWYSNLWV (SEQ ID NO:21) The multispecific polypeptide construct of any of claims 1021 to 1057, comprising: [This invention 1059] Anti-CD3 dsFv, a VH having the amino acid sequence of any of SEQ ID NOs: 14 and 32-62, or a sequence exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 14 and 32-62; and VL having an amino acid sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NOs: 15 and 63 to 81 or any of SEQ ID NOs: 14 and 32 to 62. The multispecific polypeptide construct of the present invention 1057 or 1058, comprising: [The present invention 1060] A multispecific polypeptide construct of any of claims 1057 to 1059, wherein the anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO:14 and the amino acid sequence of SEQ ID NO:15; or the amino acid sequence of SEQ ID NO:44 and the amino acid sequence of SEQ ID NO:72. [This invention 1061] The multispecific polypeptide construct of any of claims 1001 to 1060, wherein the multispecific polypeptide construct is conjugated to an agent, and optionally the agent is a therapeutic agent, an anti-tumor agent, a toxin or fragment thereof, a detectable moiety, or a diagnostic agent. [This invention 1062] A polynucleotide encoding any one of the multispecific polypeptide constructs of the present inventions 1001 to 1061. [This invention 1063] A polynucleotide encoding any one of the polypeptide chains of the multispecific polypeptide constructs of the present inventions 1001 to 1061. [The present invention 1064] A polynucleotide comprising a first nucleic acid sequence encoding a first polypeptide of a multispecific construct of any one of claims 1001 to 1061 and a second nucleic acid sequence encoding a second polypeptide of said multispecific construct, the first and second nucleic acid sequences are separated by an internal ribosome entry site (IRES) or by a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; Polynucleotide. [This invention 1065] the multispecific polypeptide construct comprises a third polypeptide chain; the polynucleotide further comprises a third nucleic acid encoding a third polypeptide of the multispecific construct; Optionally, the third nucleic acid is separated from the first and / or second polypeptide by an internal ribosome entry site (IRES) or by a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and / or the third nucleic acid sequence is operably linked to the same promoter as the first and / or second nucleic acid sequence; The polynucleotide of the present invention 1064. [The present invention 1066] A vector comprising any one of the polynucleotides of the present inventions 1062 to 1065. [This invention 1067] A cell comprising one or more polynucleotides of any one of 1062 to 1065 or a vector of the present invention. [The present invention 1068] Introducing one or more polynucleotides of any one of 1062 to 1065 of the present invention or the vector of 1066 of the present invention into a cell; and culturing said cells under conditions in which the multispecific polypeptide construct is produced. 1. A method for producing a multispecific polypeptide construct, comprising: [The present invention 1069] A method of producing a multispecific polypeptide construct, comprising culturing a cell of the invention 1067 under conditions in which the multispecific polypeptide is produced by the cell. [The present invention 1070] A multispecific polypeptide construct produced by the method of invention 1068 or invention 1069. [This invention 1071] A pharmaceutical composition comprising any one of the multispecific polypeptide constructs of the present inventions 1001 to 1061 and 1070, and a pharmaceutically acceptable carrier. [This invention 1072] A method for stimulating or inducing an immune response, comprising the step of contacting target cells and T cells with any of the multispecific polypeptide constructs of inventions 1001 to 1061 and 1070 or the pharmaceutical composition of invention 1071, wherein the target cells express a tumor-associated antigen recognized by the multispecific polypeptide construct. [This invention 1073] The method of claim 1072, wherein the target cells are tumor cells expressing a tumor-associated antigen (TAA). [This invention 1074] 1074. The method of claim 1072 or claim 1073, wherein the multispecific polypeptide construct comprises a cleavable linker that functions as a substrate for a protease, and wherein the induction or stimulation of an immune response is increased in the presence of the protease. [This invention 1075] The method of any of claims 1072 to 1074, wherein the protease is selected from matriptase, matrix metalloproteinase (MMP), granzyme B, and combinations thereof. [This invention 1076] The method of any of claims 1072 to 1075, wherein the contacting is performed ex vivo, in vitro, or in vivo in a subject. [This invention 1077] A method for stimulating or inducing an immune response in a subject, comprising the step of administering a therapeutically effective amount of any of the multispecific conjugates of the present inventions 1001 to 1061 and 1070 or the pharmaceutical composition of the present invention 1080 to a subject in need thereof. [This invention 1078] The method of claim 1077, wherein the immune response to the tumor or cancer is increased. [This invention 1079] The method of any of claims 1072 to 1078, for treating a disease or condition in a subject. [The present invention 1080] A method for treating a disease or condition in a subject, comprising administering a therapeutically effective amount of any of the multispecific conjugates of the present inventions 1001 to 1061 and 1070 or the pharmaceutical composition of the present invention 1071 to a subject in need thereof. [This invention 1081] The method of claim 1079 or claim 1080, wherein the disease or condition is a tumor or cancer. [This invention 1082] The method of any one of claims 1076 to 1081, wherein the subject is a human. [Brief explanation of the drawings]

[0113] [Figure 1] Figure 1 is a schematic diagram of the basic components of a multispecific polypeptide construct of the present disclosure with constrained CD3 binding. The antigen-binding domain is located at the amino-terminus and / or carboxy-terminus. An Fc region, such as a heterodimeric Fc region, is located N-terminal to the CD3-binding region. This positioning of the Fc in close proximity to the CD3-binding region prevents CD3 binding. [Figure 2] FIG. 2 shows an exemplary structure of a multispecific molecule of the disclosure containing a cleavable linker and having dual effector functions, where proteolytic cleavage of the cleavable linker results in activation of the multispecific polypeptide construct, producing two components each with biological activity. [Figure 3]Figure 3 is a schematic diagram of various constrained CD3 constructs targeting FRα, composed of two polypeptides, chain 1 and chain 2. The top panel provides an exemplary illustration of a cleavable multispecific polypeptide construct with a cleavable linker containing protease substrate recognition sites for, for example, one or more of MTSP1, MMP, and / or granzyme B. Chain 1 contains the FRα sdAb (antigen-binding domain) linked to a heterodimeric Fc "hole" linked via a protease-cleavable linker (cx1547: granzyme B only; cx309: MTSP1, MMP, and granzyme B) to an anti-CD3 VL domain linked to a second FRα sdAb. Chain 2 contains the FRα sdAb linked to a complementary heterodimeric Fc "knob" linked via the same protease linker to an anti-CD3 VH domain linked to a second FRα sdAb. The bottom panel of Figure 3 shows a similar arrangement to the top panel, except that the linker is a non-cleavable linker (from 3 amino acids at cx1356 to 18 amino acids at cx681). When co-expressed, the CD3-binding domains are properly assembled via VL:VH association in the hole and knob, respectively. [Figure 4]Figures 4A-4C illustrate constructs generated to compare the effect of linkers on CD3 binding constraints in the resulting constructs. Figure 4A illustrates an example of a multispecific polypeptide construct containing identical cleavable linkers in each polypeptide chain to couple each Fc polypeptide of the heterodimeric Fc to the CD3-binding domain (exemplary construct cx1762 is shown). In the format shown in Figure 4A, the construct is shown in its uncleaved state. Another version of the construct is shown in Figure 4B, in which only one cleavable linker is utilized to connect the Fc region to the CD3-binding domain, termed a half-truncated construct (exemplary construct cx3238 is shown). Figure 4C illustrates a structure representing the C-terminal portion of the construct format of Figures 4A and 4B when fully cleaved (exemplary construct cx2190 is shown). Constructs representing protein cleavage products can be recombinantly generated by coexpressing the various chains shown. The sdAb targeting FRα is positioned in the C-terminal position in each construct. [Figure 5A] Figures 5A-5E show representative EGFR-targeted and EGFR / cMET dual-targeted restricted CD3 engagers. In Figure 5A, cx2513 has an EGFR-targeted sdAb positioned at the C-terminus of each chain of the heterodimer, thereby exhibiting bivalent binding to EGFR. [Figure 5B] In Figure 5B, cx3030 has EGFR-targeting sdAbs positioned at both the N- and C-termini of each chain of the heterodimer, thereby exhibiting tetravalent binding to EGFR. [Figure 5C] Figure 5C, cx2973 has an sdAb targeting cMET positioned at the N-terminus of each chain of the heterodimer and an sdAb targeting EGFR positioned at the C-terminus, thereby exhibiting bivalent binding to each of cMET and EGFR. [Figure 5D]In Figure 5D, cx2979 has an sdAb targeting cMET positioned at the N-terminus of one chain of the heterodimer and an sdAb targeting EGFR positioned at the C-terminus of each chain of the heterodimer, thereby exhibiting monovalent binding to cMET and bivalent binding to EGFR. [Figure 5E] In Figure 5E, cx2977 has an sdAb targeting cMET positioned at the N-terminus of one chain of the heterodimer and an sdAb targeting EGFR positioned at the C-terminus of the other chain, thereby exhibiting monovalent binding to each of cMET and EGFR. [Figure 6A] Figures 6A and 6B are schematic diagrams of the component chains used to assemble exemplary B7H3-targeted restricted CD3 engagers. The B7H3-binding domains utilized in these representative constructs included sdAb, scFv, or FAB. Generally, constructs containing sdAb and scFv were configured as two heterodimeric chains, while constructs containing FAB included a third chain for the cognate light chain (VL-CL). [Figure 6B] See legend to Figure 6A. [Figure 7] Figure 7 illustrates a constrained CD3 engager targeting 5T4. The core of the molecule was generated to contain a heterodimeric Fc followed by a cleavable linker and a disulfide-stabilized anti-CD3 Fv. The TAA-binding portion of these molecules was placed at the N- or C-terminus of either heterodimeric Fc chain. In the top row, the TAA-binding unit is a Fab composed of an Fd (VH-CH1) at the N-terminus of the knob polypeptide and the C-terminus of the hole polypeptide. In cases where the Fab is the binding unit, a third chain (light chain VL-CL) was expressed to associate with the Fd. In the middle and bottom rows, the TAA-binding units are single-domain antibodies, both located at the N- and C-termini of the knob polypeptide. In the middle row, the TAA-binding sdAbs of the generated constructs are identical; in the bottom row, the TAA-binding sdAbs of the generated constructs are different sequences with different epitopes. [Figure 8] FIG. 8 is a schematic diagram of a representative CD20-targeted, constrained CD3-engaging construct, cx3309, in which the CD20-binding domain is an scFv derived from the CD20 antibody GA101. [Figure 9] Figure 9 is a schematic diagram of a representative DLL3-targeted, constrained CD3-engaging construct, cx3308, in which the DLL3-binding domain is an scFv. This exemplary construct is composed of two chains, each containing a complementary component of a heterodimeric Fc linked to a CD3-binding domain and one component of a DLL3-binding scFv. In assembled form, the construct is bivalent for DLL3, with the CD3-binding domain positioned C-terminal to the Fc heterodimer. [Figure 10A] Figure 10A is an image of SDS-PAGE of a representative FRα-targeted, constrained CD3-engaging construct, cx1547, under reducing (R) and non-reducing (NR) conditions. Expected molecular weight: 135 kDa. [Figure 10B] Figures 10B and 10C are graphs of chromatograms from size exclusion analysis of cx1547, demonstrating that it is a single species with a determined molecular weight of 137.9 kDa. [Figure 10C] Figures 10B and 10C are graphs of chromatograms from size exclusion analysis of cx1547, demonstrating that it is a single species with a determined molecular weight of 137.9 kDa. Figure 10C is a zoomed-in view of the vicinity of the major peak shown in Figure 10B. [Figure 11A] 11A and 11B are a pair of graphs demonstrating the binding ability of an exemplary multispecific polypeptide construct of the present disclosure, referred to herein as cx309, in its uncleaved or proteolytically cleaved state to bind to human T cells. Matriptase and MMP-2 were used to cleave cx309 in FIG. 11A and FIG. 11B, respectively. [Figure 11B] See legend to Figure 11A. [Figure 12A]Figures 12A-12D illustrate cell binding by representative FRα-targeting constrained CD3-engaging constructs cx1356, cx681, and cx1547. Figures 12A and 12C show binding to Ovcar5 cells (an FRα-positive ovarian cancer cell line). Figures 12B and 12D show lack of binding to T cells. Figures 12A and 12B show histograms of normalized cell number versus fluorescence at 100 nM for each construct. Full titration of each construct in various cell types is shown in Figures 12C and 12D. In Figures 12A and 12B, the secondary anti-human APC antibody-only control is shown as a black trace, binding of the positive control anti-CD3 is shown as a white trace, and cx1356, cx681, and cx1547 are shown as gray-shaded traces. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 12D] See legend to Figure 12A. [Figure 13] Figures 13A-13B illustrate cell binding by a representative EFGR-targeting constrained CD3-engaging construct, cx3030. Figure 13A shows binding to the EGFR-positive cell line Colo-205 at 100 nM. Figure 13B demonstrates the lack of binding to T cells at 100 nM. Binding is shown as a histogram of normalized cell number versus fluorescence. The secondary anti-human APC antibody-only control is shown in black, the positive control anti-CD3 binding is shown in white, and cx3030 is shown in gray shaded traces. [Figure 14A]Figures 14A-14D illustrate the binding of the B7H3-targeted restricted CD3 engager to B7H3-positive A375 (Figures 14A and 14B) and the lack of binding to CD3 on T cells (Figures 14C and 14D). The alternative format, DART-Fc, targeting both B7H3 and CD3, demonstrated strong binding to both B7H3 and CD3 on T cells. Various B7H3 antigen-binding domains were used herein, including the cx3095 sdAb, cx3313 FAB, and cx3314 scFv. The scFv and FAB contain the same anti-B7H3 VH and VL sequences as those used in the DART-Fc format. Figures 14A and 14C show comparative histograms at 100 nM concentrations for each construct. The secondary anti-human APC antibody-only control is shown in black traces, and the various B7H3-targeted CD3-engaging constructs are shown in white non-common traces. Figures 14B and 14D show titrations of binding to BH73 and CD3 by the various constructs, respectively. [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 14D] See legend to Figure 14A. [Figure 15] Figures 15A-15B illustrate cell binding by representative 5T4-targeting constrained CD3-engaging constructs cx3262 and cx3315. Figure 15A shows binding to the 5T4-positive cell line Ovcar-5 at 400 nM. Figure 15B demonstrates the lack of binding to T cells at 400 nM. Binding is shown as a histogram of normalized cell number versus fluorescence. The secondary anti-human APC antibody-only control is shown in black, the positive control anti-CD3 binding is shown in white, and cx3262 and cx3315 are shown in gray-shaded traces. [Figure 16A]Figures 16A-16D illustrate cell binding by a representative CD20-targeting constrained CD3-engaging construct, cx3309. Figures 16A and 16C show binding to Ramos cells (a CD20-positive cell line). Figures 16B and 16D demonstrate the lack of binding to T cells. Figures 16A-16B show histograms of normalized cell number versus fluorescence at 100 nM for each construct. A complete titration of each construct on various cell types is shown in Figures 16C-16D. The secondary anti-human APC antibody-only control is shown in black traces, binding of the positive control anti-CD3 is shown in white traces, and cx3309 is shown in gray shaded traces. [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. [Figure 16D] See legend to Figure 16A. [Figure 17] FIG. 17 is a graph showing the ability of cleaved or uncleaved cx309 to activate the CD3 NFAT reporter Jurkat cell line (Promega, USA) in the presence or absence of FRα-expressing cells Ovcar5. [Figure 18] Figure 18 illustrates antigen-dependent T cell activation by cx1547. Various cell lines, either FRα-positive (T47D, IGROV1, NCI-H2342, Ovcar-5, Skov-3, and A2780) or -negative (NCI-H460), were co-incubated with the Jurkat CD3 NFAT-GFP reporter cell line, and fluorescence was measured at 6 hours. This demonstrates the ability of the restricted CD3 construct to activate T cells in an antigen-dependent manner. [Figure 19-1]Figures 19A-19D illustrate the enhanced T cell activation capacity of restricted CD3 engagers when proteolysis occurs in the linker between the Fc domain and the CD3-binding domain. Shown here are the kinetics of T cell activation mediated by 20 nM cx1762, cx3238, or cx2190 in the presence of FRα-positive Ovcar-5 cells (Figure 19A) or FRα-negative CCRF-CEM cells (Figure 19B). Also shown here are the potencies of T cell activation mediated by cx1762, cx3238, or cx2190 in the presence of FRα-positive Ovcar-5 cells (Figure 19C) or FRα-negative CCRF-CEM cells (Figure 19D). A Jurkat CD3 NFAT-GFP reporter was used to monitor CD3 signaling over a 24-hour period using an Incucyte ZOOM imager. Notably, T cell activation depends on antigen expression in the target cell line and is greatly enhanced by removal of the Fc domain N-terminal to one or both CD3-binding VH:VL domains. [Figure 19-2] See description of Figure 19-1. [Figure 20-1] Figures 20A-20D are a series of graphs demonstrating the antigen-dependent T cell activation capacity of various EGFR-targeted and EGFR / cMET-targeted restricted CD3 engagers. Notably, T cell activation capacity is enhanced by increasing valency or additional target antigen binding specificity. The kinetics of T cell activation mediated by various constructs in antigen-positive A431 cells is illustrated in Figure 20A, and in antigen-negative CCRF-CEM cells is shown in Figure 20C. The efficacy of T cell activation by various constructs in antigen-positive A431 cells is shown in Figure 20B, and in negative CCRF-CEM cells is shown in Figure 20D. Here, the Jurkat CD3 NFAT-GFP reporter cell line was used. [Figure 20-2] See description of Figure 20-1. [Figure 21]Figures 21A-21B illustrate the ability to mediate target antigen-specific T cell activation by a representative B7H3-targeted, restricted CD3-engaging construct, cx3095, and alternative DART-Fc formats targeting B7H3 and CD3. Jurkat CD3 NFAT-GFP reporter cells were used to assess T cell activation in the presence of the B7H3-positive cell line, A375 (Figure 21A), and the B7H3-negative cell line, Raji (Figure 21B). [Figure 22-1] Figures 22A-22F illustrate the ability to mediate target antigen-specific T cell activation by representative B7H3-targeted restricted CD3-engaging constructs and alternative DART-Fc formats targeting B7H3 and CD3. Notably, the restricted CD3-engaging constructs utilize either a B7H3-targeted sdAb, scFv, or FAB. Jurkat CD3 NFAT-GFP reporter cells were used to assess T cell activation in the presence of the B7H3-positive cell line A375 (Figures 22A, 22C, 22E) and the B7H3-negative cell line CCRF (Figures 22B, 22D, 22F). The kinetics of T cell activation mediated by 50 nM (Figures 22A and 22B) or 2 nM (Figures 22C or 22D) of each construct are shown. The potency of T cell activation mediated by each construct in antigen-positive (Figure 22E) and -negative (Figure 22F) cell lines is also shown. [Figure 22-2] See description of Figure 22-1. [Figure 23] Figures 23A-23B are a series of graphs showing the T cell activation ability of 5T4-targeted restricted CD3-engaging constructs. This example shows that TAA targeting of a bivalent, biepitope protein can increase the activity of a restricted CD3 engager compared to a bivalent, monoepitope protein in TAA-positive cells (OVCAR5). Neither construct induced T cell activation in the presence of TAA-negative cells (CCRF). [Figure 24]Figure 24 is a graph showing the ability of a representative 5T4-targeted restricted CD3-engaging construct, cx3315, to induce antigen-dependent T cell activation. The Jurkat CD3 NFAT-GFP reporter cell line was used to monitor T cell activation by cx3315 in the presence of a 5T4-positive cell line (OVCAR5) and a 5T4-negative cell line (CCRF-CEM). [Figure 25] Figure 25 illustrates the ability of a representative CD20-targeted, restricted CD3-engaging construct, cx3309, to induce antigen-dependent T cell activation. The Jurkat CD3 NFAT-GFP reporter cell line was used to monitor T cell activation by cx3309 in the presence of the CD20-positive cell line, Ramos, and the CD20-negative cell line, CCRF-CEM. [Figure 26] Figure 26 is a graph showing the ability to induce T cell activation by a representative DLL3-targeted restricted CD3-engaging construct, cx3308. The Jurkat CD3 NFAT-GFP reporter cell line was used to monitor T cell activation by cx3309 in the presence of DLL3-positive SHP-77 cells. This demonstrates that scFv moieties target TAA in a restricted CD3 format and can be used to effectively activate T cells when bound to a cognate TAA-positive cell line. [Figure 27A]Figures 27A-27F illustrate the effect of linker length on the ability to activate T cells in the presence of FRα-positive cells IGROV1 (Figures 27A, 27C, 27E) or FRα-negative cells NCI-H460 (Figures 27B, 27D, 27F). Figures 27A-27B show the kinetics of T cell activation by 2 nM of various constructs in antigen-positive and antigen-negative cells, respectively. Figures 27C-27D show the magnitude of T cell activation ability of various constructs in antigen-positive and antigen-negative cells, respectively. Figures 27E-27F show the potency of T cell activation ability of various constructs with different linker lengths in antigen-positive and antigen-negative cells, respectively. The Jurkat CD3 NFAT-GFP reporter cell line was used to evaluate T cell activation. The restricting CD3 protein effectively engages and clusters CD3 on T cells only upon binding to a second antigen on the target cells. [Figure 27B] See legend to Figure 27A. [Figure 27C] See legend to Figure 27A. [Figure 27D] See legend to Figure 27A. [Figure 27E] See legend to Figure 27A. [Figure 27F] See legend to Figure 27A. [Figure 28A] Figures 28A-28C illustrate FRα-dependent T cell-mediated cytotoxicity by cx1547. Figure 28A demonstrates that cx1547 does not induce T cell-mediated cytotoxicity in an antigen-negative cell line (NCI-H460). Figure 28B demonstrates that cx1547 induces T cell-mediated cytotoxicity in an antigen-positive cell line (OVCAR5). Figure 28C shows the kinetics of T cell-mediated cytotoxicity against OVCAR5 cells induced by cx1547 at 3 nM. cx1547 induced T cell-mediated cytotoxicity only in the antigen-positive cell line. Cytotoxicity of differentially labeled target cells was monitored using a caspase 3 / 7 fluorogenic substrate on an Incucyte ZOOM imager. Effector cell to target cell ratios (E:T) were assessed at 20:1 and 10:1 in this assay. [Figure 28B] See legend to Figure 28A. [Figure 28C] See legend to Figure 28A. [Figure 29-1] Figures 29A-29F illustrate the kinetics of T cell-mediated cytotoxicity driven by representative B7H3-targeted, restricted CD3-engaging constructs and alternative DART-Fc formats targeting B7H3 and CD3. Titration ranges of 50 nM to 80 pM of CD3-engaging constructs in the B7H3-positive A375 cell line are shown in Figures 29A-29E. Figure 29F shows measurements of each construct at 50 nM in A549 cells in which B7H3 expression has been knocked down. Notably, all constructs demonstrate B7H3-dependent T cell-mediated cytotoxicity. [Figure 29-2] See description of Figure 29-1. [Figure 30] Figure 30 illustrates the magnitude of T cell-mediated cytotoxicity induced by 2.5 nM of B7H3-targeted restricted CD3-engaging constructs and DART-Fc B7H3xCD3 format in antigen-positive (A375) and -negative (A549-B7H3 knockdown) cell lines. [Figure 31-1]Figures 31A-31F illustrate the relative potency of two formats of FRa-targeted CD3 engagers in inducing T cell-mediated cytotoxicity against FRa-positive Ovcar-5 cells (Figures 31A-31E) and FRa-negative NCI-H60 cells (Figure 31F). cx2190 is a representative C-terminal product derived from proteolytic processing of cx1762 by granzyme B. Notably, cx2190 exhibited superior potency compared to cx1792, demonstrating substantial enhancement of CD3 binding mediated by proteolysis in the linker region between the Fc and CD3-binding domains. The kinetics of T cell-mediated cytotoxicity in FRa-positive cells at 20 nM, 32 pM, and 6 pM are shown in Figures 31A, 31B, and 31C, respectively. Figures 31D and 31E show the efficacy of the two formats of the FRα CD3 engager at 24 and 40 hours, respectively. Graph F demonstrates that in the absence of FRα expression on target cells, no substantial cytotoxicity is mediated by either construct. [Figure 31-2] See description of Figure 31-1. [Figure 32] Figure 32 illustrates T cell-mediated cytotoxicity mediated by a representative 5T4-targeted, restricted CD3-engaging construct, cx3315. cx3315 induced specific T cell cytotoxicity against the 5T4-expressing cell line, Ovcar-5, but not against the 5T4-negative cell line, CCRF-CEM. 20 nM cx3315 was used in this assay. [Figure 33] Figure 33 is a graph demonstrating T cell activation after 20 hours of co-culture of T cells and Ovcar5 cells in the presence of cleaved or uncleaved cx309. Only cleaved cx309 was able to mediate FRα-dependent T cell activation through CD3 binding. T cell activation was monitored by flow cytometry analysis of the CD25% of CD4 and CD8 populations. [Figure 34-1]Figures 34A-34H illustrate the ability of representative B7H3-targeted, restricted CD3-engaging constructs and alternative DART-Fc formats targeting B7H3 and CD3 to activate CD4 T cells (Figures 34A and 34E) and CD8 T cells (Figures 34C and 34G) in a target-dependent manner. T cells were incubated with the B7H3-positive cell line A375 (Figures 34A, 34C, 34E, 34G) or the B7H3-knockdown A549 cell line (Figures 34B, 34D, 34F, 34H), and activation markers CD25 and CD71 were assessed by flow cytometry. These data demonstrate the B7H3-dependent T cell activation capacity of the constructs used. [Figure 34-2] See description of Figure 34-1. [Figure 35] Figure 35 illustrates the ability of the B7H3-targeted, restricted CD3-engaged construct cx3095 to mediate antigen-dependent INFγ production. Cytokine production was quantified using an INFγ ELISA. A375 was used as a B7H3-positive cell line, and the B7H3 knockdown A549 cell line was used as a negative cell line. [Figure 36] Figures 36A-36B illustrate the ability of a representative FRα-targeted, restricted CD3-engaging construct, cx1547, to induce IFNγ (Figure 36A) and IL-2 (Figure 36B) from human PBMCs in an FRα-dependent manner. Cytokine production was measured using FluoroSpot cytokine capture assays. IGROV-1 and NCI-H460 were used as FRα-positive and -negative cell lines, respectively. [Figure 37] Figure 37 illustrates the ability of the B7H3-targeted, restricted CD3-engaging construct cx3095 to mediate antigen-dependent INFγ production. Cytokine production was monitored using a FluoroSpot assay. A375 and CCRF-CEM cell lines were used as B7H3-positive and -negative cell lines, respectively. [Figure 38A]Figures 38A-38D illustrate the ability of the FRα-targeted restricted CD3 construct cx1547 to activate and induce cytotoxicity in T cells present in dissociated primary human ovarian tumor samples. Figure 38A illustrates flow plots of the relative abundance of tumor cells (EpCAM+) and infiltrating lymphocytes (CD45+) in dissociated ovarian tumor samples. [Figure 38B] FIG. 38B shows the viability (CellTiterGlo) of adherent tumor cells after treatment with conventional FRα antibody or cx1547 after 6 days of incubation. [Figure 38C] Figure 38C shows INFγ production after treatment with FRα antibody or cx1547 after 6 days of incubation. [Figure 38D] Figure 38D shows representative images of remaining adherent tumor cells after 6 days of treatment with no antibody (left), conventional FRα antibody (center), or cx1547 (right). DETAILED DESCRIPTION OF THE INVENTION

