Multispecific polypeptide constructs with restricted CD3 binding and methods of use thereof

Abstract

The present invention generally relates to multispecific polypeptides with restricted CD3 binding. In some embodiments, the multispecific polypeptide contains a cleavable linker that upon cleavage produces dual effector functions. Also provided are methods of preparing such multispecific polypeptides and methods of using such multispecific polypeptides in a variety of therapeutic, diagnostic, and prophylactic indications.

Description

[0001] This application claims priority to U.S. Provisional Application No. 62 / 484,217, filed Apr. 11, 2017, entitled “Multispecific Polypeptide Constructs with Limited CD3 Binding and Methods of Use Thereof,” the entire content of which is incorporated herein by reference.

[0002] The Sequence Listing is incorporated by reference

[0003] This invention is filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 744952000141SeqList.TXT, created on Apr. 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. FIELD OF THE INVENTION

[0004] The present invention generally relates to multispecific polypeptides with limited CD3 binding. In some embodiments, the multispecific polypeptide contains a cleavable linker that, upon cleavage, results in dual effector functions. Also provided are methods of making such multispecific polypeptides and methods of using such multispecific polypeptides in a variety of therapeutic, diagnostic, and prophylactic indications. BACKGROUND OF THE INVENTION

[0005] Therapeutic antibodies that result in target cell depletion typically rely on effector functions mediated via interactions with Fc-γ-receptors (FcγR) and complement proteins. Effector cells expressing FcγR are primarily those of the innate immune system. T cells are not the direct effector cells involved in antibody-mediated target cell depletion.

[0006] The CD3 (cluster of differentiation 3) T cell co-receptor is a multimeric protein composed of four different polypeptide chains, called the ε, γ, δ, and ζ chains. The CD3 complex functions as the signaling module of the T cell receptor that non-covalently associates with the antigen-binding α / β chains of the T cell receptor.

[0007] Since direct engagement of CD3 leads to T cell activation, it is a desirable target for various therapeutic and / or diagnostic indications. Accordingly, there is a need for antibodies and therapeutic agents that target the CD3 / TCR pathway. SUMMARY OF THE INVENTION

[0008] The present invention provides multispecific polypeptide constructs that display restricted CD3 binding. This is because, in some embodiments, the multispecific polypeptide construct consists 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, wherein the Fc region is located at the N-terminus of the CD3 binding region; and one or both of the first and second components comprise an antigen-binding domain that binds to a tumor-associated antigen (TAA). In some embodiments, the multispecific polypeptide construct in an inactive state consists of a first component and a second component, wherein the first and second components are operably linked, wherein each of the first and second components comprises an antigen-binding domain that binds to a tumor-associated antigen (TAA), wherein the first component comprises an Fc region, wherein the second component comprises a CD3 binding region, and wherein the first and second components are coupled by a cleavable linker. In some embodiments, the CD3 binding region binds to CD3 (CD3ε).

[0009] In some embodiments, the antigen-binding domain is located at the amino terminus relative to the Fc region of the multispecific polypeptide construct and / or at the carboxyl terminus relative to the CD3 binding region. In some embodiments, the first component comprises a first antigen-binding domain and the second component comprises a second antigen-binding domain, wherein each of the antigen-binding domains binds to a tumor-associated antigen (TAA). In some cases, the first antigen-binding domain is located at the amino terminus of the multispecific construct and the second antigen-binding domain is located at the carboxyl terminus of the multispecific construct. In some embodiments, the first antigen-binding domain is located at the amino terminus relative to the Fc region of the multispecific polypeptide construct and / or at the carboxyl terminus relative to the CD3 binding region.

[0010] Multispecific polypeptide constructs are provided herein, wherein the multispecific construct sequentially comprises, from the N-terminus to the C-terminus: a first antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker; a CD3 binding region that binds to CD3 (CD3ε); and a second antigen-binding domain that binds to a tumor-associated antigen (TAA). Multispecific polypeptide constructs are also provided, wherein the multispecific construct sequentially comprises, from the N-terminus to the C-terminus: an immunoglobulin Fc region; a linker; a CD3 binding region that binds to CD3 (CD3ε); and an antigen-binding domain that binds to a tumor-associated antigen (TAA). Multispecific polypeptide constructs are provided, wherein the multispecific construct sequentially comprises, from the N-terminus to the C-terminus: an antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker; and a CD3 binding region that binds to CD3 (CD3ε).

[0011] The present invention provides multispecific polypeptide constructs that bind 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 that includes one or more copies of an antigen-binding domain that binds an antigen conjugated to an immunoglobulin Fc region; a second component that includes one or more copies of at least one CD3-binding domain (referred to herein as an anti-CD3 binding domain or CD3-binding region, which may be used interchangeably herein); and a linker that couples the first component to the second component, such as a cleavable linker.

[0012] Positioning the Fc region at the N-terminus of the CD3-binding region reduces or prevents the ability of the CD3-binding region to bind 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 and not permitted to bind to CD3 unless the antigen-binding domain binds to its cognate antigen. This is advantageous as it prevents systemic binding of the CD3-binding region to T cells and localizes it to the site of antigen expression. This is beneficial as it eliminates the major binding groove of peripheral T cells, thereby allowing for more favorable distribution and localization at the site of antigen expression, such as tumor cells or the tumor microenvironment. In some cases, the binding and / or engagement of CD3 can be amplified or increased by incorporating a cleavable linker that couples Component 1 to Component 2, whereupon cleavage of the cleavable linker (e.g., by proteolytic cleavage) results in increased binding by the CD3-binding region.

[0013] In the inactive, i.e., uncleaved state, the first and second components of the multispecific polypeptide construct are operably linked to CD3 and do not bind to or otherwise engage CD3 unless the antigen-binding domain binds to its cognate antigen. In some embodiments, the uncleaved multispecific polypeptide construct can interact with FcγRs and mediate innate immune effector functions, such as antibody-dependent cell cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP). In some embodiments, the uncleaved multispecific polypeptide construct can interact with complement proteins (i.e., C1q) and mediate complement-dependent cytotoxicity.

[0014] The multispecific polypeptide constructs of the present disclosure typically have more than one antigen-binding domain. In the provided aspects in which the multispecific polypeptide construct contains a cleavable linker, once the linker that couples the first and second components is cleaved by, for example, a protease, each component maintains 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.

[0015] The 1st component and the 2nd component are physically separated by cleavage within the cleavable linker, e.g., by proteolysis. Each of such components has a therapeutic use, but depends on different effector cells. The 1st component contains at least one antigen-binding domain and an Fc region. In some embodiments, the 1st component can initiate innate immune effector functions, such as ADCC, cytokine release, degranulation, and / or phagocytosis. The 2nd component contains at least a CD3-binding region and an antigen-binding domain, and the former can bind to CD3 (when separated from the 1st component). The 2nd component can form an immune synapse between antigen-expressing cells and T cells. This co-engagement mediates antigen-dependent T cell activation, cytotoxicity, cytokine release, degranulation, and proliferation. In the cleaved / activated state, the 2nd component is not operably linked to the Fc region of the 1st component and thus the 2nd component cannot interact with FcRn, and has an enhanced serum clearance rate if localized at the site of cells without antigen expression. This is advantageous as it limits the systemic exposure of the activated anti-CD3 binding domain and directly concentrates it into antigen-expressing tissues (e.g., tumor cells or tumor microenvironment).

[0016] In some embodiments, the multispecific polypeptide is in an inactive state, i.e., an uncleaved state, and the binding of the CD3-binding region to CD3 is inhibited or significantly reduced when the multispecific polypeptide construct is in an 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., a cleaved state, and the second component binds to the ε chain of CD3 (CD3ε) and a tumor-associated antigen (TAA).

[0017] In some aspects, the antigen-binding domain, or each antigen-binding domain independently, is selected from an antibody or an antigen-binding fragment, a natural cognate binding partner, an Anticalin (engineered lipocalin), a Darpin, a Fynomer, a Centyrin (engineered fibronectin type III domain), a cysteine-stub domain, an Affilin, an Affibody, or an engineered CH3 domain. In some embodiments, the natural cognate binding partner comprises an extracellular domain of the natural cognate binding partner of a TAA or a binding fragment thereof, or a variant thereof that exhibits binding activity to the TAA.

[0018] In some aspects, the antigen-binding domain, or each antigen-binding domain independently, comprises an extracellular domain of the natural cognate binding partner of a TAA or a binding fragment thereof, or a variant thereof that exhibits binding activity to the TAA.

[0019] In some embodiments, the first component comprises one or more copies of an antigen-binding domain. In some embodiments, the first component comprises 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 on the same TAA. In some cases, at least two antigen-binding domains of the first component bind to the same epitope of the same TAA. In some embodiments, at least two antigen-binding domains of the first component bind to different TAAs.

[0020] In some embodiments, the antigen-binding domain of the first component, which in some cases is the first antigen-binding domain, comprises one or more copies of an antibody or an 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 an antigen-binding fragment thereof selected from the group consisting of: Fab fragment, F(ab′)2 fragment, Fv fragment, scFv, scAb, dAb, single-domain heavy-chain antibody, and single-domain light-chain antibody. In some embodiments, the first antigen-binding domain comprises one or more single-domain antibody (sdAb) fragments (e.g., V H H, V NAR , engineered V H or V K domains) of one or more copies. V H H can be generated from camelid heavy-chain-only antibodies. V NAR can be generated from chondrichthyan heavy-chain-only antibodies. Various methods have been implemented to generate monomeric sdAbs from the common heterodimeric V H and V K domains, including surface engineering and selection of specific germline families.