[0114] Detailed Description The present disclosure provides constrained T cell-engaging fusion proteins in the form of multispecific polypeptide constructs that bind at least to CD3 and a second antigen. The multispecific polypeptide constructs provided herein include at least a first component comprising one or more copies of an antigen-binding domain that binds to the antigen operably linked to an immunoglobulin Fc region, a second component comprising one or more copies of a binding domain that binds at least CD3 (referred to herein as an anti-CD3 binding domain or CD3 binding region, terms used interchangeably herein), and a linker, such as a polypeptide linker, joining the first component and the second component. In some embodiments, the antigen is a tumor-associated antigen (TAA). In some embodiments, the linker is a cleavable linker.

[0115] The provided multispecific polypeptide constructs include an arrangement in which a first component containing an Fc region is N-terminal to a second component containing a CD3-binding region. In such embodiments, the first and second components are joined via a linker C-terminal to the end of the Fc region. In some embodiments, the antigen-binding domain is located in the amino-terminal (N-terminal) region of the multispecific polypeptide construct. In some embodiments, the antigen-binding domain is located in the carboxy-terminal (C-terminal) region of the multispecific polypeptide construct. In some embodiments, the antigen-binding domain is located in both the N-terminal and C-terminal regions of the multispecific polypeptide construct. Various arrangements of the multispecific polypeptide constructs provided herein are shown in Figure 1.

[0116] Because the provided multispecific polypeptide constructs substantially bind to CD3 only after antigen is bound via the antigen-binding domain, such constructs exhibit restricted T cell-engaging activity. This is illustrated in the Examples and Figures provided herein, which demonstrate that restricted CD3-engaging proteins can efficiently bind to TAA-positive cells but exhibit little to no binding to T cells. This unique property allows the restricted CD3-engaging proteins to distribute to sites where TAAs are present without binding to peripheral T cells. This format differs from other CD3-engaging multispecific constructs in that constitutive CD3 binding is not permitted or eliminated, providing a significant advantage by avoiding peripheral T cell binding and allowing preferential distribution to sites where the antigen recognized by the antigen-binding domain is present. For example, as shown in the Examples, the restricted CD3-engaging format allows potency similar to the DART-Fc format (e.g., published PCT application WO2017 / 030926), but with significantly attenuated peripheral T cell binding. Furthermore, while other CD3-engaging constructs mediate antigen-dependent T cell activation, the multispecific polypeptide constructs provided herein mediate both antigen-dependent T cell binding and activation.

[0117] The constrained T cell-engaging activity of the provided multispecific polypeptide constructs is, in some aspects, due to the positioning of an Fc region N-terminal to the CD3-binding region. In some embodiments, such positioning reduces, attenuates, interferes with, and / or prevents CD3 binding by the CD3-binding region. In the absence of antigen binding by the antigen-binding domain, the multispecific polypeptide constructs provided herein exhibit reduced or eliminated CD3 binding and T cell activation capabilities. In some embodiments, in the presence of an antigen binding event mediated by the antigen-binding domain of the multispecific polypeptide construct, the ability of the CD3-binding region to bind to CD3 is greatly enhanced. In some embodiments, in the presence of an antigen binding event mediated by the antigen-binding domain of the multispecific polypeptide construct, the ability to activate T cells is greatly enhanced. Engagement of its cognate antigen by the antigen-binding domain within the multispecific polypeptide construct results in subsequent T cell engagement and mediates antigen-dependent T cell activation, such as cytotoxicity, cytokine release, degranulation, and proliferation. In some embodiments, the provided multispecific polypeptide constructs can be used to increase an immune response, for example, to enhance T cell activity, including cytolytic (or cytotoxic) T cell activity. Modulation of the immune response can, in some aspects, treat a disease or condition in a subject.

[0118] In some embodiments, one or more antigen-binding domains bind to antigens of tumor cells or cells in the tumor microenvironment. In some aspects, the provided multispecific polypeptide constructs can be used to increase immune responses, such as T cell activity, e.g., cytotoxic activity, against tumors or cancer. In some embodiments, the provided multispecific polypeptide constructs can be used to treat tumors or cancer in a subject.

[0119] Because the CD3-binding region of the multispecific polypeptide constructs of the present disclosure is constrained or otherwise blocked and / or inhibited by the presence of the Fc region, these constructs ensure that T cell binding via CD3 in the peripheral blood does not occur. Thus, the multispecific polypeptide constructs of the present disclosure offer numerous advantages. In some aspects, these constructs limit the sink effect caused by binding of all T cells. In some aspects, these constructs reduce systemic toxicity.

[0120] In some embodiments, the multispecific polypeptide constructs provided herein allow for controlled biodistribution to desired sites in a subject, such as, for example, sites of tumor-associated antigen (TAA) expression, including, for example, the tumor and surrounding tumor microenvironment.

[0121] In some embodiments, the multispecific polypeptide constructs of the present disclosure exhibit specificity for CD3 and one or more other antigens. In some embodiments, the multispecific polypeptide constructs may contain multiple antigen-binding domains capable of binding to one or more TAAs, such as two, three, or four antigen-binding domains. See, for example, FIG. 1. In some embodiments, one or more antigen-binding domains bind to the same antigen. In some embodiments, the multispecific polypeptide constructs comprise multiple antigen-binding domains that bind to different epitopes of the same antigen. In some embodiments, the multispecific polypeptide constructs comprise multiple antigen-binding domains that bind to one or more different antigens. In some embodiments, the multispecific polypeptide constructs comprise multiple antigen-binding domains that bind to different epitopes of the same antigen, and also comprise additional antigen-binding domains that bind to one or more different antigens. In some aspects, the provided multispecific polypeptide constructs are bispecific polypeptide constructs that can bind to CD3 and another antigen, such as a TAA, through binding of the antigen-binding domains of the multispecific polypeptide construct. In some examples, the provided multispecific polypeptide constructs are bispecific polypeptide constructs that provide tetravalent engagement of one or more TAAs through the use of a first antigen-binding domain and a second antigen-binding domain. For example, in some embodiments, the bispecific polypeptide construct comprises a first antigen-binding single domain antibody (sdAb) and a second antigen-binding sdAb, as shown in Figures 1 and 2.

[0122] In some embodiments, the multispecific polypeptide constructs provided herein exist in two states with respect to their ability to bind CD3 and subsequently activate T cells: (1) an "inactive" or uncleaved state occurs in the absence of binding of any or all of the antigen binding domains, such that CD3 binding is restricted and T cell interaction is ablated, and (2) an "active" state occurs upon antigen binding by any or all of the antigen binding domains, such that the CD3 binding region can bind CD3 and T cell interaction is possible.

[0123] In some embodiments, the Fc region is linked to the CD3 binding domain via a linker. In some embodiments, the Fc region is linked to the CD3 binding region via a non-cleavable linker. In some embodiments, the Fc region is linked to the CD3 binding region via a cleavable or otherwise labile linker.

[0124] In some embodiments, the Fc region and the CD3 binding region are linked by a cleavable linker. In some aspects, enhanced CD3 binding occurs after cleavage of the cleavable linker. In some such aspects, the "active" state can be further amplified through several mechanisms, for example, through cleavage of the linker joining the CD3 binding region and the Fc region. In some embodiments, the cleavable linker is a linker containing a substrate recognition site for a protease. In some embodiments, where the Fc region and the CD3 binding region are linked by a cleavable linker, enhanced CD3 binding can occur after cleavage within the linker.

[0125] In some aspects, the multispecific polypeptide constructs of the present disclosure enable therapeutic efficacy in the absence of proteolysis.

[0126] In some embodiments, the Fc region is a homodimeric Fc region. In some embodiments, the Fc region is a heterodimeric Fc region. In some embodiments, the Fc region is a monomeric Fc region. In some embodiments, the Fc region of the multispecific polypeptide construct can interact with FcγR and mediate innate immune effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the Fc region of the multispecific polypeptide construct can interact with complement proteins, i.e., C1q, and mediate complement-dependent cytotoxicity. Thus, in some aspects, the multispecific polypeptide constructs of the present disclosure enable multiple immune effector mechanisms, including innate immune effectors and T cells.

[0127] In some embodiments, in which the Fc region and the CD3-binding region are operably linked by a cleavable linker, cleavage of the linker between the Fc region and the CD3-binding region can separate the multispecific polypeptide construct into a first and a second component. Depending on the composition of the multispecific polypeptide construct, the first and second components may have different functionalities. In some embodiments, the Fc region is a region that exhibits one or more effector functions, such as ADCC, CDC, or ADCP. In such examples, the multispecific polypeptide constructs of the present disclosure can be used to create a self-amplifying system. For example, the multispecific construct can be used as follows: ADCC mediated by NK cells after TAA targeting and CD16 binding of the Fc region results in the release of granzyme B, which is capable of extracellular proteolysis and cleavage of the linker between the first and second components of the multispecific polypeptide construct.

[0128] In some embodiments, the linker is a cleavable linker. The multispecific polypeptide construct provides a two-in-one therapeutic moiety with dual effector functions, where proteolytic activation of the multispecific polypeptide construct produces two components, each with biological activity. The multispecific polypeptide constructs of the present disclosure can provide Fc-mediated effector functions, such as ADCC (e.g., release of granzyme B by NK cells), ADCP, and / or CDC.

[0129] Constrained CD3-engaging constructs can be used with any TAA-binding domain and are intended to avoid interactions with peripheral T cells and mediate potent TAA-dependent T cell cytotoxicity, thereby enabling better therapeutic exposure in the tumor or tumor microenvironment. Incorporation of a protease-cleavable linker between the Fc and CD3-binding domain components allows for full exposure of the CD3-binding domain, thereby amplifying T cell activation capacity. Depending on the specific linker included, the amplification step may be mediated by tumor-associated proteases or by granzymes released after antigen-dependent T cell activation. When a tumor protease-cleavable linker is included, amplification is mediated by the tumor or tumor microenvironment. On the other hand, when a granzyme B-cleavable linker is included, amplification can be self-mediated by T cells after antigen-dependent activation. Furthermore, in cases where an effector-enabled Fc is included in the construct, amplification can be mediated by granzymes released from NK cells, which occurs through the ADCC mechanism.

[0130] In some embodiments, the protease is a protease produced in the tumor microenvironment and / or a protease produced during T cell activation induced by initial binding of the CD3-binding region to CD3 in the tumor microenvironment via binding of the antigen-binding domain to a TAA. In some embodiments, the protease is granzyme B. In some aspects, the multispecific polypeptide constructs of the present disclosure exploit the ability of proteases and / or granzyme B in the tumor microenvironment to cleave a linker within the multispecific polypeptide construct below the Fc immunoglobulin polypeptide, thereby generating two therapeutically active proteins, in some cases with different effector cell engagement. In some aspects, upon cleavage of the cleavable linker, the cleaved first portion or component retains Fc effector function and bivalent targeting of a first antigen, such as a TAA, via the first antigen-binding domain, and the second portion or component retains the ability for T cell engagement because separation of the CD3-binding region from the Fc region allows CD3 binding. The cleaved second portion or component retains the ability to bind to the TAA, which in some cases may be bivalent binding, via the second antigen binding domain.

[0131] In some embodiments, the second portion or component contains a CD3-binding region that is monovalent for CD3, such that T cell activation does not occur unless a TAA is present. In some aspects in which the multivalent polypeptide construct contains a cleavable linker, the cleaved second portion or component enables TAA-dependent T cell-mediated cytotoxicity. In some cases, the cleaved second portion or component ensures that FcRn interaction does not occur. Furthermore, the cleaved second portion or component is sufficiently small in size, e.g., only approximately 50 kDa, to ensure rapid clearance if, for some reason, the cleaved second portion or component is distributed outside the tumor site and / or is abnormally cleaved outside the tumor site.

[0132] In some embodiments, the multispecific polypeptide constructs of the present disclosure allow for simultaneous T cell- and NK cell-mediated cytotoxicity. In some cases, such activity can occur in a multispecific polypeptide construct that contains a first antigen-binding domain, e.g., a first anti-TAA antigen-binding domain, and a second antigen-binding domain, e.g., a second anti-TAA antigen-binding domain, that can target different and / or non-competing epitopes of a given TAA.

[0133] In some aspects, the multispecific polypeptide constructs of the present disclosure offer numerous advantages over current bispecific therapeutics. The multispecific polypeptide constructs of the present disclosure are smaller than conventional therapeutic antibodies, e.g., 125 kDa versus 150 kDa, which may allow for better target, e.g., tumor, penetration. First, the overall size of the multispecific polypeptide construct provides a long half-life for the uncleaved construct, and upon cleavage of the construct, the cleaved second portion or component will be small enough to ensure a short half-life. In some aspects, because CD3 binding by the CD3 binding region relies on TAA engagement before CD3 engagement occurs, the multispecific polypeptide constructs of the present disclosure exhibit reduced systemic toxicity or toxicity in areas outside the tumor and / or tumor microenvironment. In some cases, the inclusion of a cleavable linker specific for a protease in the tumor environment reduces CD3 binding by the multispecific construct until proteolytic activation and TAA engagement occur, thereby amplifying or enhancing CD3 engagement.

[0134] The multispecific polypeptide constructs of the present disclosure are designed to ensure that the protease that cleaves the cleavable linker does not need to have a tumor bias (e.g., it does not need to be differentially expressed only at the tumor site and / or in the tumor environment). Rather, these multispecific polypeptide constructs only require that the protease be co-located with the TAA. The valency of these constructs will drive biodistribution and retention in the tumor and / or tumor microenvironment.

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

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

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

[0138] As used herein, the term "antibody" refers to immunoglobulin molecules and antigen-binding portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts" or "immunospecifically binds" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with, or with a much lower affinity (K d >10 -6 ) means 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 a Fab expression library.

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

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

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

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

[0143] As used herein, the terms "specific binding," "immunological binding," and "immunological binding characteristics" refer to noncovalent interactions of the type that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction is determined by the dissociation constant (K d ) and smaller K d represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of formation and dissociation of the antigen-binding site / antigen complex, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, the "association rate constant" (K on ) and "dissociation rate constant" (K off ) can be determined by calculation of the concentration and the actual rates of association and dissociation (see Nature 361:186-87 (1993)). off / Kon The ratio of α to β allows for the cancellation of all parameters unrelated to affinity, and the dissociation constant K d (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473). The antibodies of the present disclosure may have a binding constant (K) as measured by an assay such as a radioligand binding assay or similar assay known to those of skill in the art. d ) is ≦1 μM, for example, in some embodiments, ≦100 nM, in some embodiments, ≦10 nM, and in some embodiments, ≦100 pM to about 1 pM.

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

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

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

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

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

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

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

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

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

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

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

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

[0156] As described herein, minor variations in the amino acid sequence of an antibody or immunoglobulin molecule are contemplated as being encompassed by the present disclosure, so long as the variation in the amino acid sequence is at least 75%, e.g., in some embodiments, at least 80%, 90%, 95%, and in some embodiments, 99%. Specifically, conservative amino acid substitutions are contemplated. Conservative substitutions are those that occur within a family of amino acids whose side chains are related. Genetically encoded amino acids are generally classified into the following families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other amino acid families include: (i) the aliphatic hydroxy family, serine and threonine; (ii) the amide-containing family, asparagine and glutamine; (iii) the aliphatic family, alanine, valine, leucine, and isoleucine; and (iv) the aromatic family, phenylalanine, tryptophan, and tyrosine. For example, it is reasonable to expect that isolated substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitutions between structurally related amino acids will not have a significant effect on the binding or properties of the resulting molecule, especially when the substitution does not involve an amino acid within a framework region. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative, which assays are described in detail herein.Fragments or analogs of antibodies or immunoglobulin molecules can be easily prepared by those skilled in the art. In some embodiments, the amino and carboxy termini of the fragments or analogs are located near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data with public or proprietary sequence databases. Computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains present in other proteins with known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known. Bowie et al. Science 253:164 (1991). Thus, the above examples demonstrate that those skilled in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains according to the present disclosure.

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

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

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

[0160] As used herein, the term "label" or "labeled" refers to the incorporation of a detectable marker, for example, by incorporation of a radiolabeled amino acid into a polypeptide or by attachment of a biotinyl moiety that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In certain circumstances, the label or marker may also be therapeutic. A variety of methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include radioisotopes or radionuclides (e.g.,3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., fluorophores, rhodamines, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labels are attached by spacer arms of various lengths to reduce potential steric hindrance. The term "pharmaceutical agent or drug," as used herein, refers to a chemical compound or composition capable of inducing a desired therapeutic effect when appropriately administered to a patient.

[0161] As used herein, "substantially pure" means that a species is the predominant species present (i.e., it is more abundant than other individual species in the composition on a molar basis), and a substantially purified fraction is a composition in which a species constitutes at least about 50 percent (on a molar basis) of all macromolecular species.

[0162] Generally, a substantially pure composition will comprise greater than about 80 percent of all macromolecular species present in the composition, e.g., in some embodiments, greater than about 85%, 90%, 95%, and 99%. In some embodiments, the species is purified to essential homogeneity, such that the composition consists essentially of a single macromolecular species (contaminating species cannot be detected in the composition by conventional detection methods).

[0163] The term patient includes human and veterinary subjects.

[0164] Other chemical terms herein are used in accordance with conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

[0165] II. Multispecific Polypeptide Constructs Provided herein is a multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, wherein the first and second components are coupled by a linker and the Fc region is positioned N-terminal to the CD3 binding region; and one or both of the first and second components comprises an antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0166] In some embodiments, the multispecific polypeptide construct contains, from N-terminus to C-terminus, an immunoglobulin Fc region; a linker; a CD3-binding region that binds to CD3 (CD3ε); and an antigen-binding domain that binds to a tumor-associated antigen (TAA). In some embodiments, the multispecific polypeptide construct contains, from N-terminus to C-terminus, an antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker; and a CD3-binding region that binds to CD3 (CD3ε). In some embodiments, the multispecific polypeptide construct contains at least a first antigen-binding domain that binds to a TAA and a second antigen-binding domain that binds to a TAA. In some embodiments, the multispecific polypeptide construct contains, from N-terminus to C-terminus, a first antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker; a CD3-binding region that binds to CD3 (CD3ε); and a second antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0167] Each of the components of the multispecific polypeptide constructs of the present disclosure is described in more detail below.

[0168] 1. Anti-CD3 binding domain: The multispecific polypeptide constructs of the present disclosure comprise one or more copies of an anti-CD3 binding domain. The anti-CD3 binding domain of the present disclosure activates T cells through engagement of CD3ε on T cells. The anti-CD3 binding domain of the present disclosure induces, stimulates, activates, and / or otherwise enhances CD3-mediated T cell activation. The biological activity of CD3 includes, for example, T cell activation and other signaling through the interaction between CD3 and the antigen-binding subunit of the T cell receptor (TCR). For example, the anti-CD3 binding domain of the present disclosure activates T cells fully or partially through engagement of CD3ε on T cells by partially or fully modulating CD3-mediated T cell activation, e.g., by inducing, stimulating, activating, or otherwise enhancing it.

[0169] In preferred embodiments, the anti-CD3 binding domain of the present disclosure specifically binds to the ε chain of CD3, also known as CD3ε. The anti-CD3ε binding domain of the present disclosure activates T cells through engagement of CD3ε on T cells. The anti-CD3ε binding domain of the present disclosure includes monoclonal antibodies, such as mammalian monoclonal antibodies, primate monoclonal antibodies, fully human monoclonal antibodies, as well as humanized monoclonal antibodies and chimeric antibodies, and antigen-binding fragments thereof. In some embodiments, the anti-CD3ε binding domain comprises one or more copies of an antibody or its antigen-binding fragment.

[0170] In some embodiments, the anti-CD3ε binding domain comprises one or more copies of an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, an scFv, a scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. In some embodiments, the anti-CD3ε binding domain comprises an Fv antibody fragment that binds to CD3ε (referred to herein as an anti-CD3ε Fv fragment). In some embodiments, the anti-CD3ε Fv antibody fragment is a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv). In some embodiments, the anti-CD3 binding domain is monovalent for CD3 binding.

[0171] In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO:14. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO:15. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO:14 and a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO:15. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO:44. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO:72. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO:44 and a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO:72. In some embodiments, the anti-CD3ε binding domain comprises a combination of a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence comprising an amino acid sequence selected from the group of SEQ ID NOs: 32 to 81. In some embodiments, the anti-CD3ε binding domain comprises a combination of a heavy chain variable region amino acid sequence selected from the group of SEQ ID NOs: 32 to 62 and a light chain variable region amino acid sequence comprising an amino acid sequence selected from the group of SEQ ID NOs: 63 to 81.

[0172] In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (Lv) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:14, and a variable light chain (Lv) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:15.

[0173] In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (Lv) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:44, and a variable light chain (Lv) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:72.

[0174] In some embodiments, the anti-CD3ε Fv antibody fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32 to 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 to 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 to 62 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 63 to 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.

[0175] In some embodiments, the anti-CD3ε binding domain comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence comprises at least the amino acid sequence TYAMN (SEQ ID NO: 16); VH CD2 sequence containing at least TIFF0007798942000011.tif4128; and amino acid sequence The VH CDR3 sequence is selected from VH CDR3 sequences containing at least TIFF0007798942000012.tif4128.

[0176] In some embodiments, the anti-CD3ε binding domain comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence is the amino acid sequence TIFF0007798942000013.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 comprises a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO:16); VH CD2 sequence containing at least TIFF0007798942000014.tif4128; amino acid sequence VH CDR3 sequence containing at least TIFF0007798942000015.tif4128; amino acid sequence TIFF0007798942000016.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 combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence TYAMN (SEQ ID NO:16); a VH CD2 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007798942000017.tif4128; and an amino acid sequence TIFF0007798942000018.tif4128, or a VH CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007798942000018.tif4128.

[0179] In some embodiments, the anti-CD3ε binding domain comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence is the amino acid sequence a VL CDR1 sequence comprising a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007798942000019.tif4128; a VL CDR2 sequence comprising a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence GTNKRAP (SEQ ID NO:20); and a VL CDR3 sequence comprising a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ALWYSNLWV (SEQ ID NO:21).

[0180] In some embodiments, the anti-CD3ε binding domain comprises a VH CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence TYAMN (SEQ ID NO:16); a VH CD2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007798942000020.tif4128; an amino acid sequence A VH CDR3 sequence, amino acid sequence, that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007798942000021.tif4128 a VL CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007798942000022.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).

[0181] In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence and a light chain variable amino acid sequence. In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence and a light chain variable amino acid sequence comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81. In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence and a light chain variable amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81. In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence selected from the group of SEQ ID NOs: 32-62 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81. In some embodiments, the anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62, and 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.