[0021] In some embodiments, the antigen-binding domain of the first component, such as the first antigen-binding domain, binds 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, α-4 integrin, α-V integrin, α4β1 integrin, α4β7 integrin, AGR2, Anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9 (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), Claudin-3, Claudin-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK 1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor α (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GP IIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R (wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS 1-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, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidyl-serine, 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.

[0022] In some embodiments, the Fc region is a homodimeric Fc region. In some embodiments, the Fc region is a heterodimeric Fc region.

[0023] In some embodiments, the immunoglobulin Fc region of the first component is an IgG isotype selected from the group consisting of: IgG1 isotype, IgG2 isotype, IgG3 isotype, and IgG4 subclass. In some embodiments, the Fc region is the Fc region of human IgG1, human IgG2, human IgG3, or human IgG4, or an immunologically active fragment thereof. In some embodiments, the Fc region comprises a polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1. In some cases, the Fc region comprises a polypeptide containing the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 2. In some of any such embodiments, the Fc region comprises a polypeptide containing the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 4. In some embodiments, the Fc region comprises a polypeptide containing the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 5.

[0024] In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6. In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6.

[0025] 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, which comprises 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, which comprises 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 cell cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), induce heterodimerization, prevent dimerization, stabilize homodimerization on the CH3:CH3 surface, and combinations thereof.

[0026] In some embodiments, the Fc is a heterodimeric Fc. In some cases, compared to the polypeptide of the homodimeric Fc region, optionally compared to the Fc polypeptide shown in SEQ ID NO: 1 or its immunologically active fragment, one or both Fc polypeptides of the heterodimeric Fc region comprise at least one modification to induce heterodimerization. In some embodiments, each of the Fc polypeptides of the heterodimeric Fc independently comprises at least one amino acid modification. In some cases, each of the Fc polypeptides of the heterodimeric Fc comprises a knob-into-hole modification or comprises a charge mutation to increase the electrostatic complementarity of the polypeptide. In some embodiments, the amino acid modification is a knob-into-hole modification.

[0027] In some embodiments, the first Fc polypeptide of the heterodimeric Fc comprises a modification selected from Thr366Ser, Leu368Ala, Tyr407Val, and combinations thereof, and the second Fc polypeptide of the heterodimeric Fc comprises the modification T366W. In some cases, the first and second Fc polypeptides further comprise a modification from a non-cysteine residue to a cysteine residue, wherein the modification of the first polypeptide is one of positions Ser354 and Y349, and the modification of the second Fc polypeptide is the other of positions Ser354 and Y349.

[0028] In some embodiments, the amino acid modification is a charge mutation to increase the electrostatic complementarity of the polypeptide. In some embodiments, the first and / or second Fc polypeptide comprises a modification at a complementary position, wherein the modification is replacement of a complementary amino acid of another polypeptide with an amino acid having an opposite charge. In some embodiments, the first or second polypeptide comprises a modification at a complementary position, wherein the modification is replacement of a complementary amino acid of another polypeptide with an amino acid having an opposite charge. In some embodiments, at least the first or second Fc polypeptide each comprises a modification at a complementary position, wherein the modification is replacement of a complementary amino acid of another polypeptide with an amino acid having an opposite charge. In some embodiments, the first and second Fc polypeptides each comprise a modification at a complementary position, wherein the modification is replacement of a complementary amino acid of another polypeptide with an amino acid having an opposite charge.

[0029] In some embodiments, one of the first or second Fc polypeptides of the heterodimeric Fc further comprises a modification of 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 of residue His435. In some cases, the modification is His435Arg. In some embodiments, the Fc region comprises a polypeptide lacking Lys447.

[0030] In some embodiments, the modification within the Fc region reduces binding to the Fc-receptor-γ receptor but has a minimal effect on binding to the neonatal Fc receptor (FcRn). In some embodiments, the mutated 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).

[0031] In some embodiments, the Fc region comprises a polypeptide that comprises at least one modification to enhance FcRn binding. In some embodiments, the modification is at a position selected from the group consisting of: Met252, Ser254, Thr256, Met428, Asn434, and combinations thereof. In some cases, the modification is at a position selected from the group consisting of: Met252Y, Ser254T, Thr256E, Met428L, Met428V, Asn434S, and combinations thereof. In some particular 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.

[0032] In some embodiments, the first polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NO: 82, 86, 94 or 96, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NO: 83, 87, 90, 92, 98 or 100.

[0033] In some embodiments, the Fc region comprises a polypeptide containing at least one amino acid modification that reduces effector function and / or reduces binding to an effector molecule selected from Fcγ receptors or C1q. In some examples, one or more of the amino acid modifications are deletions of one or more of Glu233, Leu234 or Leu235. In some aspects, the first polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NO: 84, 88, 95 or 97, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NO: 85, 89, 91, 93, 99 or 101.

[0034] In some embodiments, the Fc region comprises a polypeptide containing at least one modification to enhance FcγR binding. In some cases, the modification is a modification of 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 embodiments, the Fc region has no fucose content or has a reduced fucose content.

[0035] In some embodiments, the CD3 binding region is an anti-CD3 antibody or antigen-binding fragment. In some embodiments, the anti-CD3 antibody or antigen-binding fragment comprises a variable heavy chain region (VH) and a variable light chain region (VL). In some of any such embodiments, the CD3 binding region is monovalent.

[0036] In some embodiments, the anti-CD3 antibody or antigen-binding fragment is not a single-chain antibody, optionally not a single-chain variable fragment (scFv). In some embodiments, the Fc is a heterodimeric Fc, and the VH and VL of the anti-CD3 antibody or antigen-binding fragment are linked to opposite polypeptides of the heterodimeric Fc. In some embodiments, the CD3 binding region cannot or substantially cannot bind or engage CD3 unless at least one of the antigen-binding domains binds to its TAA. In some aspects, the CD3 binding region cannot or substantially cannot bind or engage CD3 unless at least two of the antigen-binding domains bind to their TAAs.

[0037] In some embodiments, the multispecific polypeptide construct contains a linker, which is a polypeptide linker. In some embodiments, the linker is a polypeptide having a length of at most 25 amino acids. In some cases, the linker is a polypeptide of or about 2 to 24 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to 24 amino acids, 6 to 20 amino acids, 6 to 18 amino acids, 6 to 14 amino acids, 6 to 12 amino acids, 6 to 10 amino acids, 6 to 8 amino acids, 8 to 24 amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14 amino acids, 8 to 12 amino acids, 8 to 10 amino acids, 10 to 24 amino acids, 10 to 20 amino acids, 10 to 18 amino acids, 10 to 14 amino acids, 10 to 12 amino acids, 12 to 24 amino acids, 12 to 20 amino acids, 12 to 18 amino acids, 12 to 14 amino acids, 14 to 24 amino acids, 14 to 20 amino acids, 14 to 18 amino acids, 18 to 24 amino acids, 18 to 20 amino acids, or 20 to 24 amino acids. In some embodiments, the linker is a polypeptide having a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some cases, the linker is a cleavable linker.

[0038] In some embodiments, the first antigen-binding domain and the immunoglobulin Fc polypeptide are operably linked via an amino acid linker. In some embodiments, the linker within such components consists mainly of the amino acids glycine and serine, herein referred to as a GS-linker. The GS-linkers of the fusion proteins of the present invention have different lengths, such as lengths of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids.

[0039] In some embodiments, the GS-linker comprises an amino acid sequence selected from the group consisting of: GGSGGS, i.e., (GGS)2 (SEQ ID NO: 10); GGSGGSGGS, i.e., (GGS)3 (SEQ ID NO: 11); GGSGGSGGSGGS, i.e., (GGS)4 (SEQ ID NO: 12); and GGSGGSGGSGGSGGS, i.e., (GGS)5 (SEQ ID NO: 13).

[0040] In some embodiments, the second component also includes one or more copies of an anti-CD3 binding domain. In some embodiments, the anti-CD3 binding domain includes one or more copies of an antibody or an antigen-binding fragment thereof. In some embodiments, the anti-CD3 binding domain includes one or more copies of an antibody or an antigen-binding fragment thereof selected from the group consisting of: Fab fragment, F(ab′)2 fragment, Fv fragment, scFv, scAb, dAb, single-domain heavy-chain antibody, and single-domain light-chain antibody. In some embodiments, the anti-CD3 binding domain includes 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 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81. In some embodiments, the anti-CD3ε Fv antibody fragment includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81. In some embodiments, the anti-CD3ε Fv antibody fragment includes a combination of an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81. In some embodiments, the anti-CD3ε Fv antibody fragment includes a combination of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62 and an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81.

[0041] In some embodiments, the anti-CD3ε Fv antibody fragment is a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv).

[0042] In some embodiments, the second component comprises one or more copies of an antigen-binding domain. In some embodiments, the second component comprises 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 on the same TAA. In some embodiments, at least two antigen-binding domains of the second component bind to different TAAs.