[0182] 2. Immunoglobulin Fc Polypeptides: The first component of the multispecific polypeptide construct of the present disclosure comprises an immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is an IgG isotype selected from the group consisting of IgG1 isotype, IgG2 isotype, IgG3 isotype, and IgG4 subclass. In some embodiments, the Fc region is human Fc. In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6. In some embodiments, the immunoglobulin Fc region contains an Fc chain that is an immunologically active fragment of any of SEQ ID NOs: 1-6. In some embodiments, the immunoglobulin Fc region contains an Fc polypeptide chain that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any of SEQ ID NOs: 1-6, or an immunologically active fragment thereof.

[0183] In some embodiments, the multispecific polypeptide construct is a dimer formed by polypeptides each containing an Fc. In some specific embodiments, identical or substantially identical polypeptides will dimerize to create a homodimer. In some embodiments, the dimer is a homodimer in which the two polypeptides of the multispecific polypeptide construct are identical. In other cases, the Fc region is formed by an Fc domain that has been mutated or modified to promote heterodimerization, in which different polypeptides dimerize to give a heterodimer. Thus, in some embodiments, the dimer is a heterodimer in which the two polypeptide chains of the multispecific polypeptide construct are different. Exemplary modifications to promote heterodimerization are known and include the following:

[0184] Generally, the Fc region is responsible for effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in addition to antigen-binding ability, which is the primary function of immunoglobulins. Furthermore, the FcRn sequence present in the Fc region plays a role in regulating serum IgG levels by increasing in vivo half-life through conjugation with the in vivo FcRn receptor. In some embodiments, such functions may be altered, for example, reduced or enhanced, in the Fc for use with the provided multispecific polypeptide constructs.

[0185] In some embodiments, the Fc region of the provided multispecific polypeptide constructs exhibits one or more effector functions. In some cases, the Fc region can provide an Fc-mediated effector function, such as, for example, ADCC (e.g., release of granzyme B by NK cells), ADCP, and / or CDC. Thus, in some embodiments in which the multispecific polypeptide construct contains a cleavable linker, cleavage of the linker can produce two components, each with biological activity: a CD3-binding region capable of binding and engaging CD3 on a T cell, and an Fc region linked to a TAA antigen-binding domain capable of exhibiting target-specific effector function.

[0186] In some embodiments, the Fc region comprises an Fc polypeptide that has been mutated or modified to alter one or more effector functions. Various examples of mutations in Fc polypeptides to alter, e.g., reduce, effector function are known and include those described below. In some embodiments, unless described with reference to a specific SEQ ID NO, references to amino acid substitutions in the Fc region are in accordance with EU numbering according to Kabat (also referred to as Kabat numbering). EU numbering is known and is in accordance with the EU index reported in the latest IMGT Scientific Chart (IMGT®, the international ImMunoGeneTics information system®, http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created: 17 May 2001, last updated: 10 January 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).

[0187] In some embodiments, provided multispecific polypeptide constructs containing an Fc region exhibiting reduced effector function may be desirable candidates for applications in which constrained CD3 binding is desired, but certain effector functions (such as CDC and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the multispecific polypeptide construct and / or truncated components thereof lack FcγR binding (and thus likely lack ADCC activity) but retain FcRn binding ability. The primary cells for mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063(1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502(1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361(1987)). Alternatively, non-radioactive assay methods may be utilized (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96™ Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the multispecific polypeptide construct or a truncated component thereof is unable to bind C1q and therefore lacks CDC activity. See, for example, the C1q binding ELISA and C3c binding ELISA of WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0188] In some embodiments, the immunoglobulin Fc region or immunologically active fragment thereof is of the IgG isotype. For example, the immunoglobulin Fc region of the fusion protein is of the human IgG1 isotype, having the following amino acid sequence: TIFF0007798942000023.tif33132

[0189] In some embodiments, the immunoglobulin Fc region or immunologically active fragment thereof comprises a human IgG1 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:1.

[0190] In some embodiments, the human IgG1 Fc region is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). For example, Natsume et al.,2008 Cancer Res,68(10):3863-72;Idusogie et al.,2001 J Immunol,166(4):2571-5;Moore et al.,2010 mAbs,2(2):181-189;Lazar et al.,2006 PNAS,103(11):4005-4010;Shields et al.,2001 JBC,276(9):6591-6604;Stavenhagen et al.,2007 Cancer Res,67(18):8882-8890;Stavenhagen et al.,2008 Advan. Enzyme Regul.,48: 152-164;Alegre et al,1992 J Amino acid modifications described in Immunol, 148:3461-3468; and reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11, the contents of each of which are incorporated herein by reference in their entirety.

[0191] In some embodiments, an Fc region, such as a human IgG1 Fc region, is modified to enhance ADCC or CDC activity. Examples of mutations that enhance ADCC include modifications at Ser239 and Ile332, such as Ser239Asp and Ile332Glu (S239D, I332E). Examples of mutations that enhance CDC include modifications at Lys326 and Glu333. In some embodiments, the Fc region is modified at one or both of these positions using the Kabat numbering system (e.g., Lys326Ala and / or Glu333Ala (K326A and E333A)).

[0192] In some embodiments, the human IgG1 Fc region fusion protein of the present disclosure lacks or has reduced fucose attached to the N-linked glycan chain at N297. There are numerous ways to prevent fucosylation, including, but not limited to, production in a FUT8-deficient cell line; addition of inhibitors, such as castanospermine, to mammalian cell culture medium; and metabolic engineering of the production cell line. In some embodiments, the human IgG1 Fc region is modified at amino acid Asn297 (boxed, Kabat numbering) (e.g., Asn297Ala (N297A) or Asn297Asp (N297D)) to prevent glycosylation of the fusion protein.

[0193] In some embodiments, the Fc region of the fusion protein is altered at one or more of the following positions to reduce Fc receptor binding: Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325), or Ala327 (A327). For example, Leu234Ala (L234A), Leu235Ala (L235A), Asp265Asn (D265N), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Asn325Glu (N325E), or Ala327Ser (A327S). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu235 (within the box of SEQ ID NO:1, Kabat numbering) to alter Fc-receptor interactions (e.g., Leu235Glu (L235E) or Leu235Ala (L235A)). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (in the box of SEQ ID NO:1, Kabat numbering) to alter Fc receptor interactions (e.g., Leu234Ala (L234A)). In some embodiments, the Fc region of the fusion protein is altered at both amino acids 234 and 235 (e.g., Leu234Ala and Leu235Ala (L234A / L235A) or Leu234Val and Leu235Ala (L234V / L235A)). In preferred embodiments, modifications within the Fc region reduce binding to the Fc receptor gamma receptor but have minimal effect on binding to the neonatal Fc receptor (FcRn).

[0194] In some embodiments, the human IgG Fc region is modified to enhance FcRn binding. Examples of Fc mutations that enhance FcRn binding are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E, respectively) (Kabat numbering, Dall'Acqua et al. 2006, J. Biol Chem Vol. 281(33)23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al. 2010 Nature Biotech, Vol. 28(2)157-159) (Kabat et al. 1991 EU Index of Sequences of Proteins of Immunological Interest). In some embodiments, the variant or modified Fc polypeptide comprises the following mutations, using the Kabat numbering system: Met252Tyr and Met428Leu or Met252Tyr and Met428Val (M252Y, M428L or M252Y, M428V).

[0195] In some embodiments, the Fc region of the fusion protein lacks amino acids at one or more of the following positions to reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (L235). In these embodiments, the Fc deletion of these three amino acids reduces complement protein C1q binding.

[0196] TIFF0007798942000024.tif31131

[0197] In some embodiments, the Fc region of the fusion protein is altered at Gly236 (within the box of SEQ ID NO:1) to reduce Fc receptor binding. For example, Gly236 is deleted from the fusion protein. In some embodiments, a human IgG1 Fc region is modified at amino acid Gly236 (e.g., Gly236Ala (G236A)) to enhance interaction with CD32A.

[0198] In some embodiments, the human IgG1 Fc region lacks Lys447 (Kabat et al 1991 EU Index of Sequences of Proteins of Immunological Interest).

[0199] In some embodiments, the fusion or immunologically active fragment thereof comprises a human IgG2 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:2.

[0200] In some embodiments, the immunoglobulin Fc region or immunologically active fragment thereof of the fusion protein is of the human IgG2 isotype, having the following amino acid sequence: TIFF0007798942000025.tif31131

[0201] In some embodiments, the fusion or immunologically active fragment thereof comprises a human IgG2 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:3.

[0202] In some embodiments, the human IgG2 Fc region is modified at amino acid Asn297 (intrabox, to prevent glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D)). In some embodiments, the human IgG2 Fc region lacks Lys447 (Kabat et al 1991 Sequences of Proteins of Immunological Interest, EU Index).

[0203] In some embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG3 isotype, having the following amino acid sequence: TIFF0007798942000026.tif33131

[0204] In some embodiments, the antibody or immunologically active fragment thereof comprises a human IgG3 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:4.

[0205] In some embodiments, the human IgG3 Fc region is modified at amino acid Asn297 (in box, Kabat numbering) to prevent glycosylation of the antibody (e.g., Asn297Ala (N297A) or Asn297Asp (N297D)). In some embodiments, the human IgG3 Fc region is modified at amino acid 435 (e.g., Arg435His (R435H)) to extend half-life. In some embodiments, the human IgG3 Fc region lacks Lys447 (Kabat et al 1991 Sequences of Proteins of Immunological Interest, EU Index).

[0206] 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: TIFF0007798942000027.tif33131

[0207] In some embodiments, the antibody or immunologically active fragment thereof comprises a human IgG4 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:5.

[0208] 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: TIFF0007798942000028.tif31131

[0209] In some embodiments, the antibody or immunologically active fragment thereof comprises a human IgG4 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:6.

[0210] 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 (in box, Kabat numbering) to prevent glycosylation of the antibody (e.g., Asn297Ala(N297A) or Asn297Asp(N297D)). In some embodiments, the human IgG4 Fc region lacks Lys447 (Kabat et al 1991 Sequences of Proteins of Immunological Interest EU Index).

[0211] 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).

[0212] In some embodiments, the human IgG Fc region is modified to induce heterodimerization. Various methods are known for promoting heterodimerization of complementary Fc polypeptides. For example, Ridgway et al,Protein Eng. 9:617-621(1996);Merchant et al,Nat. Biotechnol. 16(7):677-81(1998);Moore et al. (2011)MAbs,3:546-57;Von Kreudenstein et al. MAbs,(2013)5:646-54;Gunasekaran et al. (2010)J. Biol. Chem. ,285:19637-46;Leaver-Fay et al. (2016)Structure,24:641-51;Ha et al. (2016)Frontiers in Immunology,7:1;Davis et al. (2010) Protein Eng Des Sel,23:195-202;Published International PCT Application No. WO 1998 / 050431, WO2009 / 089004, WO2011143545, WO2014 / 067011, WO2012 / 058768, WO2018027025; published U.S. patent application numbers US20140363426, US20150307628, US20180016354, US20150239991; and U.S. Patent Nos. 5,731,168, 7,183,076, 9,701,759, 9,605,084, and 9,650,446. Methods for promoting heterodimerization of the Fc chain include mutagenesis of the Fc region, for example, by including a set of "knob-into-hole" mutations or mutations to achieve electrostatic steering of the Fc to favor attractive interactions between different polypeptide chains.For example, in some embodiments, heterodimeric Fc polypeptides contain mutations to alter the charge polarity at the Fc dimer interface so that coexpression of electrostatically matched Fc chains favors favorable attractive interactions, thereby promoting the formation of desired Fc heterodimers, and unfavorable repulsive charge interactions suppress the formation of undesired Fc homodimers (Guneskaran et al., (2010) JBC, 285:19637-19646). When coexpressed in cells, interchain association is possible, but due to charge repulsion, the chains do not substantially self-associate. Another strategy for generating heterodimeric Fc involves mixing CH3 domain segments from human IgG and IgA to create complementary CH3 heterodimers, referred to as SEED Fc.

[0213] In some embodiments, both polypeptides of the Fc heterodimer contain paired or complementary amino acid modifications to promote heterodimerization. Exemplary pairs of amino acid modifications for polypeptides of Fc fusions are shown in Table 1.

[0214] Table 1. Amino acid pairs of heterodimeric Fc TIFF0007798942000029.tif49138

[0215] In some embodiments, the modification comprises introducing a protuberance (knob) into a first Fc polypeptide and a cavity (hole) into a second Fc polypeptide, such that the protuberance is positioned in the cavity to facilitate complexation of the first and second Fc-containing polypeptides. The amino acids targeted for substitution and / or modification to create the protuberance or cavity in the polypeptide are typically interface amino acids that interact with or contact one or more amino acids at the interface of the second polypeptide.

[0216] In some embodiments, the first Fc polypeptide modified to contain a protuberance (hole) amino acid comprises a substitution of a native or initial amino acid with an amino acid having at least one side chain that protrudes from the interface of the first Fc polypeptide and thus is positioned in a compensatory cavity (hole) in the adjacent interface of the second polypeptide. The replacement amino acid most often has a side chain volume larger than that of the initial amino acid residue. Methods for determining and / or evaluating the properties of amino acid residues to identify ideal replacement amino acids for creating a protuberance are known to those skilled in the art. In some embodiments, the replacement residue for forming the protuberance is a naturally occurring amino acid residue, including, for example, arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). In some examples, the initial residue identified for substitution is an amino acid residue with a small side chain, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0217] In some embodiments, the second Fc polypeptide to be modified to contain a cavity (hole) is recessed from the interface of the second polypeptide and thus includes a substitution of the native or initial amino acid with an amino acid having at least one side chain that can accommodate a corresponding protrusion from the interface of the first polypeptide. The substituted amino acid most often has a smaller side chain volume than the initial amino acid residue. Methods for determining and / or evaluating the characteristics of amino acid residues to identify ideal replacement residues for cavity formation are known to those skilled in the art. Generally, replacement residues for cavity formation are naturally occurring amino acids, including, for example, alanine (A), serine (S), threonine (T), and valine (V). In some examples, the initial amino acid identified for substitution is an amino acid with a large side chain, such as, for example, tyrosine, arginine, phenylalanine, or tryptophan.

[0218] The CH3 interface of human IgG1, for example, comprises 16 residues from each domain located on four antiparallel β-strands buried 1090 Å from each surface (see, for example, Deisenhofer et al. (1981) Biochemistry, 20:2361-2370; Miller et al., (1990) J Mol. Biol., 216, 965-973; Ridgway et al., (1996) Prot. Engin., 9:617-621; U.S. Patent No. 5,731,168). Modifications of the CH3 domain to create protuberances or cavities are described, for example, in U.S. Patent No. 5,731,168; International Patent Applications WO98 / 50431 and WO2005 / 063816; and Ridgway et al., (1996) Prot. Engin. 617-621. In some instances, modifications of the CH3 domain to create a bulge or cavity are typically targeted to residues located in the two central antiparallel β-strands, with the goal being to minimize the risk that the resulting bulge will be accommodated by protruding into the surrounding solvent rather than being accommodated in a compensatory cavity in the partner CH3 domain.

[0219] For example, in some embodiments, the heterodimeric Fc comprises a polypeptide having an amino acid modification at Thr366 in a CH3 domain, which, when replaced with a bulkier amino acid, e.g., Try (T366W), can preferentially pair with a second CH3 domain having an amino acid modification at positions Thr366, Leu368, and Tyr407 to a less bulky amino acid, e.g., Ser, Ala, or Val (T366S / L368A / Y407V). Heterodimerization via CH3 modifications can be further stabilized by the introduction of disulfide bonds, e.g., by changing Ser354 to Cys (S354C) and Tyr349 to Cys (Y349C) in the opposing CH3 domain (reviewed in Carter, 2001 Journal of Immunological Methods, 248:7-15).

[0220] The resulting multispecific polypeptide construct can be purified by an appropriate method, such as affinity chromatography on a Protein A or Protein G column. When two nucleic acid molecules encoding different polypeptides are transformed into a cell, homodimers and heterodimers will form. Expression conditions can be adjusted so that heterodimer formation is favored over homodimer formation.

[0221] Techniques for recovering heterodimers from homodimers based on the differential affinity of the heterodimers for affinity reagents are known. In some aspects, such techniques involve designing heterodimers such that one of the Fc polypeptide chains does not bind to the affinity reagent Protein A. In some cases, one of the polypeptide chains may contain one or more amino acid substitutions to eliminate or reduce the affinity of one of the polypeptides of the Fc heterodimer for the Protein A reagent. See, e.g., WO2017134440, WO2010151792, and Jendeberg et al. (Jendeberg et al., (1997) J. Immunol. Methods, 201(1): 25-34). In some of these embodiments, the Fc region may be modified in the Protein A binding site of one member of the heterodimer to prevent Protein A binding, thereby allowing for more efficient purification of the heterodimeric fusion protein. An exemplary modification at this binding site is Ile253, e.g., Ile253Arg (I253R). In some embodiments, the modification can be H435R or H435R / Y436F. In some embodiments, the Fc polypeptide of the Fc heterodimer can contain a modification (pA+ / pG-) that allows it to bind to Protein A but not Protein G. Exemplary pA+ / pG- amino acid modifications include, for human IgG1, an Fc containing serine at position 428, serine at position 434, and optionally, histidine at position 436, or those residues at the corresponding positions in human IgG2, 3, or 4. In some aspects, such amino acid modifications at positions 428, 434, and optionally, at position 436 of an IgG Fc polypeptide reduce or prevent Protein G binding and enhance protein purification.