[0043] In some embodiments, the first component comprises 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 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 a different epitope on 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 an epitope on the same antigen as the first antigen-binding domain of the first component.

[0044] 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 an antigen-binding fragment thereof. In some embodiments, the second antigen-binding domain comprises one or more copies of an antibody or an antigen-binding fragment thereof selected from the group consisting of: Fab fragment, F(ab′)2 fragment, Fv fragment, scFv, scAb, dAb, single-domain heavy-chain antibody, and single-domain light-chain antibody. In some embodiments, the second antigen-binding domain comprises one or more single-domain antibody (sdAb) fragments (e.g., V H H, V NAR , engineered V H or V K domains) of one or more copies. V H H can be generated from camelid heavy-chain-only antibodies. V NAR can be generated from cartilaginous fish heavy-chain-only antibodies. Various methods have been implemented to generate monomeric sdAbs from common heterodimeric V H and V K domains, including surface engineering and selection of specific germline families.

[0045] 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, α-4 integrin, α-V integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis-Y, Ephrin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9 (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), Claudin-3, Claudin-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor α (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptors, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R (wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, 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, nacasteroin protein, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidyl-serine, 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.

[0046] 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, such internal linkers within such components consist primarily of the amino acids glycine and serine, herein referred to as GS-linkers. The GS-linkers of the fusion proteins of the present invention can have different lengths, such as lengths of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids.

[0047] In some embodiments, the GS-linker comprises an amino acid sequence selected from the group consisting of: GGSGGS, i.e., (GGS)2 (SEQ ID NO: 10); GGSGGSGGS, i.e., (GGS)3 (SEQ ID NO: 11); GGSGGSGGSGGS, i.e., (GGS)4 (SEQ ID NO: 12); and GGSGGSGGSGGSGGS, i.e., (GGS)5 (SEQ ID NO: 13).

[0048] The present disclosure provides a multispecific polypeptide construct comprising a first component containing a heterodimeric Fc region and a second component comprising an anti-CD3 antibody or antigen-binding fragment comprising a variable heavy chain region (VH) and a variable light chain region (VL), wherein: the VH and VL constituting the anti-CD3 antibody or antigen-binding fragment are linked to opposite polypeptides of the heterodimeric Fc; the first and second components are coupled by a cleavable linker, wherein the heterodimeric Fc region is located at the N-terminus of the anti-CD3 antibody; and one or both of the first and second components comprise an antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0049] In some embodiments, the binding of the CD3-binding region to CD3 is significantly reduced when the multispecific polypeptide construct is in an uncleaved state compared to the cleaved state. In some embodiments, in the cleaved state, the first and second components are unlinked.

[0050] 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 an immune effector cell, by a tumor, or by a cell present in the tumor microenvironment. In some embodiments, the protease is produced by a tumor that is proximal to a cell expressing CD3ε and / or by a tumor that is co-localized with a cell expressing CD3ε in a tissue, and 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 that is proximal to a cell expressing one or more tumor-associated antigens (TAAs) and / or by a tumor that is co-localized with a cell expressing a target TAA in a tissue, and 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 an immune effector cell. In some embodiments, the protease is produced by an immune effector cell that is proximal to a cell expressing a TAA. 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 NKT cell. In some embodiments, 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 an immune effector cell that is proximal to a cell expressing a TAA, and wherein when the multispecific polypeptide construct is exposed to the protease, the protease cleaves the cleavable linker in the multispecific polypeptide construct.

[0051] In some embodiments, the cleavable linker is a polypeptide having a length of up to 50 amino acids. In some embodiments, the cleavable linker is a polypeptide having a length of up to 25 amino acids. In some embodiments, the cleavable linker is a polypeptide having a length of up to 15 amino acids.

[0052] 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, asparaginyl endopeptidase, matriptase (also referred to herein as MT-SP1 or MTSP1), ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-9, MMP-12, MMP-13, MMP-14, and any combination thereof. In some embodiments, the cleavable linker is a substrate for a protease selected from the group consisting of: uPA, asparaginyl endopeptidase, and matriptase. In some embodiments, the protease is selected from matriptase, matrix metalloproteinase (MMP), granzyme B, and combinations thereof.

[0053] In some embodiments, the protease is granzyme B. In some embodiments, the cleavable linker comprises an amino acid sequence of the general formula P4 P3 P2 P1 ↓ P1’ (SEQ ID NO: 150), where P4 is amino acid I, L, Y, M, F, V or A; P3 is amino acid A, G, S, V, E, D, Q, N or Y; P2 is amino acid H, P, A, V, G, S or T; P1 is amino acid D or E; and P1’ is amino acid I, L, Y, M, F, V, T, S, G or A. In some embodiments, the cleavable linker comprises an amino acid sequence of the general formula P4 P3 P2 P1 ↓ P1’ (SEQ ID NO: 151), where P4 is amino acid I or L; P3 is amino acid E; P2 is amino acid P or A; P1 is amino acid D; and P1’ is amino acid I, V, T, S or G. In some embodiments, the cleavable linker comprises the amino acid sequences IEPDI (SEQ ID NO: 136), LEPDG (SEQ ID NO: 152), LEADT (SEQ ID NO: 137), IEPDG (SEQ ID NO: 138), IEPDV (SEQ ID NO: 139), IEPDS (SEQ ID NO: 140), IEPDT (SEQ ID NO: 141) or LEADG (SEQ ID NO: 153). In some cases, the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 105 - 112, 136 - 141, 148, 150 - 153.

[0054] In some embodiments, the protease is a stromelysin. In some cases, the cleavable linker comprises the sequence P1QAR ↓ (A / V) (SEQ ID NO: 154), where P1 is any amino acid; or the cleavable linker comprises the sequence RQAR(A / V) (SEQ ID NO: 155). In some embodiments, 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 NO: 23, 154 - 156.

[0055] In some embodiments, the protease is MMP. 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), where P3 is P, V, or A; P2 is Q or D; P1 is A or N; and P1’ is L, I, or M. In some cases, the cleavable linker comprises the general formula P3 P2 P1 ↓ P1’ (SEQ ID NO: 158), where P3 is P; P2 is Q or D; P1 is A or N; and P1’ is L or I. In some embodiments, the cleavable linker comprises the sequence PAGL (SEQ ID NO: 24). In some embodiments, the cleavable linker is a substrate for a matrix metalloproteinase (MMP).

[0056] 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 comprise at least one antigen-binding domain that binds to a tumor-associated antigen (TAA). In some cases, only one of the first or second polypeptides comprises at least one antigen-binding domain that binds to a TAA.

[0057] In some of any provided embodiments, the antigen-binding domain produces monovalent, bivalent, trivalent, or tetravalent binding to the TAA. In some embodiments, one or more antigen-binding domains that bind to a TAA are independently selected from sdAb, scFv, or Fab. In some embodiments, one or more antigen-binding domains that bind to a TAA are single-chain molecules, such as single-chain antibody fragments containing VH and VL, e.g., sdAb or scFv. In some embodiments, at least one of the antigen-binding domains is a Fab containing a first chain comprising VH-CH1 (Fd) and a second chain comprising VL-CL.

[0058] In some embodiments, at least one antigen-binding domain is located amino-terminal relative to the Fc region and / or carboxyl-terminal relative 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 located amino-terminal relative to the Fc region of the multispecific construct, and a second antigen-binding domain is located carboxyl-terminal relative to the CD3-binding region of the multispecific construct.

[0059] In some embodiments, at least one of the antigen-binding domains is a Fab. In some embodiments, the multispecific polypeptide construct comprises: (i) a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and the VH domain of an anti-CD3 antibody or antigen-binding fragment; (ii) a second polypeptide comprising a second Fc polypeptide of a heterodimeric Fc region, a linker, and the VL domain of an anti-CD3 antibody or antigen-binding fragment, and (iii) a third polypeptide comprising the VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen, wherein the first and / or second polypeptide further comprises the other of the VH-CH1 (Fd) or VL-CL of a Fab antibody fragment. In some cases, only one of the first or second polypeptides comprises the other of the VH-CH1 (Fd) or VL-CL of a Fab antibody fragment. In some embodiments, both the first and second polypeptides comprise the other of the VH-CH1 (Fd) or VL-CL of a Fab antibody fragment. In some cases, the other of the VH-CH1 (Fd) or VL-CL of a Fab antibody fragment is not at the amino terminus relative to the Fc region and / or is not at the carboxyl terminus relative to the CD3-binding region in 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 a Fab antibody fragment is at the amino terminus relative to the Fc region of the first or second polypeptide and is at the carboxyl terminus relative to the CD3-binding region in the other of the first or second polypeptides.

[0060] In some embodiments, the antigen-binding domain or each of the antigen-binding domains independently binds to a tumor antigen selected from the following: 1-92-LFA-3, 5T4, α-4 integrin, α-V integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis-Y, Ephrin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9 (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), Claudin-3, Claudin-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor α (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, 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, 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, Lv6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, nakasteroin protein, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidyl-serine, 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.

[0061] 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 on 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.

[0062] In some embodiments, the multispecific polypeptide construct comprises a first linker 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 linker 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 linker peptide (LP1) between the first antigen-binding domain and the immunoglobulin Fc polypeptide region (Fc region) and a second linker peptide (LP2) between the anti-CD3 binding domain (CD3 binding region) and the second antigen-binding domain.

[0063] In some embodiments, the multispecific polypeptide construct in the uncleaved state has the following structural configuration from the N-terminus to the C-terminus: first antigen-binding domain - LP1 - immunoglobulin Fc polypeptide linker region (Fc region) - linker (e.g., cleavable linker) - anti-CD3 binding domain - LP2 - second antigen-binding domain. In some embodiments, the multispecific polypeptide construct in the uncleaved state has the following structural configuration from the N-terminus to the C-terminus: second antigen-binding domain - LP2 - anti-CD3 binding domain (CD3 binding region) - linker (e.g., cleavable linker) - immunoglobulin Fc polypeptide linker region - LP1 - first antigen-binding domain. In some embodiments, the linker is a cleavable linker. In some embodiments, the two linker peptides are different from each other. In some cases, LP1 or LP2 is independently a peptide having a length of about 1 to 20 amino acids. In some embodiments, LP1 or LP2 independently comprises a peptide that is or comprises any Gly-Ser linker as shown in SEQ ID NOs: 10 - 13, 119, 135, 147, 149.

[0064] In some embodiments, the multispecific construct is a construct having Figure 1 any of the structural configurations shown in Figure 2Bispecific constructs of the structural configuration shown in the figure. In some embodiments, the bispecific construct has the following structural configuration from the N-terminus to the C-terminus. The N-terminus of the bispecific construct includes a first antigen-binding domain that binds to a tumor-associated antigen (TAA). The first binding domain binds to a first epitope on the TAA target. A central immunoglobulin Fc polypeptide region that regulates FcγR interaction and / or FcRn interaction is coupled to the first antigen-binding domain. In some embodiments, the central immunoglobulin Fc polypeptide region is a heterodimer. The immunoglobulin Fc polypeptide region is coupled to a cleavable linker that contains one or more proteolytic cleavage sites located at the C-terminal positions at the end of the immunoglobulin Fc polypeptide region. In some embodiments, one or more proteolytic cleavage sites are substrates of stromelysin, matrix metalloproteinase (MMP), or granzyme B. The cleavable linker is attached to the C-terminus of the Fc and, in some cases, to the anti-CD3 binding sequence distal to the second component.

[0065] 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 that is engineered to include a disulfide bond between the variable heavy chain (VH) region and the variable light chain (VL) region, thereby resulting in a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv). In some embodiments, the VH and VL domains of the anti-CD3 Fv are operably linked to the opposing members of a heterodimeric Fc region. In such embodiments, the anti-CD3 Fv binds to CD3 in a monovalent manner. When the cleavable linker is intact, i.e., in an uncleaved or inactive state, the anti-CD3 dsFv does not engage CD3. The C-terminus of the bispecific construct includes a second antigen-binding domain that binds to the TAA. In some embodiments, the second antigen-binding domain binds to the same TAA as the first antigen-binding domain located within the first component. In some embodiments, the second antigen-binding domain binds to a second epitope on the TAA, where the second epitope is non-competitive with the first epitope on the TAA. In some embodiments, the second antigen-binding domain binds to a different TAA than the first antigen-binding domain.

[0066] In some embodiments, each of the first antigen-binding domain and the second antigen-binding domain of the bispecific construct comprises one or more copies of an antibody or an antigen-binding fragment thereof. In some embodiments, each of the first antigen-binding domain and the second antigen-binding domain of the bispecific construct comprises one or more copies of an antibody or an antigen-binding fragment thereof selected from the group consisting of: Fab fragment, F(ab′)2 fragment, Fv fragment, scFv, scAb, dAb, single domain heavy chain antibody, and single domain light chain antibody. In some embodiments, each of the antigen-binding domains, or independently each of the antigen-binding domains, is an antibody or an antigen-binding fragment thereof selected from the group consisting of: Fab fragment, F(ab′)2 fragment, Fv fragment, scFv, scAb, dAb, single domain heavy chain antibody, and single domain light chain antibody. In some embodiments, each of the first antigen-binding domain and the second antigen-binding domain of the bispecific construct comprises one or more single domain antibody (sdAb) fragments (e.g., V H H, V NAR L, engineered V H H or V K L domains) of one or more copies. V H H can be generated from natural camelid heavy-chain only antibodies, genetically modified rodents that produce heavy-chain only antibodies, or naive / synthetic camelid or humanized camelid single domain antibody libraries. M NAR L can be generated from cartilaginous fish heavy-chain only antibodies. Various methods have been implemented to generate monomeric sdAbs from common heterodimeric V H H and V K L domains, including surface engineering and selection of specific germline families.

[0067] 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 V H H, V NAR L, an engineered VH domain, or an engineered VK domain. In some embodiments, the antibody or its antigen-binding fragment is an scFv. In some cases, the antibody or its antigen-binding fragment is a Fab.

[0068] In some embodiments, the anti-CD3 antibody or antigen-binding fragment comprises a VH CDR1 containing the amino acid sequence TYAMN (SEQ ID NO: 16); a VH CD2 containing the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 17); a VH CDR3 containing the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18); a VL CDR1 containing the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 19); a VL CDR2 containing the amino acid sequence GTNKRAP (SEQ ID NO: 20); and a VL CDR3 containing the amino acid sequence ALWYSNLWV (SEQ ID NO: 21).

[0069] In some embodiments, the anti-CD3 dsFv comprises: a VH having the amino acid sequence of any one 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 with any one of SEQ ID NOs: 14, 44, and 32 - 62; and a VL having the amino acid sequence of any one 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 with any one 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.

[0070] In some embodiments, the immunoglobulin Fc region of the first component is an IgG isotype selected from the group consisting of: IgG1 isotype, IgG2 isotype, IgG3 isotype, and IgG4 subclass. In some 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 having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 - 6.

[0071] In some embodiments, the immunoglobulin Fc region is a polypeptide having an amino acid sequence comprising an amino acid sequence derived from the group consisting of SEQ ID NOs: 1-6. In some embodiments, the immunoglobulin Fc region is a polypeptide having an amino acid sequence comprising an amino acid sequence derived from the group consisting of SEQ ID NOs: 1-6, which comprises one or more modifications. In some embodiments, the immunoglobulin Fc region is a polypeptide having an amino acid sequence comprising an amino acid sequence derived from the group consisting of SEQ ID NOs: 1-6, which comprises 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 cell cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), induce heterodimerization, prevent dimerization, stabilize homodimerization at the CH3:CH3 surface, and combinations thereof. In some embodiments, the modification within the Fc region reduces binding to the Fc-receptor-gamma receptor while having a minimal impact on binding to the neonatal Fc receptor (FcRn). In some embodiments, the mutated 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).

[0072] In some embodiments, the first antigen-binding domain and the immunoglobulin Fc polypeptide are operably linked via an amino acid linker. In some embodiments, such an internal linker within such components consists primarily of the amino acids glycine and serine, herein referred to as a GS-linker. The GS-linker of the fusion protein of the present invention can have different lengths, for example, lengths of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids.

[0073] In some embodiments, the GS-linker comprises an amino acid sequence selected from the group consisting of: GGSGGS, i.e., (GGS)2 (SEQ ID NO: 10); GGSGGSGGS, i.e., (GGS)3 (SEQ ID NO: 11); GGS GGSGGSGGS, i.e., (GGS)4 (SEQ ID NO: 12); and GGSGGSGGSGGSGGS, i.e., (GGS)5 (SEQ ID NO: 13).

[0074] In some embodiments, the anti-CD3ε dsFv antibody fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32 - 81. In some embodiments, the anti-CD3ε dsFv antibody fragment comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32 - 81. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of an amino acid sequence selected from the group of SEQ ID NOs: 32 - 62 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 63 - 81. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32 - 62 and an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 63 - 81.

[0075] 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, such an intra-component linker consists mainly of the amino acids glycine and serine, herein referred to as a GS-linker. The GS-linker of the fusion protein of the present invention can have different lengths, for example, a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids.

[0076] In some embodiments, the GS-linker comprises an amino acid sequence selected from the group consisting of: GGSGGS, i.e., (GGS)2 (SEQ ID NO: 10); GGSGGSGGS, i.e., (GGS)3 (SEQ ID NO: 11); GGSGGSGGSGGS, i.e., (GGS)4 (SEQ ID NO: 12); and GGSGGSGGSGGSGGS, i.e., (GGS)5 (SEQ ID NO: 13).

[0077] 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 that is proximal to a cell expressing CD3ε and / or is produced by a tumor that is co-localized with a cell expressing CD3ε in a tissue, and 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 that is proximal to a cell expressing one or more tumor-associated antigens (TAAs) and / or is produced by a tumor that is co-localized with a cell expressing a target TAA in a tissue, and wherein when the multispecific polypeptide construct is exposed to the protease, the protease cleaves the cleavable linker in the multispecific polypeptide construct.