[0222] In some embodiments, any of these modifications that confer differential affinity to affinity reagents can be combined with one or more of the other amino acid modifications described above. For example, the I253R modification can be combined with either the T366S / L368A / Y407V or T366W modification. The T366S / L368A / Y407V modified Fc can form homodimers because there is no steric hindrance at the dimerization interface that exists in the case of the T366W modified Fc. Thus, in some embodiments, the I253R modification is combined with the T366S / L368A / Y407V modified Fc to disallow purification of any homodimeric Fc that may have formed. A similar modification can be utilized by combining T366S / L368A / Y407V with H453R.

[0223] In some embodiments, the Fc region of the heterodimeric molecule may further comprise one or more other Fc mutations, such as any of those described above, hi some embodiments, the heterodimeric molecule comprises an Fc region comprising a mutation that reduces effector function.

[0224] In some embodiments, one Fc polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs: 82, 86, 94, or 96, and the other Fc polypeptide of the heterodimeric Fc contains the sequence of amino acids set forth in any of SEQ ID NOs: 83, 87, 90, 92, 98, or 100. In some embodiments, one Fc polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs: 84, 88, 95, or 97, and the other Fc polypeptide of the heterodimeric Fc comprises the sequence of amino acids set forth in any of SEQ ID NOs: 85, 89, 91, 93, 99, or 101.

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

[0226] In some embodiments, the Fc region of the fusion protein is altered at one or more of the following positions to reduce Fc receptor binding: Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325), or Ala327 (A327). For example, Leu234Ala (L234A), Leu235Ala (L235A), Asp265Asn (D265N), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Asn325Glu (N325E), or Ala327Ser (A327S). In preferred embodiments, modifications within the Fc region reduce binding to the Fc receptor gamma receptor but have minimal effect on binding to the neonatal Fc receptor (FcRn).

[0227] In some embodiments, the fusion protein contains a polypeptide derived from an immunoglobulin hinge region. The hinge region can be selected from any of the human IgG subclasses. For example, the fusion protein can contain a modified IgG1 hinge having the sequence EPKSSDKTHTCPPC (SEQ ID NO:7), in which Cys220, which forms a disulfide with the C-terminal cysteine ​​of the light chain, is mutated to serine (e.g., Cys220Ser (C220S)). In other embodiments, the fusion protein contains a shortened hinge having the sequence DKTHTCPPC (SEQ ID NO:8).

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

[0229] 3. Linker The provided multispecific polypeptide constructs contain a linker that joins or couples a first component containing an immunoglobulin Fc region to a second component containing a CD3-binding region. In some embodiments, the linker is positioned at the end of the C-terminal region of the Fc region, such that the Fc region is N-terminal to the CD3-binding region. Because the provided multispecific polypeptide constructs are multimeric, such as dimers, the provided constructs include a linker that joins a first Fc polypeptide and a first domain (e.g., VH) of a CD3-binding region of a first polypeptide to a second domain (e.g., VL) of a CD3-binding region of a second polypeptide. Typically, the linkers present in the first and second polypeptides of the multispecific polypeptide construct are identical. Thus, in some embodiments, each domain of the CD3-binding domain is linked to the opposite polypeptide of the Fc, such as a heterodimeric Fc, via a linker, such as the same linker.

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

[0231] In some embodiments, the linker is selected so that, when the CD3-binding region is conjugated to the Fc region of the multispecific polypeptide conjugate, the CD3-binding region is constrained and is unable or substantially unable to bind or engage with CD3 on the surface of cells, e.g., T cells, when the multispecific polypeptide construct contacts the cells. Various assays can be used to evaluate CD3 binding or engagement by the multispecific polypeptide construct, including assays to assess T cell binding, NFAT activation using reporter systems, cytolytic T cell activity, cytokine production, and / or expression of T cell activation markers. Exemplary assays are shown in the provided examples. Typically, the linker ensures correct folding of the polypeptide construct, does not exhibit charges inconsistent with the activity or function of the linked polypeptide, and does not form bonds or other interactions with amino acid residues in one or more of the domains that would weaken or alter the activity of the linked polypeptide. In some embodiments, the linker is a polypeptide linker. The polypeptide linker can be a flexible linker or a non-flexible linker, or a combination of both. In some aspects, the linker is a short, medium, or long linker. In some embodiments, the linker is up to 40 amino acids in length. In some embodiments, it is up to 25 amino acids in length. In some embodiments, the linker is at least about 2 amino acids in length. In some aspects, a suitable length is, for example, at least 1 amino acid residue, typically less than about 40 amino acid residues, e.g., 2 to 25 amino acid residues, 5 to 20 amino acid residues, 5 to 15 amino acid residues, or 8 to 12 amino acids in length.In some embodiments, the linker is about 2 to 24 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to 24 amino acids, 6 to 20 amino acids, 6 to 18 amino acids, 6 to 14 amino acids, 6 to 12 amino acids, 6 to 10 amino acids, 6 to 8 amino acids, 8 to 24 amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14 amino acids, 8 to 12 amino acids, 8 to 10 amino acids, 10 to 24 amino acids, 10 to 20 amino acids, 10 to 18 amino acids, 10 to 14 amino acids, 10 to 12 amino acids, 12 to 24 amino acids, 12 to 20 amino acids, 12 to 18 amino acids, 12 to 14 amino acids, 14 to 24 amino acids, 14 to 20 amino acids, 14 to 18 amino acids, 18 to 24 amino acids, 18 to 20 amino acids, or 20 to 24 amino acids. In some embodiments, the linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0232] In certain aspects, the longer the linker length, the greater the CD3 binding when the multispecific polypeptide conjugate is bound to its antigen, e.g., a TAA. Thus, in some aspects, the linker is greater than 12 amino acids in length, e.g., greater than 13, 14, 15, 16, 17, or 18 amino acids in length. In some embodiments, the linker is 12-40 amino acids in length, 12-30 amino acids, 12-24 amino acids, 12-18 amino acids, 12-15 amino acids, 15-40 amino acids, 15-30 amino acids, 15-24 amino acids, 15-18 amino acids, 18-40 amino acids, 18-30 amino acids, 18-24 amino acids, 24-40 amino acids, 24-30 amino acids, or 30-40 amino acids.

[0233] The linker may be naturally occurring, synthetic, or a combination of both. Particularly suitable linker polypeptides primarily comprise amino acid residues selected from glycine (Gly), serine (Ser), alanine (Ala), and threonine (Thr). For example, the linker may contain at least 75%, e.g., at least 80%, at least 85%, or at least 90% (calculated based on the total number of residues present in the peptide linker) of amino acid residues selected from Gly, Ser, Ala, and Thr. The linker may consist exclusively of Gly, Ser, Ala, and / or Thr residues. In some embodiments, the linker contains 1-25 glycine residues, 5-20 glycine residues, 5-15 glycine residues, or 8-12 glycine residues. In some aspects, suitable peptide linkers typically contain at least 50% glycine residues, e.g., at least 75% glycine residues. In some embodiments, the peptide linker comprises only glycine residues. In some embodiments, the peptide linker comprises only glycine and serine residues.

[0234] In some embodiments, these linkers are composed primarily of the amino acids glycine and serine, and are referred to herein as GS linkers. In some embodiments, the linker contains (GGS)n (n is 1-10, e.g., 1-5, e.g., 1-3), e.g., GGS(GGS)n (SEQ ID NO:171) (n is 0-10). In a specific embodiment, the linker contains the sequence (GGGGS)n (SEQ ID NO:173) (n is 1-10 or n is 1-5, e.g., 1-3). In a further embodiment, the linker contains (GGGGGS)n (SEQ ID NO:172) (n is 1-4, e.g., 1-3). The linker may include any combination of the above, e.g., 2, 3, 4, or 5 repeats of the GS linker, GGS linker, GGGGS linker, and / or GGGGGS linker may be combined. In some embodiments, such linkers are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids in length.

[0235] In some embodiments, the linker is as follows (single letter amino acid code): GGS, GGGGS (SEQ ID NO: 149), or GGGGGS (SEQ ID NO: 135). In some embodiments, the GS linker is TIFF0007798942000030.tif39159. In some embodiments, the linker is GGGG (SEQ ID NO: 103). In some of the above examples, serine may be substituted with alanine (e.g., (Gly4Ala) or (Gly3Ala)).

[0236] In some embodiments, the linker comprises the amino acid sequence Gly x -Xaa-Gly y -Xaa-Gly z(SEQ ID NO:174) wherein each Xaa is independently selected from alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp), proline (Pro), glycine (Gly), serine (Ser), threonine (Thr), cysteine ​​(Cys), tyrosine (Tyr), asparagine (Asn), glutamine (Gln), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), and glutamic acid (Glu); and wherein x, y, and z are each integers ranging from 1 to 5. In some embodiments, each Xaa is independently selected from the group consisting of Ser, Ala, and Thr. In a particular variation, x, y, and z each equal 3, thus providing a peptide linker having the amino acid sequence Gly-Gly-Gly-Xaa-Gly-Gly-Gly-Xaa-Gly-Gly-Gly (SEQ ID NO:175), where each Xaa is selected as described above.

[0237] In some embodiments, the linker is a serine-rich linker based on repeating (SSSSG)y (SEQ ID NO:185) motifs, where y is at least 1, but can also be 2, 3, 4, 5, 6, 7, 8, and 9.

[0238] In some cases, it may be desirable to provide a peptide linker with a degree of immobility. This can be achieved by including proline residues in the amino acid sequence of the peptide linker. Thus, in some embodiments, the linker includes at least one proline residue within the amino acid sequence of the peptide linker. For example, the peptide linker may have an amino acid sequence in which at least 25% (e.g., at least 50% or at least 75%) of the amino acid residues are proline residues. In one specific embodiment, the peptide linker includes only proline residues.

[0239] In some aspects, the peptide linker comprises at least one cysteine ​​residue, e.g., one cysteine ​​residue. For example, in some embodiments, the linker comprises at least one cysteine ​​residue and an amino acid residue selected from the group consisting of Gly, Ser, Ala, and Thr. In some such embodiments, the linker comprises only glycine and cysteine ​​residues, e.g., only glycine and cysteine ​​residues. Typically, only one cysteine ​​residue is included in each peptide linker. An example of a specific linker containing a cysteine ​​residue is the amino acid sequence Gly m -Cys-Gly n (n and m are each an integer from 1 to 12, e.g., 3 to 9, 4 to 8, or 4 to 7.) In a particular variation, such a peptide linker has the amino acid sequence GGGGG-C-GGGGG (SEQ ID NO:177).

[0240] In some embodiments, the linker of the fusion protein is a structured or constrained linker. In specific embodiments, the structured linker contains the sequence (AP)n or (EAAAK)n (SEQ ID NO:178) (n is 2 to 20, preferably 4 to 10), such as, but not limited to, AS-(AP)n-GT (SEQ ID NO:179) or AS-(EAAAK)n-GT (SEQ ID NO:180) (n is 2 to 20, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). In other embodiments, the linker contains the sequence TIFF0007798942000031.tif18151 (n is 2 to 20). In some embodiments, the linker TIFF0007798942000032.tif4170. In some embodiments, such linkers, due to their structure, may be more resistant to proteolytic cleavage and therefore may offer advantages when injected in vivo.

[0241] In some embodiments, the linker is not a cleavable linker, but is also referred to as a non-cleavable linker. In some embodiments, the linker is not cleavable by a protease. In some embodiments, a linker that is not cleavable or not cleavable by a protease is generally stable for in vivo delivery or recombinant production. In some aspects, a linker that is not cleavable by a protease includes one that does not contain at least one peptide bond that is preferably located within a cleavable peptide sequence or protease recognition site. In specific embodiments, a non-cleavable linker is not a target substrate of a protease, and therefore is not preferentially or specifically cleaved by a protease compared to a linker that contains a substrate recognition site for the same protease.

[0242] In some embodiments, the linker is a cleavable linker. In some aspects, the cleavable linker is a linker that contains a sequence that is a substrate for a protease due to the presence of at least one bond that can be degraded under physiological conditions. In some cases, the cleavable linker is susceptible or hypersensitive to cleavage under certain conditions present in vivo, for example, after exposure to extracellular proteases, including those present in the cellular environment in vivo. In some cases, the proteases may be present in a specific physiological microenvironment, such as a tumor microenvironment, thereby limiting the sites at which cleavage can occur.

[0243] Proteases typically exhibit specificity or preference for cleaving a particular target substrate compared to other non-target substrates. The degree of such specificity can be determined based on the rate constant of cleavage of a sequence, e.g., a linker, which is a measure of the preference of the protease for the substrate and the efficiency of the enzyme. A method of 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 that is released when cleaved by the protease. By determining the rate of cleavage at different protease concentrations, the cleavage specificity constant (k cat / K m In some embodiments, the cleavable linker has a molecular weight of approximately at least 1×10 4 M -1 S -1 , or at least 5 × 10 4 M -1 S, at least 10 x 10 4 M -1 S, at least 10 x 10 5 M -1 S or faster.

[0244] Cleavable Linker In some embodiments, the multispecific polypeptide construct of the present disclosure comprises a cleavable linker joining the first and second components. In some embodiments, the cleavable linker comprises an amino acid sequence that can serve as a substrate for a protease, typically an extracellular protease. For example, the cleavable linker may comprise a cleavage sequence containing at least one peptide bond, preferably located within the protease-cleavable peptide sequence. Suitable proteases include matrix metalloproteinases (MMPs), cysteine ​​proteases, serine proteases, and plasmin activators, which are formed or activated in an enhanced manner in diseases such as rheumatoid arthritis or cancer, resulting in excessive tissue degradation, inflammation, and metastasis. In specific embodiments, the protease is a protease produced by tumors, activated immune effector cells (e.g., T cells or NK cells), or cells of the tumor microenvironment. In some embodiments, the protease is an MMP, such as granzyme B, matriptase, or MMP-2.

[0245] The cleavable linker can be selected based on the proteases produced by tumors in the vicinity of cells expressing the target and / or co-localized in tissue with the desired target of the multispecific polypeptide construct. Increased levels of proteases with known substrates in many cancers, such as solid tumors, have been reported in the literature. See, for example, La Rocca et al., (2004) British J. of Cancer 90(7):1414-1421.

[0246] In some embodiments, the cleavable linker joining the first and second components of the multispecific polypeptide construct is cleaved by a protease produced by an immune effector cell activated by one of the components. For example, a multispecific polypeptide construct comprising an effector-enabled or enhanced IgG Fc region can induce ADCC upon engagement with a target antigen. Central to ADCC is the release of granzyme B and perforin from effector cells, i.e., NK cells and cytotoxic T cells. Upon release, granzyme B enters target cells in a perforin-dependent manner, where it mediates apoptosis. Importantly, granzyme B is active at the extracellular synapse between effector and target cells. In some embodiments, the cleavable linker joining the first and second components of the multispecific polypeptide construct is cleaved by granzyme B. Granzyme B is released during effector cell activation mediated by one of the components of the multispecific polypeptide construct. In some embodiments, granzyme B and other proteases can be produced by immune effector cells, including activated T cells or NK cells. In some embodiments, activation of T cells by CD3 engagement upon TAA binding by the multispecific polypeptide construct releases such proteases, which can then cleave the specific cleavable linker, thereby enhancing or increasing the CD3-engaging activity of the CD3-binding molecule. In some embodiments, cleavage can amplify or increase the activity achieved by the multispecific construct when bound to a TAA in its uncleaved state.

[0247] Exemplary substrates include, but are not limited to, substrates cleavable by one or more of the following enzymes or proteases: ADAMS, ADAMTS, e.g., ADAM8; ADAM9; ADAM10; ADAM12; ADAM15; ADAM17 / TACE; ADAMDECl; ADAMTS1; ADAMTS4; ADAMTS5; aspartic proteases, e.g., BACE or renin; aspartic cathepsins, e.g., cathepsin D or cathepsin E; caspases, e.g., cathepsins caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, or caspase 14; cysteine ​​cathepsins, for example, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P; cysteine ​​proteinases, for example, cruzipain; legumain; otubain 2; KLKs, for example, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, or KLK14; metalloproteinases, e.g., meprin; neprilysin; PSMA; BMP-1; MMPs, e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, or MMP27, serine proteases, e.g., activated protein C, cathepsin A, cathepsin G, chymase, coagulation factor proteases (e.g., 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 (TTSPs), such as DESC1, DPP-4, FAP, hepsin, matriptase 2, matriptase, TMPRSS2, TMPRSS3, or TMPRSS4;and any combination thereof.;

[0248] In some embodiments, the cleavable linker is cleaved by multiple proteases, eg, two or more proteases, three or more proteases, four or more proteases, etc.