[0078] In some embodiments, the cleavable linker is a polypeptide having a length of up to 50 amino acids. In some embodiments, the cleavable linker is a polypeptide having a length of up to 25 amino acids. In some embodiments, the cleavable linker is a polypeptide having a length of up to 15 amino acids. 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, asparaginyl endopeptidase, matriptase (also referred to herein as MT-SP1 or MTSP1), ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-9, MMP-12, MMP-13, MMP-14, and any combination thereof. In some embodiments, the cleavable linker is a substrate for a protease selected from the group consisting of: uPA, asparaginyl endopeptidase, and matriptase. In some embodiments, the cleavable linker is a substrate for a matrix metalloproteinase (MMP).

[0079] In some embodiments, the multispecific construct also includes a reagent conjugated to the multispecific construct. In some embodiments, the reagent is a therapeutic agent. In some embodiments, the reagent is a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent. In some embodiments, the reagent 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.

[0080] In some embodiments, the anti-multispecific constructs described herein are used in combination with one or more additional reagents or combinations of additional reagents. Suitable additional reagents include current medical and / or surgical therapies for the intended application (e.g., cancer). For example, the multispecific construct can be used in combination with additional chemotherapeutic agents or anti-tumor agents.

[0081] In some embodiments, the multispecific construct and the additional reagent are formulated into a single therapeutic composition and the multispecific construct and the additional reagent are administered simultaneously. In some embodiments, the multispecific construct and the additional reagent are separate from each other; for example, each is formulated into a separate therapeutic composition and the multispecific construct and the additional reagent are administered simultaneously, or the multispecific construct and the additional reagent are administered at different times during a treatment regimen. By way of example, the multispecific construct is administered before the additional reagent, the multispecific construct is administered after the additional reagent, or the multispecific construct and the additional reagent are administered in an alternating fashion. As described herein, the multispecific construct and the additional reagent are administered in a single dose or in multiple doses.

[0082] In some embodiments, the multispecific construct naturally contains one or more disulfide bonds. In some embodiments, the multispecific construct can be engineered to include one or more disulfide bonds.

[0083] The present disclosure also provides an isolated nucleic acid molecule or polynucleotide encoding at least a portion of a multispecific construct described herein and / or one or more nucleic acid molecules encoding a multispecific construct described herein, such as a first nucleic acid encoding at least a portion of a first component of the multispecific construct and a second nucleic acid encoding at least a portion of a second component of the multispecific construct, and vectors comprising such isolated nucleic acid sequences.

[0084] The provided embodiments are polynucleotides encoding any of the provided multispecific polypeptide constructs. Also provided are polynucleotides encoding a polypeptide chain of any of the provided multispecific polypeptide constructs. 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 ribosomal 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 ribosomal 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 embodiments, the nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosomal skipping is selected from T2A, P2A, E2A, or F2A (SEQ ID NOs: 159-164, or encoded by the sequence shown in SEQ ID NO: 165).

[0085] The present disclosure provides a vector comprising any one of the provided polynucleotides. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector or a eukaryotic vector, optionally wherein the eukaryotic vector is a mammalian vector.

[0086] The present disclosure provides a cell comprising any one of the provided polynucleotides or vectors. In some cases, the cell is recombinant or isolated. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a HEK293 or CHO cell.

[0087] The present disclosure provides a method for producing a multispecific construct by culturing a cell under conditions that result in the expression of the multispecific construct, wherein the cell comprises the (an) nucleic acid molecule. In some embodiments, the cell comprises the vector.

[0088] The present disclosure provides a method for producing a bispecific polypeptide construct, the method comprising introducing any one of the provided polynucleotides or vectors into a cell and culturing the cell under conditions that result in the expression of the multispecific construct to produce a multispecific polypeptide construct. Also provided is a method for producing a multispecific polypeptide construct, the method comprising culturing any one of the provided cells under conditions in which the multispecific polypeptide is expressed or produced by the cell. In some cases, the cell is a mammalian cell. In some embodiments, the cell is a HEK293 or CHO cell. In some embodiments, the method further comprises isolating or purifying the multispecific polypeptide construct from the cell. In some cases, the multispecific polypeptide construct is a heterodimer.

[0089] The present disclosure provides a multispecific polypeptide construct produced by any one of the provided methods.

[0090] The present disclosure provides a method for stimulating or inducing an immune response, the method comprising contacting a target cell and a T cell with any one of the provided multispecific polypeptide constructs or a pharmaceutical composition, wherein the target cell expresses a tumor-associated antigen recognized by the multispecific polypeptide construct. In some embodiments, the target cell is a tumor cell expressing a tumor-associated antigen (TAA).

[0091] In some embodiments, the multispecific polypeptide construct comprises a cleavable linker that serves as a substrate for a protease and enhances the induction or stimulation of an immune response in the presence of the protease. In some cases, the protease is produced by an immune effector cell, by a tumor, or by a cell present in the tumor microenvironment.

[0092] 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 proximity to a cell expressing an antigen. In some embodiments, the protease is produced by a tumor in proximity to a cell expressing a TAA in a tissue and / or is produced by a tumor co-localized with a TAA in a tissue, and wherein upon exposure of the multispecific polypeptide construct to the protease, the protease cleaves a cleavable linker in the multispecific polypeptide construct. In some embodiments, the protease is selected from among interstitial proteases, matrix metalloproteinases (MMPs), granzyme B, and combinations thereof. In some cases, the protease is granzyme B.

[0093] In some embodiments, the contacting is performed ex vivo or in vitro. In some embodiments, the contacting is performed in a subject.

[0094] Provided are methods of stimulating or inducing an immune response in a subject, the methods comprising administering to a subject in need thereof a therapeutically effective amount of either the provided multispecific conjugate or the pharmaceutical composition. In some cases, the method increases cell-mediated immunity. In some embodiments, the method increases T cell activity. In some embodiments, the method increases cytotoxic T lymphocyte (CTL) activity. In some embodiments, an immune response against a tumor or cancer is increased. In some embodiments, the method treats a disease or condition in a subject.

[0095] The present invention also provides methods of treating, preventing, or otherwise ameliorating the symptoms of one or more pathologies, delaying their progression, or alleviating symptoms associated with such pathologies, which are accomplished by administering to a subject desiring such treatment or prevention a multispecific polypeptide construct of the present disclosure. Provided herein are methods of treating a disease or condition in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of either the provided multispecific conjugate or the pharmaceutical composition. In some embodiments, the disease or condition is a tumor or cancer.

[0096] In some embodiments of any of the provided methods, the subject (e.g., the subject to be 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 companion animal (e.g., a cat, dog, horse), a farm animal, a draft animal, or a zoo animal. In some embodiments, the subject is a rodent.

[0097] The multispecific polypeptide constructs of the present disclosure used in any of the embodiments of such methods and uses can be administered at any stage of the disease. For example, the multispecific polypeptide construct can be administered to a patient having cancer at any stage (from early to metastatic). The terms subject and patient are used interchangeably herein.

[0098] The multispecific polypeptide constructs of the present disclosure used in any of the embodiments of such methods and uses can be used in treatment regimens that include a lead therapy.

[0099] The multispecific polypeptide constructs of the present disclosure used in any of the embodiments of such methods and uses can be administered alone or in combination with one or more additional agents, such 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 construct is administered in combination with one or more additional agents, such additional agents being, for example (by non-limiting example), chemotherapeutic agents such as alkylating agents, antimetabolites, antimicrotubule agents, topoisomerase inhibitors, cytotoxic antibiotics, and any other nucleic acid-damaging agents. In some embodiments, the additional agent is a taxane, such as paclitaxel (e.g., ). In some embodiments, the additional agent is an antimetabolite, such as gemcitabine. In some embodiments, the additional agent is an alkylating agent, such as platinum-based chemotherapy, such as carboplatin or cisplatin. In some embodiments, the additional agent is a targeting agent, such as a kinase inhibitor, such as sorafenib or erlotinib. In some embodiments, the additional agent is a targeting agent, such as another antibody, such as 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 immunomodulator, such as lenolidomide or IL-2. In some embodiments, the additional agent is radiation. In some embodiments, the additional agent is an agent considered to be the standard of care by those skilled in the art. In some embodiments, the additional agent is a chemotherapeutic agent well known to those skilled in the art. In some embodiments, the multispecific polypeptide construct and the additional agent are formulated in a single composition. In some embodiments, the multispecific polypeptide construct and the additional agent are administered in 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.

[0100] 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., albumin-bound paclitaxel), doxorubicin, oxaliplatin, carboplatin, cisplatin, irinotecan, and gemcitabine.

[0101] In some embodiments, the additional agent is a checkpoint inhibitor, a kinase inhibitor, an inhibitor of an agent targeted in the tumor microenvironment, and / or a T cell or NK agonist. In some embodiments, the additional agent is radiotherapy, which is alone or in combination with another additional agent (such as a chemotherapeutic agent or an anti-tumor agent). In some embodiments, the additional agent is a vaccine, an oncolytic virus, and / or a DC activator (such as by way of non-limiting exemplary toll-like receptor (TLR) agonists and / or α-CD40). In some embodiments, the additional agent is a tumor-targeting antibody designed to kill tumors via ADCC or via direct binding to a toxin (such as an antibody-drug conjugate (ADC)).