[0249] In some embodiments, the cleavable linker is selected for use with a particular protease, for example, a protease known to be produced by a tumor in the vicinity of cells expressing the target and / or co-localized with the target of the multispecific polypeptide construct.

[0250] In some embodiments, the cleavable linker contains a substrate recognition site or cleavage site of a specific protease, which is a sequence recognized by the active site of the protease that is cleaved by the protease. Typically, for example, for a serine protease, the cleavage sequence is composed of P1-P4 and P1'-P4' amino acids in the substrate, where cleavage occurs after the P1 position. Typically, the cleavage sequence of a serine protease is six residues long to match the extended substrate specificity of many proteases, but may be longer or shorter depending on the protease. Typically, the cleavable linker includes a cleavable P1-P1' bond sequence recognized by the protease. In some aspects, the cleavable linker is modified to introduce a peptide bond that can be cleaved by a specific protease, for example, by introducing a substrate recognition site sequence or cleavage sequence of the protease.

[0251] In some embodiments, the cleavable linker comprises a combination of two or more substrate sequences. In some embodiments, each substrate sequence is cleaved by the same protease. In some embodiments, at least two of the substrate sequences are cleaved by different proteases. In some embodiments, the cleavable linker comprises amino acids that are substrates for Granzyme B. In some embodiments, the Granzyme B cleavable linker contains an amino acid sequence having the general formula P4 P3 P2 P1↓P1' (SEQ ID NO:150) (P4 is amino acid I, L, Y, M, F, V, or A; P3 is amino acid A, G, S, V, E, D, Q, N, or Y; P2 is amino acid H, P, A, V, G, S, or T; P1 is amino acid D or E; and P1' is amino acid I, L, Y, M, F, V, T, S, G, or A). In some embodiments, the granzyme B cleavable linker contains an amino acid sequence having the general formula P4 P3 P2 P1↓P1' (SEQ ID NO:151) (P4 is amino acid I or L; P3 is amino acid E; P2 is amino acid P or A; P1 is amino acid D; and P1' is amino acid I, V, T, S, or G).

[0252] In some embodiments, the substrate for granzyme B comprises the amino acid sequence LEAD (SEQ ID NO:22), LEPG (SEQ ID NO:142), or LEAE (SEQ ID NO:143). In some embodiments, the cleavable linker comprises the amino acid sequence IEPDI (SEQ ID NO:136), LEPDG (SEQ ID NO:152), LEADT (SEQ ID NO:137), IEPDG (SEQ ID NO:138), IEPDV (SEQ ID NO:139), IEPDS (SEQ ID NO:140), IEPDT (SEQ ID NO:141), IEPDP (SEQ ID NO:144), LEPDG (SEQ ID NO:152), or LEADG (SEQ ID NO:153).

[0253] In some embodiments, the cleavable linker comprises an amino acid that is a substrate for matriptase. In some embodiments, the cleavable linker comprises the sequence P4QAR↓(A / V) (SEQ ID NO:154) (P4 is any amino acid). In some embodiments, the cleavable linker comprises the sequence RQAR(A / V) (SEQ ID NO:155). In some embodiments, a substrate for matriptase comprises the amino acid sequence RQAR (SEQ ID NO:23). In some embodiments, the cleavable linker comprises the amino acid sequence RQARV (SEQ ID NO:156).

[0254] In some embodiments, the cleavable linker comprises an amino acid that is a substrate for one or more matrix metalloproteases (MMPs). In some embodiments, the MMP is MMP-2. In some embodiments, the cleavable linker comprises the general formula P3 P2 P1↓P1′ (SEQ ID NO:157) (P3 is P, V, or A; P2 is Q or D; P1 is A or N; P1′ is L, I, or M). In some embodiments, the cleavable linker comprises the general formula P3 P2 P1↓P1′ (SEQ ID NO:158) (P3 is P; P2 is Q or D; P1 is A or N; P1′ is L or I). In some embodiments, the substrate for an MMP comprises the amino acid sequence PAGL (SEQ ID NO:24).

[0255] In some embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for granzyme B and an amino acid sequence that is a substrate for matriptase. In some embodiments, the cleavable linker comprises a combination of the amino acid sequence LEAD (SEQ ID NO:22) and the amino acid sequence RQAR (SEQ ID NO:23).

[0256] In some embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for Granzyme B and an amino acid sequence that is a substrate for an MMP. In some embodiments, the cleavable linker comprises a combination of the amino acid sequence LEAD (SEQ ID NO:22) and the amino acid sequence PAGL (SEQ ID NO:24).

[0257] In some embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for matriptase and an amino acid sequence that is a substrate for an MMP, hi some embodiments, the cleavable linker comprises a combination of the amino acid sequence RQAR (SEQ ID NO:23) and the amino acid sequence PAGL (SEQ ID NO:24).

[0258] In some embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for Granzyme B, an amino acid sequence that is a substrate for matriptase, and an amino acid sequence that is a substrate for an MMP. In some embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for Granzyme B and an amino acid sequence that is a substrate for an MMP. In some embodiments, the cleavable linker comprises a combination of the amino acid sequence LEAD (SEQ ID NO:22), the amino acid sequence RQAR (SEQ ID NO:23), and the amino acid sequence PAGL (SEQ ID NO:24).

[0259] The cleavable linker can include known linkers. Examples of cleavable linkers are described in Be'liveau et al. (2009) FEBS Journal, 276; published U.S. application numbers US20160194399; US20150079088; US20170204139; US20160289324; US20160122425; US20150087810; US20170081397; U.S. Patent No. 9,644,016.

[0260] In some embodiments, the cleavable linker is TIFF0007798942000033.tif94151

[0261] 4. Antigen-binding domain: The multispecific polypeptide constructs of the present disclosure comprise at least one antigen-binding domain, e.g., at least a first antigen-binding domain and a second antigen-binding domain. In some aspects, the antigen-binding domain, or each of the antigen-binding domains, is independently selected from an antibody or antigen-binding fragment, a natural cognate binding partner, anticalin (a modified lipocalin), phapin, fynomer, centirin (a modified fibronectin III domain), a cystine-knot domain, affilin, affibody, or a modified CH3 domain. In some embodiments, the natural cognate binding partner comprises the extracellular domain of the native cognate binding partner of the TAA or a binding fragment thereof, or a variant thereof that exhibits binding activity with the TAA.

[0262] 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, comprises one or more copies of an antibody or antigen-binding fragment thereof. In some embodiments, the antigen-binding domain, or each of the antigen-binding domains, such as the first antigen-binding domain and the second antigen-binding domain, independently, comprises one or more copies of an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, an scFv, an scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody ... H H, V NAR , Modified V H domain, or modified V K Contains domain. V HH can be generated from natural camelid heavy chain-only antibodies, genetically modified rodents producing heavy chain-only antibodies, or from naive / synthetic camelid or humanized camelid single domain antibody libraries. NAR can be generated from cartilaginous fish heavy chain-only antibodies. Various methods, including interface engineering and specific germline family selection, have been used to generate V-heterodimeric antibodies. H Domains and V K In some embodiments, the antigen-binding domain of a 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 or scFv. In some embodiments, the antigen-binding domain of a 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 binding domain as a single-domain antibody (sdAb).

[0263] In some embodiments, the antigen binding domain, or each of the antigen binding domains independently, is or comprises the extracellular domain of the native cognate binding partner of the TAA, or a binding fragment thereof, or a variant thereof that exhibits binding activity with the TAA.

[0264] In some embodiments, the antigen binding domain, or each of the antigen binding domains, such as the first and second antigen binding domains, independently bind to the same antigen. In some embodiments, the antigen binding domain, or each of the antigen binding domains, such as the first and second antigen binding domains, independently bind to different antigens. In some embodiments, the antigen binding domain, or each of the antigen binding domains, such as the first and second antigen binding domains, independently bind to the same tumor-associated antigen (TAA). In some embodiments, the antigen binding domain, or each of the antigen binding domains, such as the first and second antigen binding domains, independently bind to different TAAs. In some embodiments, the antigen binding domain, or each of the antigen binding domains, such as the first and second antigen binding domains, independently bind to different epitopes of the same TAA. In some embodiments, the antigen binding domain, or each of the antigen binding domains, such as the first and second antigen binding domains, independently bind to the same epitope of the same TAA.

[0265] In some embodiments, the antigen binding domain that binds to the TAA, or each of the antigen binding domains independently, provides monovalent, bivalent, trivalent, or tetravalent binding to the TAA.

[0266] In some embodiments, the TAA is 1-92-LFA-3, 5T4, alpha4 integrin, alphaV integrin, alpha4beta1 integrin, alpha4beta7 integrin, AGR2, anti-Lewis Y, apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD 20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD 64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD16 6, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), claudin 3, claudin 4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EG FR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL2 1, IL23, IL23R, IL27 / IL27R(wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin 16(MUC16, CA-125), Na / K ATPase, NGF, nicastrin, Notch receptor, Notch1, Notch2, Notch3, Notch4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine-1-phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, 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.

[0267] In some embodiments, at least one antigen-binding domain, or each antigen-binding domain independently, binds to the tumor-associated antigen (TAA), folate receptor alpha (FRα). For example, the antigen-binding domain contains a binding domain as an sdAb that binds to FRα. Exemplary FRα-binding sdAbs are shown in SEQ ID NOs: 120, 121, and 122.

[0268] In some embodiments, at least one antigen-binding domain, or each antigen-binding domain independently, binds to cMET, a tumor-associated antigen (TAA). For example, the antigen-binding domain contains a binding domain as an sdAb that binds to cMET. An exemplary cMET-binding sdAb is shown in SEQ ID NO: 123 (U.S. Patent No. 9,346,884).

[0269] In some embodiments, at least one antigen-binding domain, or each antigen-binding domain independently, binds to the tumor-associated antigen (TAA) B7H3. For example, the antigen-binding domain contains the binding domain as an scFv that binds to B7H3. An exemplary B7H3-binding scFv is set forth in SEQ ID NO: 124. In some embodiments, the antigen-binding domain is or contains a Fab antibody fragment comprising a VH-CH1 (Fd) and an LC. An exemplary B7H3 Fd is set forth in SEQ ID NO: 127, and an exemplary B7H3 LC is set forth in SEQ ID NO: 128 (PCT Publication No. WO2017 / 030926).

[0270] In some embodiments, at least one antigen-binding domain, or each antigen-binding domain independently, binds to the tumor-associated antigen (TAA), CD20. For example, the antigen-binding domain contains a binding domain as an scFv that binds to CD20. Exemplary CD20-binding scFvs are set forth in SEQ ID NOs: 125, 189, and 190 (U.S. Publication No. US2005 / 0123546).

[0271] In some embodiments, at least one antigen-binding domain, or each antigen-binding domain independently, binds to DLL3, a tumor-associated antigen (TAA). For example, the antigen-binding domain contains a binding domain as an scFv that binds to DLL3. Exemplary DLL3-binding scFvs are set forth in SEQ ID NOs: 126 and 189 (U.S. Publication No. US2017 / 0037130). In some embodiments, the antigen-binding domain is or contains a Fab antibody fragment comprising an Fd and an LC that binds to DLL3. An exemplary DLL3 Fd is set forth in SEQ ID NO: 133, and an exemplary DLL3 LC is set forth in SEQ ID NO: 134 (U.S. Publication No. US8,044,178).

[0272] In some embodiments, at least one antigen binding domain, or each antigen binding domain independently, binds to the tumor-associated antigen (TAA), 5T4. An exemplary 5T4 Fd is set forth in SEQ ID NO: 129, and an exemplary 5T4 LC is set forth in SEQ ID NO: 130. In some embodiments, the antibody binding domain comprises a VH-CH1(Fd) or VL-CL set forth in SEQ ID NOs: 167 and 168 (U.S. Patent No. 8,044,178).

[0273] In some embodiments, at least one antigen-binding domain, or each antigen-binding domain independently, binds to the tumor-associated antigen (TAA) gpNMB. In some embodiments, the antigen-binding domain is or contains a Fab fragment comprising an Fd chain and an LC chain. An exemplary gpNMB Fd is set forth in SEQ ID NO:131, and an exemplary gpNMB LC is set forth in SEQ ID NO:132.

[0274] In some embodiments, the antigen-binding domain is linked to the Fc region and / or the CD3-binding region directly or indirectly via a linker. In some embodiments, the linkage is via a linker. In some embodiments, the linker is a connecting peptide (LP), which may include a flexible or non-flexible linker as described in Section II.3, although generally the peptide linking the antigen-binding domains is not a cleavable linker.

[0275] In some embodiments, the multispecific polypeptide construct comprises a first connecting peptide (LP1) between the first antigen-binding domain and the Fc region. In some embodiments, the multispecific polypeptide construct comprises a second connecting peptide (LP2) between the CD3-binding region and the second antigen-binding domain. In some embodiments, the multispecific polypeptide construct comprises a first connecting peptide (LP1) between the first antigen-binding domain and the Fc region and a second connecting peptide (LP2) between the CD3-binding region and the second antigen-binding domain. In some aspects, the multispecific polypeptide construct has the following structural arrangement from N-terminus to C-terminus: first antigen-binding domain - LP1 - Fc region - linker - CD3-binding region - LP2 - second antigen-binding domain. In some embodiments, the two connecting peptides are not identical to each other.

[0276] In some embodiments, LP1 or LP2 are independently peptides of about 1 to 20 amino acids in length. In some embodiments, LP1 or LP2 are independently peptides that are or include a Gly-Ser linker set forth in SEQ ID NOs: 10-13, 119, 135, 147, 149, or GGS.

[0277] III. Pharmaceutical Compositions Compositions of any of the provided multispecific polypeptide constructs are provided herein. It will be understood that administration of therapeutic entities according to the present disclosure will be administered with appropriate carriers, excipients, and other agents incorporated into the formulation to provide improved entry, delivery, tolerance, etc. 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)), particularly Chapter 87 therein by Blaug and Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as Lipofectin™), 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 may be suitable in treatments and therapies according to the present disclosure, provided that the active ingredients in the formulation are not inactivated by the formulation and the formulation is physiologically compatible and acceptable for the route of administration.For additional information regarding formulations, excipients, and carriers well known to pharmacists, see also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. Toxicol Pharmacol. 32(2):210-8 (2000); Wang W. "Lyophilization and development of solid protein pharmaceuticals." Int. J. Pharm. 203(1-2):1-60 (2000); Charman WN "Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts." J Pharm Sci. 89(8):967-78 (2000); Powell et al. "Compendium of excipients for parenteral formulations." PDA J Pharm Sci Technol. 52:238-311 (1998), and citations therein.

[0278] In some embodiments, the multispecific polypeptide constructs, conjugated multispecific polypeptide constructs, and compositions thereof, as well as derivatives, fragments, analogs, and homologs thereof, collectively referred to herein as therapeutic agents, can be incorporated into pharmaceutical compositions suitable for administration. The principles and considerations involved in the preparation of such compositions, as well as guidance in the selection of components, are provided, for example, in Remington's Pharmaceutical Sciences: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0279] Such compositions typically comprise the multispecific polypeptide construct or a conjugate thereof and a pharmaceutically acceptable carrier. When the multispecific polypeptide construct comprises an antibody fragment, the smallest fragment of the antibody that specifically binds to the target protein can be used. For example, based on the variable region sequence of the antibody, a peptide molecule can be designed that retains the antibody's ability to bind to the target protein sequence. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)).

[0280] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in this field, which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmacologically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated.

[0281] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0282] The pharmaceutical compositions of the present disclosure are formulated to be compatible with their intended route of administration. Examples of administration routes include parenteral administration, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium sulfite; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetic acid, citric acid, or phosphate, and an agent for adjusting osmolality such as sodium chloride or dextrose. pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0283] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (water soluble) or dispersions, or sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be appropriate to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0284] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent, optionally with one or a combination of the above-listed ingredients, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution. If the composition is lyophilized, sterilization using this method can be carried out either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. Furthermore, parenteral compositions are generally placed in a container with a sterile access port, such as an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle.

[0285] In some embodiments, the pharmaceutical composition is administered to a subject through any route, including orally, transdermally, by inhalation, intravenously, intraarterially, intramuscularly, directly to a wound site, applied to a surgical site, intraperitoneally, by suppository, subcutaneously, intradermally, transdermally, by spray, intrapleurally, intraventricularly, intraarticularly, intraocularly, or intraspinally.