[0102] In some embodiments, the checkpoint inhibitor is an inhibitor of a target selected from the group consisting of: CTLA-4, LAG-3, PD-1, PDL1, TIGIT, TIM-3, B7H3, B7H4, and Vista. In some embodiments, the kinase inhibitor is selected from the group consisting of: B-RAFi, MEKi, and Btk inhibitors, such as ibrutinib. In some embodiments, the kinase inhibitor is carfilzomib. In some embodiments, the tumor microenvironment inhibitor is selected from the group consisting of: IDO inhibitors, α-CSF1R inhibitors, α-CCR4 inhibitors, TGF-β, myeloid-derived suppressor cells, or T-regulatory cells. In some embodiments, the agonist is selected from the group consisting of: OX40, GITR, CD137, CD28, ICOS, CD27, and HVEM. In some embodiments, the checkpoint inhibitor is an antibody that binds to a target selected from CTLA-4, PD-1, and / or PD-L1. In some embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody, an anti-PD-1 antibody, and an anti-PD-L1 antibody and / or a combination thereof. In some embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody, such as Yervoy TM . In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody, such as Opdivo TM and / or Keytruda TM .

[0103] 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 α-CSF1R inhibitor. In some embodiments, the inhibitor is an α-CCR4 inhibitor. In some embodiments, the inhibitor is TGF-β. In some embodiments, the inhibitor is myeloid-derived suppressor cells. In some embodiments, the inhibitor is T-regulatory cells.

[0104] 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.

[0105] In some embodiments, the multispecific polypeptide construct is administered during and / or after combination therapy with one or more additional agents (such as chemotherapeutic agents, anti-inflammatory agents, and / or immunosuppressive agents). In some embodiments, the multispecific polypeptide construct and the additional agent are formulated into a single therapeutic composition, and the multispecific polypeptide construct and the additional agent are administered simultaneously. Alternatively, the multispecific polypeptide construct and the additional agent are separate from each other, for example, each is formulated into a separate therapeutic composition, and the multispecific polypeptide construct and the additional agent are administered simultaneously, or the multispecific polypeptide construct and the additional agent are administered at different times during the treatment regimen. For example, the multispecific polypeptide construct is administered before the additional agent, the multispecific polypeptide construct is administered after the additional agent, or the multispecific polypeptide construct and the additional agent are administered in an alternating fashion. As described herein, the multispecific polypeptide construct and the additional agent are administered in a single dose or in multiple doses.

[0106] In some embodiments, the multispecific polypeptide construct and the additional reagent are administered simultaneously. For example, the multispecific polypeptide construct and the additional reagent can be formulated in a single composition or administered in two or more compositions. In some embodiments, the multispecific polypeptide construct and the additional reagent are administered sequentially, or the multispecific polypeptide construct and the additional reagent are administered at different times during a treatment regimen.

[0107] In addition to the above elements, the multispecific polypeptide construct can also contain additional elements, such as amino acid sequences at the N- or C-terminus of the multispecific polypeptide construct. For example, the multispecific polypeptide construct can include targeting moieties to facilitate delivery to a cell or tissue of interest. The multispecific polypeptide construct can be conjugated to a reagent such as a therapeutic agent, a detectable moiety, or a diagnostic agent. Examples of reagents are shown herein.

[0108] The multispecific polypeptide construct can also include any one of the conjugation reagents, linkers, and other components described herein together with the multispecific polypeptide construct of the present disclosure.

[0109] The present disclosure also relates to immunoconjugates comprising a multispecific polypeptide construct conjugated to a cytotoxic agent (e.g., a toxin (e.g., an enzymatically active toxin or fragment thereof from bacteria, fungi, plants, or animals)) or a radioisotope (i.e., a radio conjugate). Cytotoxic agents suitable for targeting, for example, diseased T cells in T cell-derived lymphoma include, for example, dolastatin and its derivatives (e.g., auristatin E, AFP, MMAD, MMAF, MMAE). In some embodiments, the reagent is dolastatin. In some embodiments, the reagent is auristatin or its derivative. In some embodiments, the reagent is a maytansinoid or a maytansinoid derivative. In some embodiments, the reagent is DM1 or DM4. In some embodiments, the reagent is duocarmycin or its derivative. In some embodiments, the reagent is calicheamicin or its derivative. In some embodiments, the reagent is pyrrolobenzodiazepine.

[0110] 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 the same, for example, both cleavable or non-cleavable, or the two or more linkers are different, for example, at least one is cleavable and at least one is non-cleavable.

[0111] Multispecific polypeptide constructs and conjugates thereof can be used in methods for treating a variety of conditions 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, and pancreatic cancer, etc.), rheumatoid arthritis, Crohn's disease, SLE, cardiovascular damage, ischemia, etc. For example, indications will include leukemias (including T-cell acute lymphoblastic leukemia (T-ALL)), lymphoblastic diseases (including multiple myeloma), and solid tumors (including lung tumors, colorectal tumors, prostate tumors, pancreatic tumors, and breast tumors (including triple-negative breast cancer)). For example, indications include bone disease or cancer metastasis, which is unrelated to the source of the primary tumor; breast cancer, including (by way of non-limiting embodiment) ER / PR+ breast cancer, Her2+ breast cancer, triple-negative breast cancer; colorectal cancer; endometrial cancer; gastric cancer; glioblastoma; head and neck cancer, such as esophageal cancer; lung cancer, such as by way of non-limiting embodiment, non-small cell lung cancer; multiple myeloma, ovarian cancer; pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; kidney cancer, such as by way of non-limiting embodiment, renal cell carcinoma; and / or skin cancer, such as by way of non-limiting embodiment, squamous cell carcinoma, basal cell carcinoma, or melanoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is squamous cell carcinoma of the skin. In some embodiments, the cancer is squamous cell carcinoma of the esophagus. In some embodiments, the cancer is squamous cell carcinoma of the head and neck. In some embodiments, the cancer is squamous cell carcinoma of the lung.

[0112] Also provided are pharmaceutical compositions comprising any of the multispecific polypeptide constructs provided herein and a pharmaceutically acceptable carrier. In some cases, the pharmaceutical composition is sterile. The pharmaceutical compositions of the present disclosure may include the multispecific polypeptide constructs of the present disclosure and a carrier. Such pharmaceutical compositions may be included in a kit (e.g., a diagnostic kit).

[0113] Those skilled in the art will appreciate 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 conditions. The antibodies of the present disclosure are also used as reagents in diagnostic kits or as diagnostic tools, or such antibodies can be used in competitive assays to generate therapeutic agents.

[0114] Brief description of the attached figure

[0115] Figure 1 Schematic diagram of the basic components of a multispecific polypeptide construct of the invention with restricted CD3 binding. The antigen binding domain is located at the amino and / or carboxyl terminus. The Fc region (e.g., a heterodimeric Fc region) is located at the N-terminus of the CD3 binding region. This positioning of the Fc in close proximity to the CD3 binding region hinders CD3 binding.

[0116] Figure 2 It is an illustrative diagram showing an exemplary structure of the multi - specific molecule of the present disclosure containing a cleavable linker and having dual effector functions, wherein proteolytic cleavage of the cleavable linker activates the multi - specific polypeptide construct to produce two components each having biological activity.

[0117] Figure 3 It is a schematic diagram of various FRα - targeting restricted CD3 constructs composed of two polypeptides (i.e., chain 1 and chain 2). The upper figure provides an exemplary display of a cleavable multi - specific polypeptide construct that contains a cleavable linker having one or more protease substrate recognition sites for one or more of (e.g.,) MTSP1, MMP, and / or granzyme B. Chain 1 contains an FRα sdAb (antigen - binding domain) that is linked to a heterodimeric Fc “mortar”, linked to an anti - CD3 VL domain via a protease - cleavable linker (cx1547: only granzyme B, cx309: MTSP1, MMP, and granzyme B), and linked to a second FRα sdAb. Chain 2 contains an FRα sdAb that is linked to a complementary heterodimeric Fc “pestle”, linked to an anti - CD3 VH domain via the same protease linker as above, and linked to a second FRα sdAb. The lower figure of Figure 6 shows a configuration similar to the upper figure, except that the linker is an uncleavable linker (ranging from 3 amino acids in cx1356 to 18 amino acids in cx681). Upon co - expression, the CD3 - binding domains are properly assembled via VL:VH association with the mortar and pestle respectively.

[0118] Figures 4A - 4C Shows constructs generated to compare the effects of linker restriction on CD3 binding in the generated constructs. Figure 4A Shows an embodiment of a multi - specific polypeptide construct in which each Fc polypeptide containing the same cleavable linker to couple the heterodimeric Fc to the CD3 - binding domain (showing the exemplary construct cx1762). In Figure 4A the form shown, the construct is shown in an uncleaved state. Figure 4B An alternative form of the construct is shown, in which only a single cleavable linker is used to link the Fc region to the CD3 - binding domain, named semi - cleaved (showing the exemplary construct cx3238). Figure 4C Shows representing Figure 4A and 4B the structure of the constructs in

[0119] Figures 5A - 5E when fully cleaved (showing the exemplary construct cx2190). The constructs representing the proteolytic cleavage products can be generated by co - expressing the various chains shown. The FRα - targeting sdAb is located at the C - terminal position of each construct.

[0119] Figures 5A - 5EShown are restricted CD3 conjugates that are representative of EGFR targeting and EGFR / cMET dual targeting. In Figure 5A , cx2513 has EGFR-targeting sdAbs located at the C-terminus of each chain of the heterodimer and thus exhibits bivalent binding to EGFR. In Figure 5B , cx3030 has EGFR-targeting sdAbs located at the N- and C-termini of each chain of the heterodimer and thus exhibits tetravalent binding to EGFR. In Figure 5C , cx2973 has cMET-targeting sdAbs located at the N-terminus of each chain of the heterodimer and EGFR-targeting sdAbs located at the C-terminus and thus exhibits bivalent binding to each cMET and EGFR. In Figure 5D , cx2979 has a cMET-targeting sdAb located at the N-terminus of one chain of the heterodimer and an EGFR-targeting sdAb located at the C-terminus and thus exhibits monovalent binding to cMET and bivalent binding to EGFR. In Figure 5E , cx2977 has a cMET-targeting sdAb located at the N-terminus of one or the other chain of the heterodimer and an EGFR-targeting sdAb located at the C-terminus and thus exhibits monovalent binding to each cMET and EGFR.

[0120] Figure 6A and 6B are schematic diagrams of the component chains used to assemble exemplary B7H3-targeted restricted CD3 conjugates. The B7H3-binding domains utilized in such representative constructs include sdAbs, scFvs, or Fabs. Generally, constructs containing sdAbs and scFvs consist of two heterodimeric chains, while constructs containing Fabs include a third chain of homologous light chains (VL-CL).

[0121] Figure 7 Shown are 5T4-targeted restricted CD3 conjugates. The core of the generated molecule consists of a heterodimeric Fc, followed by a cleavable linker and a disulfide-stabilized anti-CD3 Fv. The TAA-binding portion of such molecules is placed at the N- or C-terminus of the heterodimeric Fc chains. In the top column, the TAA-binding unit is a Fab, which consists of Fd (VH-CH1) at the N-terminus of the pestle polypeptide and the C-terminus of the mortar polypeptide. In the case where the Fab is the binding unit, a third chain (light chain - VL-CL) is expressed to associate with the Fd. In the middle and bottom columns, the TAA-binding unit is a single-domain antibody on the pestle polypeptides at the N- and C-termini. In the middle column, the TAA-binding sdAbs of the generated constructs are the same, while in the bottom column, the TAA-binding sdabs of the generated constructs are different sequences with different epitopes.

[0122] Figure 8Schematic of the representative CD20 - targeted restricted CD3 - engaging construct cx3309, where the CD20 - binding domain is an scFv derived from the CD20 antibody GA101.

[0123] Figure 9 Schematic of the representative DLL3 - targeted restricted CD3 - engaging construct cx3308, where the DLL3 - binding domain are scFvs. This exemplary construct comprises two chains, each having a complementary component of a heterodimeric Fc that is linked to one component of the CD3 - binding domain and the DLL3 - binding scFv. The construct in assembled form is bivalent for DLL3 and has the CD3 - binding domain at the C - terminus of the Fc heterodimer.

[0124] Figure 10A Image of SDS - PAGE of the representative FRα - targeted restricted CD3 - engaging construct cx1547 under reducing (R) and non - reducing (NR) conditions. The expected molecular weight is 135 kDa. Figure 10B and Figure 10C Are graphs of chromatograms from size - exclusion analysis of cx1547, confirming it is a single species with a determined molecular weight of 137.9 kDa. Figure 10C Is Figure 10B An enlarged view near the main peak shown in

[0125] Figure 11A and 11B Are a pair of graphs showing the binding ability of an exemplary multispecific polypeptide construct of the present disclosure (referred to herein as cx309) to human T cells in the uncleaved or proteolytically cleaved state. Stromelysin and MMP - 2 were used to cleave cx309 in Figure 11A and Figure 11B respectively.

[0126] Figures 12A - 12D Shows cell binding by the representative FRα - targeted restricted CD3 - engaging constructs cx1356, cx681, and cx1547. Figure 12A and Figure 12C Show binding to Ovcar5 cells (FRa - positive ovarian cancer cell line). Figure 12B and Figure 12D Show no binding to T cells. Figure 12A and Figure 12B Show histograms of normalized cell counts versus fluorescence at 100 nM for each construct. Complete titrations of each construct on various cell types are shown in Figure 12C and Figure 12D respectively. In Figure 12A and Figure 12BAmong them, only the secondary anti-human APC antibody control is shown in the filled black trace, while the positive control anti-CD3 binding is shown in the open trace, and cx1356, cx681, and cx1547 are shown in the gray shaded traces.

[0127] Figures 13A - 13B Shows cell binding by the representative EFGR-targeted restricted CD3 engagement construct cx3030. Figure 13A Shows binding to the EGFR-positive cell line Colo-205 at 100 nM. Figure 13B Demonstrates no binding to T cells at 100 nM. The binding is shown as a histogram of normalized cell counts versus fluorescence. Only the secondary anti-human APC antibody control is shown in the filled black trace, while the positive control anti-CD3 binding is shown in the open trace, and cx3030 is shown in the gray shaded trace.

[0128] Figures 14A - 14D Shows binding of the B7H3-targeted restricted CD3 conjugate to B7H3-positive A375 ( Figure 14A and 14B ) and no binding to CD3 on T cells ( Figure 14C and 14D ). The alternative form DART-Fc targeting B7H3 and CD3 shows strong binding to B7H3 and CD3 on T cells. Various B7H3 antigen-binding domains are used herein, including cx3095sdAb, cx3313FAB, and cx3314scFv. The scFv and FAB contain the same anti-B7H3 VH and VL sequences used in the DART-Fc form. Figure 14A and 14C Show comparison histograms of each construct at 100 nM concentration. Only the secondary anti-human APC antibody control is shown in the filled black trace, and the various B7H3-targeted CD3 engagement constructs are shown in the white non-shared traces. Figure 14B and 14D Show the titration of binding of various constructs to BH73 and CD3, respectively.

[0129] Figures 15A - 15B Shows cell binding by the representative 5T4-targeted restricted CD3 engagement constructs cx3262 and cx3315. Figure 15A Shows binding to the 5T4-positive cell line Ovcar-5 at 400 nM. Figure 15B Demonstrates no binding to T cells at 400 nM. The binding is shown as a histogram of normalized cell counts versus fluorescence. Only the secondary anti-human APC antibody control is shown in the filled black trace, while the positive control anti-CD3 binding is shown in the open trace, and cx3262 and cx3315 are shown in the gray shaded traces.

[0130] Figures 16A - 16D Show the cell binding of the representative CD20-targeted restricted CD3 conjugate cx3309. Figure 16A and Figure 16C Show binding to Ramos cells, a CD20-positive cell line. Figure 16B and Figure 16D Demonstrate no binding to T cells. Figures 16A - 16B Show histograms of normalized cell counts versus fluorescence at 100 nM for each construct. Complete titrations of each construct on various cell types are shown in Figures 16C - 16D . Only the secondary anti-human APC antibody control is shown in the filled black trace, while the positive control anti-CD3 binding is shown in the open trace, and cx3309 is shown in the gray shaded trace.

[0131] Figure 17 Is a graph showing the ability of cx309 to activate the CD3 NFAT reporter Jurkat cell line (Promega, USA) lysed or unlysed in the presence or absence of FRα-expressing cells Ovcar5.

[0132] Figure 18 Show antigen-dependent T cell activation by cx1547. Various cell lines that are FRα-positive (T47D, IGROV1, NCI-H2342, Ovcar-5, Skov-3, and A2780) or negative (NCI-H460) are co-incubated with the Jurkat CD3 NFAT-GFP reporter cell line, and fluorescence is measured at 6 hours. This demonstrates the ability of the restricted CD3 construct to activate T cells in an antigen-dependent manner.

[0133] Figures 19A - 19D Show that the T cell activation ability of the restricted CD3 conjugate is enhanced if proteolysis occurs within the linker between the Fc domain and the CD3 domain. Shown here is 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 is the kinetics of T cell activation mediated by 20 nM cx1762, cx3238, or cx2190 in the presence of FRα-positive Ovcar-5 cells ( Figure 19C ) or FRα-negative CCRF-CEM cells ( Figure 19D)Efficacy of T cell activation mediated by cx1762, cx3238, or cx2190 in the presence of . Using the Jurkat CD3 NFAT-GFP reporter gene, CD3 signaling was monitored over 24 hours using an Incucyte ZOOM imager. Notably, T cell activation is dependent on antigen expression on the target cell line and is greatly enhanced by removing the Fc domain at the N-terminus of the CD3-binding domain on one or both sides of the VH:VL domain that binds CD3.

[0134] Figures 20A - 20D is a series of figures showing the antigen-dependent T cell activation ability of various EGFR and EGFR / cMET-targeted restricted CD3 conjugates. Notably, the T cell activation ability is enhanced with increasing valence or additional target antigen binding specificities. The T cell activation kinetics mediated by various constructs on antigen-positive A431 cells are shown in Figure 20A or the T cell activation kinetics mediated by various constructs on antigen-negative CCRF-CEM cells are shown in Figure 20C . The T cell activation efficacy of various constructs on antigen-positive A431 cells is shown in Figure 20B or the T cell activation efficacy of various constructs on antigen-negative CCRF-CEM cells is shown in Figure 20D . The Jurkat CD3 NFAT-GFP reporter gene cell line was used here.