[0286] Oral compositions generally contain inert diluents or edible carriers.They can be enclosed in gelatin capsules or compressed into tablets.For oral therapeutic administration, active compounds can be incorporated with excipients and used in the form of tablets, lozenges or capsules.Oral compositions can also be prepared using liquid carriers for use as mouthwash, where the compound in the liquid carrier is applied to the mouth, sipped, expectorated or swallowed.Pharmaceutically compatible binders and / or auxiliary materials can be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a flow aid such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavor.

[0287] For administration by inhalation, the multispecific polypeptide constructs are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0288] Systemic administration can also be via transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, for transmucosal administration, surfactants, bile acids, and fusidic acid derivatives.Transmucosal administration can be achieved through the use of nasal sprays or suppositories.For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams generally known in the art.

[0289] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0290] In one embodiment, the therapeutic agent is prepared with a carrier that protects the compound from rapid elimination from the body, such as sustained / controlled release formulations, including implants and microencapsulated delivery systems.Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used.Methods for preparing such formulations will be clear to those skilled in the art.

[0291] For example, therapeutic agents may be entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization, e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions.

[0292] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, such as a filler, binder, coating, preservative, lubricant, flavoring agent, sweetener, coloring agent, solvent, buffer, chelating agent, or stabilizer. Examples of pharmaceutically acceptable fillers include cellulose, dibasic calcium phosphate, calcium carbonate, crystalline cellulose, sucrose, lactose, glucose, mannitol, sorbitol, maltol, pregelatinized starch, corn starch, or potato starch. Examples of pharmaceutically acceptable binders include polyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol, gelatin, sucrose, polyethylene glycol, methylcellulose, or cellulose. Examples of pharmaceutically acceptable coatings include hydroxypropylmethylcellulose (HPMC), shellac, corn protein zein, or gelatin. Examples of pharmaceutically acceptable disintegrants include polyvinylpyrrolidone, carboxymethylcellulose, or sodium starch glycolate. Examples of pharmaceutically acceptable lubricants include polyethylene glycol, magnesium stearate, or stearic acid. Examples of pharmaceutically acceptable preservatives include methylparaben, ethylparaben, propylparaben, benzoic acid, or sorbic acid. Examples of pharmaceutically acceptable sweeteners include sucrose, saccharin, aspartame, or sorbitol. Examples of pharmaceutically acceptable buffers include carbonate, citric acid, gluconic acid, acetic acid, phosphoric acid, or tartaric acid.

[0293] Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, in the form of shaped articles, e.g., films, or microcapsules. In some embodiments, the pharmaceutical composition further comprises an agent for controlled or sustained release of the product, such as injectable microspheres, bioerodible particles, polymeric compounds (polylactic acid, polyglycolic acid), beads, or liposomes. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

[0294] Materials may be commercially obtained from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells containing monoclonal antibodies against viral antigens) may also be used as pharmaceutically acceptable carriers. These may be prepared by methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0295] For ease of administration and dosage uniformity, it is particularly advantageous to prepare oral or parenteral compositions in dosage unit form.Dosage unit form as used herein refers to a physically discrete unit that is suitable as a unit dosage for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce desired therapeutic effect together with required pharmaceutical carrier.The details of dosage unit form of the present disclosure are dictated by and directly depend on the unique characteristics of active compound and the specific therapeutic effect to be achieved, and the inherent limitations in the field of synthesis of this active compound for individual treatment.

[0296] Kits containing the pharmaceutical compositions (or articles of manufacture) described herein are further provided. The pharmaceutical compositions may be included in a container, pack, or dispenser together with instructions for administration. The kits described herein may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for practicing the methods described herein.

[0297] The formulation may contain multiple multispecific polypeptide constructs as needed for the particular indication being treated, e.g., those with complementary activities that do not adversely affect each other. In some embodiments, or in addition, the composition may include an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent. Such molecules are suitably present in combination in amounts effective for the intended purpose.

[0298] In some embodiments, the pharmaceutical composition is administered in a single dose or multiple doses. In some embodiments, the administration is given to a subject once a day, twice a day, three times a day, or four or more times a day. In some embodiments, about one or more doses (e.g., about two or more, about three or more, about four or more, about five or more, about six or more, or about seven or more) are given per week. In some embodiments, multiple doses are given over several days, weeks, months, or years. In some embodiments, a course of treatment is about one or more doses (e.g., about two or more, about three or more, about four or more, about five or more, about seven or more, about ten or more, about fifteen or more, about twenty-five or more, about forty or more, about fifty or more, or about one hundred or more).

[0299] In some embodiments, a pharmaceutical composition is administered to a subject. Generally, the dosage and route of administration of a pharmaceutical composition are determined according to standard pharmaceutical practice based on the size and condition of the subject. For example, a therapeutically effective dose can be initially evaluated in cell culture assays or animal models such as mice, rats, rabbits, dogs, pigs, or monkeys. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes for administration in humans. The exact dosage will be determined taking into account factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active compound or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, the subject's health condition, the subject's age, weight, and sex, the time and frequency of administration, drug combinations, reaction sensitivities, and response to treatment. The optimal dosage and treatment regimen for a particular patient can be easily determined by those skilled in the art of medicine by monitoring the patient for symptoms of disease and adjusting treatment accordingly.

[0300] IV. Methods of Use and Therapeutic Administration Methods and uses of the multispecific polypeptide constructs are provided. Such methods and uses include, for example, therapeutic methods and uses comprising administering a molecule or a composition containing the same to a subject having a disease, condition, or disorder, such as a tumor or cancer. In some embodiments, the molecule and / or composition is administered in an amount effective to achieve treatment of the disease or disorder. Uses include the use of the multispecific polypeptide construct in such methods and treatments, as well as in the preparation of a medicament for carrying out such therapeutic methods. In some embodiments, the method is carried out by administering the multispecific polypeptide construct or a composition comprising the same to a subject having or suspected of having a disease or condition. In some embodiments, the method thereby treats the disease, condition, or disorder in the subject.

[0301] In one embodiment, the multispecific polypeptide constructs of the present disclosure can be used as therapeutic agents. Such agents will generally be utilized to diagnose, prognose, monitor, treat, alleviate, and / or prevent a disease or pathology in a subject. A treatment regimen is implemented by identifying a subject, e.g., a human patient or other mammal, suffering from (or at risk of developing) a disorder using standard methods. The multispecific polypeptide construct is administered to the subject. The multispecific polypeptide construct is administered to the subject and will generally exert its effect by binding to the target.

[0302] In some embodiments, provided herein are methods for modulating an immune response in a subject by administering a therapeutically effective amount of any of the provided multispecific conjugates or pharmaceutical compositions. In some embodiments, the method for modulating an immune response increases or enhances the immune response in the subject. For example, the increased or enhanced response can be an increase in cellular immunity. In some examples, the method increases T cell activity, such as cytolytic T cell (CTL) activity. In some embodiments, the modulated (e.g., increased) immune response is against tumors or cancer.

[0303] Administration of a multispecific polypeptide construct can activate innate immune cells through engagement of FcγRs via the Fc region of the multispecific polypeptide construct. Administration of a multispecific polypeptide construct can induce, stimulate, activate, and / or enhance innate immune cell effector functions, including ADCC, cytokine release, degranulation, and / or ADCP. Administration of a multispecific polypeptide construct can activate T cells after the linker joining the first and second components is cleaved by a protease, thereby allowing the anti-CD3 binding moiety to bind to CD3ε on the T cells. Administration of a multispecific polypeptide construct can induce, stimulate, activate, and / or enhance CD3-mediated T cell activation, cytotoxicity, cytokine release, and / or proliferation.

[0304] In some embodiments, the provided methods are for treating a disease or condition in a subject by administering a therapeutically effective amount of either a provided multispecific conjugate or pharmaceutical composition. In some embodiments, the disease or condition is a tumor or cancer. Generally, alleviating or treating a disease or disorder includes reducing one or more symptoms or medical problems associated with the disease or disorder. For example, in the case of cancer, a therapeutically effective amount of a drug can achieve one or a combination of the following: reducing the number of cancer cells; reducing tumor size; inhibiting (i.e., reducing and / or ceasing to some extent) cancer cell invasion into peripheral organs; inhibiting tumor metastasis; inhibiting tumor growth to some extent; and / or alleviating to some extent one or more symptoms associated with cancer. In some embodiments, the compositions of the present disclosure can be used to prevent the onset or recurrence of a disease or disorder in a subject, e.g., a human or other mammal, such as a non-human primate, a pet (e.g., cat, dog, horse), livestock, laboratory animal, or zoo animal. The terms subject and patient are used interchangeably herein.

[0305] In some embodiments, the pharmaceutical compositions can be used to inhibit the growth of mammalian cancer cells (such as human cancer cells). Methods of treating cancer can include administering an effective amount of any of the pharmaceutical compositions described herein to a subject with cancer. An effective amount of the pharmaceutical composition can be administered to inhibit, stop, or reverse the progression of cancer. Human cancer cells can be treated in vivo or ex vivo. In ex vivo treatment of human patients, tissues or fluids containing cancer cells are treated outside the body, and then the tissues or fluids are reintroduced back into the patient. In some embodiments, cancer is treated in a human patient in vivo by administering a therapeutic composition to the patient.

[0306] Non-limiting examples of diseases include all types of cancer (breast cancer, lung cancer, colorectal cancer, prostate cancer, melanoma, head and neck cancer, pancreatic cancer, etc.), rheumatoid arthritis, Crohn's disease, SLE, cardiovascular disorders, ischemia, etc. For example, indications would include leukemias, including T-cell acute lymphoblastic leukemia (T-ALL), lymphoblastic disorders, including multiple myeloma, and solid tumors, including lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, and breast cancer, including triple-negative breast cancer. For example, indications include bone disease or metastasis in cancer, regardless of primary tumor origin; breast cancer, including, but not limited to, ER / PR+ breast cancer, Her2+ breast cancer, and triple-negative breast cancer; colorectal cancer; endometrial cancer; gastric cancer; glioblastoma; head and neck cancer, such as esophageal cancer; lung cancer, including, but not limited to, non-small cell lung cancer; multiple myeloma; ovarian cancer; pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; kidney cancer, including, but not limited to, renal cell carcinoma; and / or skin cancer, including, but not limited to, squamous cell carcinoma, basal cell carcinoma, or melanoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is cutaneous squamous cell carcinoma. In some embodiments, the cancer is esophageal squamous cell carcinoma. In some embodiments, the cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is lung squamous cell carcinoma.

[0307] A therapeutically effective amount of a multispecific polypeptide construct of the present disclosure generally relates to the amount required to achieve a therapeutic goal. As discussed above, this may be a binding interaction between the multispecific polypeptide construct and its target antigen that, in certain cases, induces, stimulates, activates, and / or enhances FcγR-mediated innate immune cell activation or CD3-mediated T cell activation. The amount that needs to be administered will further depend on the binding affinity of the multispecific polypeptide construct for a particular antigen and the rate at which the administered multispecific polypeptide construct is depleted from the free volume of the subject to which it is administered. A typical range for a therapeutically effective dose of a multispecific polypeptide construct may be, for example, but not limited to, about 0.01 μg / kg body weight to about 10 mg / kg body weight. In some embodiments, a therapeutically effective dose of a multispecific polypeptide construct of the present disclosure may be, for example, but not limited to, about 0.01 mg / kg body weight to about 5-10 mg / kg body weight. A typical dosing frequency may be, for example, twice daily to once weekly.

[0308] The effectiveness of treatment is determined in conjunction with any known method for diagnosing or treating a particular disorder. Methods for screening multispecific polypeptide constructs possessing the desired specificity include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs) and other immunologically mediated techniques known in the art. A variety of means are known for determining whether administration of a provided multispecific polypeptide construct sufficiently modulates immune activity by eliminating, sequestering, or inactivating immune cells that mediate or are capable of mediating an unwanted immune response; inducing, generating, or activating immune cells that mediate or are capable of mediating a protective immune response; changing the physical or functional properties of immune cells; or a combination of these effects.Examples of measurements of modulation of immune activity include examining the presence or absence of immune cell populations (using flow cytometry, immunohistochemistry, histology, electron microscopy, polymerase chain reaction (PCR)); measuring the functional capacity of immune cells, including their ability or resistance to proliferate or divide in response to signals (e.g., T cell proliferation assays and 3H-thymidine binding assays following stimulation with anti-CD3 antibodies, anti-T cell receptor antibodies, anti-CD28 antibodies, calcium ionophore, PMA (phorbol 12-myristate 13-acetate), or antigen-presenting cells loaded with peptide or protein antigens). use of pepscan analysis based on gene uptake; use of B cell proliferation assays; measurement of the ability to kill or lyse other cells (such as in cytotoxic T cell assays); measurement of cytokines, chemokines, cell surface molecules, antibodies, and other products of cells (e.g., by flow cytometry, enzyme-linked immunosorbent assay, Western blot analysis, protein microarray analysis, immunoprecipitation analysis); measurement of biochemical markers of activation of immune cells or signaling pathways within immune cells (e.g., Western blot, and tyrosine, serine, or thymine). immunoprecipitation analysis of onin phosphorylation, polypeptide cleavage, and protein complex formation or dissociation; protein array analysis; DNA transcriptional profiling using DNA arrays or subtractive hybridization; measurement of cell death by apoptosis, necrosis, or other mechanisms (e.g., Annexin V staining, TUNEL assay, gel electrophoresis measuring DNA ladder formation, histology; fluorogenic caspase assay, Western blot analysis of caspase substrates); measurement of genes, proteins, and other molecules produced by immune cells (e.g., Northern blot analysis, polymerase chain reaction, DNA microarray, protein microarray, two-dimensional gel electrophoresis, Western blot analysis, enzyme-linked immunosorbent assay, flow cytometry); and measurement of clinical symptoms or outcomes, such as improvement of autoimmune diseases, neurodegenerative diseases, and other diseases involving self-proteins or self-polypeptides, for example, by measuring relapse rate or disease severity (clinical scores, need for additional treatment, functional status, imaging studies).

[0309] The multispecific polypeptide constructs are also useful in a variety of diagnostic and prophylactic formulations. In one embodiment, the multispecific polypeptide constructs are administered to patients at risk of developing one or more of the above-mentioned disorders. A patient's or organ's predisposition to one or more of the disorders can be determined using genotypic, serological, or biochemical markers.

[0310] In another aspect of the present disclosure, the multispecific polypeptide construct is administered to a human individual diagnosed with a clinical indication associated with one or more of the above disorders. Upon diagnosis, the multispecific polypeptide construct is administered to alleviate or reverse the effects of the clinical indication.

[0311] Combination therapy In some embodiments, the multispecific polypeptide constructs, conjugated multispecific polypeptide constructs, and compositions thereof, collectively referred to herein as therapeutic agents, are administered with one or more additional agents or combinations of additional agents. Suitable additional agents include current pharmaceutical and / or surgical treatments for the intended application. For example, the therapeutic agent may be used with an additional chemotherapeutic or anti-tumor agent. For example, the therapeutic agent and the additional agent are formulated into a single therapeutic composition, and the therapeutic agent and the additional agent are administered simultaneously. In some embodiments, the therapeutic agent and the additional agent are separate from each other, e.g., each formulated into a separate therapeutic composition, and the therapeutic agent and the additional agent are administered simultaneously, or the therapeutic agent and the additional agent are administered at different times during the treatment regimen. For example, the therapeutic agent is administered before the administration of the additional agent, the therapeutic agent is administered after the administration of the additional agent, or the therapeutic agent and the additional agent are administered alternately. As described herein, the therapeutic agent and the additional agent are administered in a single dose or multiple doses. In some embodiments, an additional agent is coupled to or otherwise attached to the therapeutic agent. Suitable additional agents are selected depending on the intended purpose of the application (i.e., killing, preventing cell proliferation, hormone therapy, or gene therapy). Such agents may include, but are not limited to, pharmaceutical agents, toxins, toxin fragments, alkylating agents, enzymes, antibiotics, antimetabolites, antiproliferative agents, hormones, neurotransmitters, DNA, RNA, siRNA, oligonucleotides, antisense RNA, aptamers, diagnostic agents, radiopaque dyes, radioisotopes, fluorogenic compounds, magnetic labels, nanoparticles, marker compounds, lectins, compounds that alter cell membrane permeability, photochemical compounds, small molecules, liposomes, micelles, gene therapy vectors, viral vectors, and the like. Finally, combinations of agents or combinations of agents from different classes may be used.

[0312] In one embodiment, the multispecific polypeptide construct is administered in combination therapy, i.e., combined with other agents, e.g., therapeutic agents useful for treating pathological conditions or disorders such as autoimmune disorders and inflammatory diseases. In this context, the term "combination" means that the agents are given substantially contemporaneously, simultaneously, or sequentially. When given sequentially, the first of the two compounds is still detectable at effective concentrations at the site of treatment at the start of administration of the second compound.

[0313] For example, a combination therapy may include one or more multispecific polypeptide constructs of the present disclosure formulated and / or co-administered with one or more additional therapeutic agents, such as one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxic or cytostatic agents, as described in more detail below. Additionally, one or more multispecific polypeptide constructs described herein may be used in combination with two or more of the therapeutic agents described herein. Such combination therapy may advantageously utilize lower doses of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with various monotherapies.

[0314] In other embodiments, one or more multispecific polypeptide constructs of the present disclosure may be formulated and / or co-administered with one or more anti-inflammatory drugs, immunosuppressants, metabolic inhibitors, or enzyme inhibitors. Non-limiting examples of drugs or inhibitors that may be used in combination with the antibodies described herein include nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, tenidap, naproxen, meloxicam, piroxicam, diclofenac, and indomethacin; sulfasalazine; corticosteroids such as prednisolone; cytokine suppressive anti-inflammatory drugs (CSAIDs); inhibitors of nucleotide biosynthesis, such as inhibitors of purine biosynthesis, folate antagonists (e.g., and inhibitors of pyrimidine biosynthesis, such as one or more of dihydroorotate dehydrogenase (DHODH) inhibitors. Suitable therapeutic agents for use in combination with the antibodies of the present disclosure include NSAIDs, CSAIDs, (DHODH) inhibitors (e.g., leflunomide), and folate antagonists (e.g., methotrexate).

[0315] Examples of additional inhibitors include corticosteroids (oral, inhaled, and local injection); immunosuppressants, e.g., cyclosporine, tacrolimus (FK-506); and mTOR inhibitors, e.g., sirolimus (rapamycin - RAPAMUNE™) or rapamycin derivatives, e.g., soluble rapamycin derivatives (e.g., ester rapamycin derivatives, e.g., CCI-779); agents that interfere with signaling by proinflammatory cytokines such as TNFα or IL-1 (e.g., IRAK, NIK, IKK, p38, or MAP kinase inhibitors); COX2 inhibitors, e.g., celecoxib, rofecoxib, and variants thereof; phosphodiesterase inhibitors, e.g., R973401 (phosphodiesterase type IV inhibitors); phospholipase inhibitors, e.g., inhibitors of cytosolic phospholipase 2 (cPLA2) (e.g., trifluoromethyl ketone analogs); inhibitors of vascular endothelial growth factor or growth factor receptors, e.g., VEGF inhibitors and / or VEGF-R inhibitors; and inhibitors of angiogenesis. Suitable therapeutic agents for use in combination with the antibodies of the disclosure are immunosuppressants, e.g., cyclosporine, tacrolimus (FK-506); mTOR inhibitors, e.g., sirolimus (rapamycin) or rapamycin derivatives, e.g., soluble rapamycin derivatives (e.g., ester rapamycin derivatives, e.g., CCI-779); COX2 inhibitors, e.g., celecoxib and variants thereof; and phospholipase inhibitors, e.g., inhibitors of cytosolic phospholipase 2 (cPLA2), e.g., trifluoromethyl ketone analogs.Additional examples of therapeutic agents that may be combined with the multispecific polypeptide constructs include one or more of 6-mercaptopurine (6-MP); azathioprine sulfasalazine; mesalazine; olsalazine; chloroquine / hydroxychloroquine (PLAQUENIL®); penicillamine; aurothiomalate (intramuscular and oral); azathioprine; colchicine; beta-2 adrenergic receptor agonists (salbutamol, terbutaline, salmeterol); xanthines (theophylline, aminophylline); cromoglycate; nedocromil; ketotifen; ipratropium and oxitropium; mycophenolate mofetil; adenosine agonists; antithrombotic agents; complement inhibitors; and adrenergic agents.

[0316] V. Illustrative Embodiments Aspects provided include: 1. A multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, the first and second components are coupled by a linker, and the Fc region is positioned N-terminal to the CD3 binding region; A multispecific polypeptide construct, wherein one or both of the first and second components comprises an antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0317] 2. The multispecific polypeptide construct of embodiment 1, wherein the CD3 binding region binds to CD3 (CD3ε).

[0318] 3. The multispecific construct of embodiment 1 or embodiment 2, wherein the antigen-binding domain is positioned amino terminal to the Fc region and / or carboxy terminal to the CD3 binding region of the multispecific polypeptide construct.

[0319] 4. The multispecific polypeptide construct of any of embodiments 1-3, wherein the first component comprises a first antigen-binding domain and the second component comprises a second antigen-binding domain, each of the antigen-binding domains binding to a tumor-associated antigen (TAA).

[0320] 5. The multispecific polypeptide construct of embodiment 4, wherein the first antigen-binding domain is positioned amino terminal to the Fc region of the multispecific construct and the second antigen-binding domain is positioned carboxy terminal to the CD3-binding region of the multispecific construct.

[0321] 6. A multispecific polypeptide construct comprising, in order from N-terminus to C-terminus: a first antigen-binding domain that binds to a tumor-associated antigen (TAA); immunoglobulin Fc region; Linker; a CD3-binding region that binds to CD3 (CD3ε); and A second antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0322] 7. A multispecific polypeptide construct comprising, in order from N-terminus to C-terminus: immunoglobulin Fc region; Linker; a CD3-binding region that binds to CD3 (CD3ε); and An antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0323] 8. A multispecific polypeptide construct comprising, in order from N-terminus to C-terminus: an antigen-binding domain that binds to a tumor-associated antigen (TAA); immunoglobulin Fc region; a linker; and CD3 binding region that binds to CD3 (CD3ε).

[0324] 9. The multispecific polypeptide construct of any of aspects 1 to 8, wherein the Fc region is a homodimeric Fc region.

[0325] 10. The multispecific polypeptide construct of any of aspects 1-9, wherein the Fc region is the Fc region of human IgG1, human IgG2, human IgG3, or human IgG4, or an immunologically active fragment thereof.

[0326] 11. The multispecific polypeptide construct of any of aspects 1 to 10, wherein the Fc region comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, or a sequence of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:1.

[0327] 12. the Fc region comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2 or a sequence of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2; the Fc region comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4, or a sequence of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:4; or the Fc region comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:5 or a sequence of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:5; 11. The multispecific polypeptide construct of any of embodiments 1 to 10.

[0328] 13. The multispecific polypeptide construct of any of embodiments 1 to 6, 9, and 12, wherein the Fc region is a heterodimeric Fc region.

[0329] 14. The multispecific polypeptide construct of embodiment 13, wherein one or both Fc polypeptides of the heterodimeric Fc region comprise at least one modification to induce heterodimerization compared to the polypeptide of the homodimeric Fc region, and optionally compared to the Fc polypeptide set forth in SEQ ID NO:1, or an immunologically active fragment thereof.

[0330] 15. The multispecific polypeptide construct of embodiment 14, wherein each of the Fc polypeptides of the heterodimeric Fc independently comprises at least one amino acid modification.

[0331] 16. The multispecific polypeptide construct of embodiment 15, wherein each of the Fc polypeptides of the heterodimeric Fc comprises a knob-into-hole modification or comprises a charge mutation to increase electrostatic complementarity of the polypeptides.

[0332] 17. The multispecific polypeptide construct of embodiment 16, wherein the amino acid modifications are knobs-into-holes modifications.

[0333] 18. The multispecific fusion polypeptide of any of embodiments 13-17, wherein a first Fc polypeptide of said heterodimeric Fc comprises a modification selected from Thr366Ser, Leu368Ala, Tyr407Val, and a combination thereof, and wherein a second Fc polypeptide of said heterodimeric Fc comprises the modification T366W.

[0334] 19. The multispecific fusion polypeptide of embodiment 18, wherein the first and second Fc polypeptides further comprise a modification of a non-cysteine ​​residue to a cysteine ​​residue, wherein the modification in the first polypeptide is at one of positions Ser354 and Y349, and the modification in the second Fc polypeptide is at the other of positions Ser354 and Y349.

[0335] 20. The multispecific polypeptide construct of embodiment 16, wherein the amino acid modifications are charge mutations to increase electrostatic complementarity of the polypeptide.

[0336] 21. The multispecific polypeptide construct of any of embodiments 13-16 and 20, wherein the first and / or second Fc polypeptide, or the first and second Fc polypeptide, each comprises a modification at a complementary position, wherein the modification is a substitution of an amino acid having an opposite charge to the complementary amino acid of the other polypeptide.

[0337] 22. The multispecific polypeptide construct of any of embodiments 14 to 21, wherein one of the first or second Fc polypeptides of the heterodimeric Fc further comprises a modification at residue Ile253.

[0338] 23. The multispecific polypeptide construct of embodiment 22, wherein the modification is Ile253Arg.

[0339] 24. The multispecific polypeptide construct of any of embodiments 14 to 23, wherein one of the first or second Fc polypeptides of the heterodimeric Fc further comprises a modification at residue His435.

[0340] 25. The multispecific polypeptide construct of embodiment 24, wherein the modification is His435Arg.

[0341] 26. The multispecific polypeptide construct of any of aspects 1-25, wherein the Fc region comprises a polypeptide lacking Lys447.

[0342] 27. The multispecific polypeptide construct of any of embodiments 1-26, wherein the Fc region comprises a polypeptide comprising at least one modification to enhance FcRn binding.

[0343] 28. The multispecific fusion polypeptide of embodiment 27, wherein the modification is at a position selected from the group consisting of Met252, Ser254, Thr256, Met428, Asn434, and combinations thereof.

[0344] 29. The multispecific fusion polypeptide of embodiment 28, wherein the modification is at a position selected from the group consisting of Met252Y, Ser254T, Thr256E, Met428L, Met428V, Asn434S, and a combination thereof.

[0345] 30. The multispecific fusion polypeptide of embodiment 28, wherein the modifications are at positions Met252 and Met428.

[0346] 31. The multispecific fusion polypeptide of embodiment 30, wherein the modifications are Met252Y and Met428L.

[0347] 32. The multispecific fusion polypeptide of embodiment 30, wherein the modifications are Met252Y and Met428V.

[0348] 33. The multispecific polypeptide construct of any of embodi...

Claims

1. 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 first and second components are coupled by a linker, and the Fc region is positioned amino-terminal to the 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), wherein the VH of the dsFv is linked to one polypeptide of a heterodimeric Fc region and the VL of the dsFv is linked to the other polypeptide of the heterodimeric Fc region; and one or both of the first and second components comprises an antigen-binding domain that binds to a tumor-associated antigen (TAA), the antigen-binding domain being a single-chain variable fragment (scFv); the CD3 binding region is incapable or substantially incapable of binding to or engaging with cell surface CD3 unless the antigen binding domain is bound to its TAA; Multispecific polypeptide constructs.

2. 2. The multispecific polypeptide construct of claim 1, wherein the linker is a polypeptide linker.

3. 3. The multispecific polypeptide construct of claim 1 or 2, wherein the linker is a cleavable linker.

4. A multispecific polypeptide construct described in any one of claims 1 to 3, wherein at least one antigen-binding domain is positioned amino-terminal to the Fc region and / or carboxy-terminal to the CD3-binding region.

5. A multispecific polypeptide construct described in any one of claims 1 to 4, wherein the first component comprises a first antigen-binding domain and the second component comprises a second antigen-binding domain, each of the antigen-binding domains binding to a tumor-associated antigen (TAA).

6. A multispecific polypeptide construct as described in claim 5, wherein the first antigen-binding domain is positioned amino-terminal to the Fc region and the second antigen-binding domain is positioned carboxy-terminal to the CD3-binding region.

7. 7. The multispecific polypeptide construct of any one of claims 3 to 6, wherein the cleavable linker is a polypeptide that functions as a substrate for a protease.

8. 8. The multispecific polypeptide construct of claim 7, wherein the protease is selected from the group consisting of matriptase, matrix metalloproteinase (MMP), granzyme B, and combinations thereof.

9. 9. The multispecific polypeptide construct of any one of claims 1 to 8, wherein the CD3 binding region binds to CD3 (CD3ε).

10. Anti-CD3 dsFv, a VH having the amino acid sequence of SEQ ID NO: 44 or a sequence exhibiting at least 90% sequence identity with SEQ ID NO: 44; and VL having the amino acid sequence of SEQ ID NO: 72 or a sequence showing at least 90% sequence identity with SEQ ID NO: 72 10. The multispecific polypeptide construct of any one of claims 1 to 9, comprising:

11. 11. The multispecific polypeptide construct of any one of claims 1 to 10, wherein the Fc region is the Fc region of human IgG1, human IgG2, human IgG3, or human IgG4, or an immunologically active fragment thereof.

12. 12. The multispecific polypeptide construct of any one of claims 1 to 11, wherein one or both Fc polypeptides of the heterodimeric Fc region comprise at least one amino acid modification to induce heterodimerization compared to the polypeptide of the homodimeric Fc region.

13. 13. The multispecific polypeptide construct of any one of claims 1 to 12, wherein each of the Fc polypeptides of the heterodimeric Fc region comprises a knobs-into-holes modification or a charge mutation to increase electrostatic complementarity of the polypeptides.

14. 14. The multispecific polypeptide construct of any one of claims 1 to 13, wherein one Fc polypeptide of the heterodimeric Fc region comprises at least one amino acid modification to promote heterodimerization compared to the Fc polypeptide set forth in SEQ ID NO: 1 or an immunologically active fragment thereof.

15. 15. The multispecific polypeptide construct of any one of claims 1 to 14, wherein the Fc region comprises a polypeptide comprising at least one amino acid modification to enhance FcRn binding.

16. The Fc region Polypeptides containing at least one amino acid modification that reduces effector function or reduces binding to Fcγ receptors or C1q 16. The multispecific polypeptide construct of any one of claims 1 to 15, comprising:

17. The antigen-binding domain, or each antigen-binding domain 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, CD3, CD6, CD9, and CD11. a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52 , CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD13 8, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), claudin 3, claudin 4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB , EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE , IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL 23, IL23R, IL27 / IL27R (wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin 16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch1, Notch2, Notch3, Notch4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine-1-phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR 17. The multispecific polypeptide construct of any one of claims 1 to 16, wherein the multispecific polypeptide construct binds to a tumor antigen selected from: 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.

18. A multispecific polypeptide construct described in any one of claims 1 to 17, 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.

19. A multispecific polypeptide construct described in any one of claims 1 to 17, 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.

20. A multispecific polypeptide construct according to any one of claims 1 to 19, further comprising a drug.

21. The multispecific polypeptide construct of claim 20, wherein the agent is a therapeutic agent, an anti-tumor agent, a toxin or fragment thereof, a detectable moiety, or a diagnostic agent.

22. A polynucleotide encoding the multispecific polypeptide construct of any one of claims 1 to 21.

23. 22. A polynucleotide comprising a first nucleic acid sequence encoding a first polypeptide of a multispecific polypeptide construct according to any one of claims 1 to 21, and a second nucleic acid sequence encoding a second polypeptide of said multispecific polypeptide construct, the first and second nucleic acid sequences are separated by an internal ribosome entry site (IRES) or by a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; Polynucleotide.

24. 24. A vector comprising one or more polynucleotides of claim 22 or 23.

25. 25. A cell comprising the polynucleotide of claim 22 or 23 or the vector of claim 24.

26. introducing the polynucleotide of claim 22 or 23 or the vector of claim 24 into a cell; and culturing said cells under conditions to produce a multispecific polypeptide construct.

1. A method for producing a multispecific polypeptide construct, comprising:

27. 22. A pharmaceutical composition comprising the multispecific polypeptide construct of any one of claims 1 to 21 and a pharmaceutically acceptable carrier.

28. 28. An ex vivo or in vitro method for stimulating or inducing an immune response comprising contacting target cells and T cells with the multispecific polypeptide construct of any one of claims 1 to 21 or the pharmaceutical composition of claim 27, wherein the target cells express a tumor-associated antigen recognized by the multispecific polypeptide construct, and wherein the contacting is performed ex vivo or in vitro.

29. 28. The pharmaceutical composition of claim 27, for stimulating or inducing an immune response in a subject or for treating a disease or condition in a subject.

30. A pharmaceutical composition for stimulating or inducing an immune response in a subject or for treating a disease or condition in a subject, comprising a multispecific polypeptide construct described in any one of claims 1 to 21.

31. 30. Use of the multispecific polypeptide construct of any one of claims 1 to 21 or the pharmaceutical composition of claim 29 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.

32. A pharmaceutical composition as described in claim 29 or 30 for stimulating or inducing an immune response in a subject, wherein the immune response against a tumor or cancer is increased.

33. 32. The use of claim 31, wherein the medicament is for stimulating or inducing an immune response in a subject, and the immune response against the tumor or cancer is increased.

34. 30. The pharmaceutical composition of claim 29 for treating a disease or condition in a subject, wherein the disease or condition is a tumor or cancer.

35. 32. The use according to claim 31, wherein the medicament is for treating a disease or condition in a subject, wherein the disease or condition is a tumor or cancer.

Images