[0135] Figures 21A - 21B Shows the ability of a representative B7H3-targeted restricted CD3 conjugate construct cx3095 and an alternative DART-Fc form targeting B7H3 and CD3 to mediate target antigen-specific T cell activation. Using the Jurkat CD3 NFAT-GFP reporter gene cells, T cell activation was evaluated in the presence of B7H3-positive cell line A375 ( Figure 21A ) and B7H3-negative cell line Raji ( Figure 21B ).

[0136] Figures 22A - 22F Shows the ability of a representative B7H3-targeted restricted CD3 conjugate construct and an alternative DART-Fc form targeting B7H3 and CD3 to mediate target antigen-specific T cell activation. Notably, the restricted CD3 conjugate construct utilizes a B7H3-targeted sdAb, scFv, or FAB. Using the Jurkat CD3 NFAT-GFP reporter gene cells, T cell activation was evaluated in the presence of B7H3-positive cell lines A375 ( Figure 22A , 22C , 22E) and B7H3-negative cell line CCRF ( Figure 22B , 22D, T cell activation in the presence of 22F). Shown is the kinetics of T cell activation mediated by each construct at 50 nM ( Figure 22A and 22B ) or 2 nM ( Figure 22C or 22D). Also shown is the efficacy of T cell activation mediated by each construct on antigen-positive ( Figure 22E ) and negative ( Figure 22F ) cell lines.

[0137] Figures 23A - 23B is a series of graphs showing the T cell activation ability of 5T4-targeted restricted CD3 engagement constructs. This embodiment shows how bivalent bispecific TAA targeting increases the activity of restricted CD3 conjugates on TAA-positive cells (OVCAR5) relative to bivalent monospecific proteins. In the presence of TAA-negative cells (CCRF), none of the constructs induced T cell activation.

[0138] Figure 24 is a graph showing the ability of the representative 5T4-targeted restricted CD3 engagement construct cx3315 to induce antigen-dependent T cell activation. T cell activation by cx3315 was monitored using the Jurkat CD3 NFAT-GFP reporter cell line in the presence of 5T4-positive cell line (OVCAR5) and 5T4-negative cell line (CCRF-CEM).

[0139] Figure 25 Shows the ability of the representative CD20-targeted restricted CD3 engagement construct cx3309 to induce antigen-dependent T cell activation. T cell activation by cx3309 was monitored using the Jurkat CD3 NFAT-GFP reporter cell line in the presence of CD20-positive cell line Ramos and CD20-negative cell line CCRF-CEM.

[0140] Figure 26 is a graph showing the ability of the representative DLL3-targeted restricted CD3 engagement construct cx3308 to induce T cell activation. T cell activation by cx3309 was monitored using the Jurkat CD3 NFAT-GFP reporter cell line in the presence of SHP-77 cells (which are DLL3-positive). This demonstrates that the scFv portion can be used to target TAAs in the form of restricted CD3 and effectively activate T cells when bound to homologous TAA-positive cell lines.

[0141] Figures 27A - 27F Shows the effect of linker length on FRα-positive cells IGROV1 ( Figure 27A , 27C , 27E) or FRα-negative NCI-H460 ( Figure 27B , 27D, the effect on the ability to activate T cells in the presence of 27F). Figures 27A - 27B Respectively show the kinetics of T cell activation by various constructs at 2 nM on antigen - positive and antigen - negative cells. Figures 27C - 27D Respectively show the magnitude of the ability of various constructs at 2 nM on antigen - positive and antigen - negative cells to activate T cells. Figures 27E - 27F Respectively show the efficacy of the ability of various constructs with different linker lengths on antigen - positive and antigen - negative cells to activate T cells. T cell activation was evaluated using the Jurkat CD3NFAT - GFP reporter cell line. When binding to a second antigen on the target cell, the restricted CD3 protein only effectively engages and clusters CD3 on T cells.

[0142] Figures 28A - 28C Show the antigen - dependent T cell - mediated cytotoxicity by cx1547. Figure 28A Demonstrate that cx1547 does not induce cytotoxicity of T cell - mediated antigen - negative cell line (NCI - H460). Figure 28B Demonstrate that cx1547 induces cytotoxicity of T cell - mediated antigen - positive cell line (Ovcar5). Figure 28C Show the kinetics of T cell - mediated cytotoxicity for OVCAR5 cells induced by 3 nM cx1547. cx1547 only induces T cell - mediated cytotoxicity in antigen - positive cell lines. Cytotoxicity was monitored using a caspase - 3 / 7 fluorescent probe substrate of differentially labeled target cells on an Incucyte ZOOM imager. The effector - to - target cell ratio (E∶T) was evaluated at 20∶1 and 10∶1 in this assay.

[0143] Figures 29A - 29F Show the kinetics of T cell - mediated cytotoxicity driven by a representative B7H3 - targeted restricted CD3 engagement construct and an alternative DART - Fc form targeting B7H3 and CD3. The titration range from 50 nM to 80 pM of the CD3 engagement construct on B7H3 - positive A375 cell line is shown in Figures 29A - 29E below. Figure 29F Show the measurement of each construct at 50 nM on A549 cells with knocked - down B7H3 expression. Notably, all constructs exhibit B7H3 - dependent T cell - mediated cytotoxicity.

[0144] Figure 30 Show the magnitude of T cell - mediated cytotoxicity induced by 2.5 nM B7H3 - targeted restricted CD3 engagement construct and DART - Fc B7H3 x CD3 form on antigen - positive (A375) and antigen - negative (A549 - B7H3 knocked - down) cell lines.

[0145] Figures 31A - 31FShowing the comparative efficacy of two forms of FRa-targeted CD3 conjugates in mediating T cell cytotoxicity against FRα-positive Ovcar-5 cells ( Figures 31A - 31E ) and FRα-negative NCI-H60 cells ( Figure 31F ). cx2190 is a representative C-terminal product resulting from proteolytic processing of granzyme B that can be derived from cx1762. Notably, cx2190 demonstrates superior efficacy compared to cx1792, which is shown by a significant enhancement of proteolysis-mediated CD3 binding in the linker region between the Fc and CD3-binding domains. The kinetics of T cell-mediated cytotoxicity on FRa-positive cells at 20 nM, 32 pM, and 6 pM are shown in Figure 31A , 31B and 31C. Figure 31D and Figure 31E show the efficacy of two forms of the FRαCD3 conjugate at 24 and 40 hours, respectively. Panel F shows that in the absence of FRα expression on the target cells, no construct mediates significant cytotoxicity.

[0146] Figure 32 show T cell-mediated cytotoxicity mediated by the representative 5T4-targeted restricted CD3 conjugate construct cx3315. cx3315 induces 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 analysis.

[0147] Figure 33 is a graph showing T cell activation after co-culture of T cells and Ovcar5 cells for 20 hr in the presence of lysed or unlysed cx309. Only lysed cx309 is capable of mediating FRa-dependent T cell activation via CD3 binding. T cell activation was monitored by flow cytometric analysis of the CD4 and CD8 populations for CD25%.

[0148] Figures 34A - 34H show the ability to activate CD4 ( Figure 34A and 34E ) and CD8 ( Figure 34C and 34G ) T cells in a target-dependent manner by the representative B7H3-targeted restricted CD3 conjugate construct and an alternative DART-Fc form targeting B7H3 and CD3. T cells were incubated with the B7H3-positive cell line A375 ( Figure 34A , 34C , 34E, 34G) or the B7H3 knockdown A549 cell line ( Figure 34B , 34D , 34F, 34H), and activation markers CD25 and CD71 were evaluated by flow cytometry. Such data demonstrate the B7H3-dependent T cell activation ability of the constructs used.

[0149] Figure 35 Demonstrate the ability of the B7H3-targeted restricted CD3 conjugate construct cx3095 to mediate antigen-dependent INFγ production. Quantify cytokine production using INFγ ELISA. Use A375 as the B7H3-positive cell line and the B7H3 knockdown A549 cell line as the negative cell line.

[0150] Figures 36A - 36B Demonstrate the ability of the representative FRα-targeted restricted CD3 conjugate construct cx1547 to induce FRα-dependent IFNγ ( Figure 36A ) and IL-2 ( Figure 36B ) from human PBMCs. Measure cytokine production using the FluoroSpot cytokine capture assay. Use IGROV-1 and NCI-H460 as the FRα-positive and negative cell lines, respectively.

[0151] Figure 37 Demonstrate the ability of the B7H3-targeted restricted CD3 conjugate construct cx3095 to mediate antigen-dependent INFγ production. Monitor cytokine production using the FluoroSpot assay. Use A375 and CCRF-CEM cell lines as the B7H3-positive and negative cell lines, respectively.

[0152] Figures 38A - 38D Demonstrate the ability of the FRα-targeted restricted CD3 construct cx1547 to activate T cells present in dissociated primary human ovarian tumor samples and elicit cytotoxicity. Figure 38A Flow chart showing the relative prevalence of tumor cells (EpCAM+) and infiltrating lymphocytes (CD45+) in dissociated ovarian tumor samples. Figure 38B Show the viability (CellTiterGlo) of adherent tumor cells after 6-day incubation following treatment with a common FRα antibody or cx1547. Figure 38C Show INFγ production after 6-day incubation following treatment with the FRa antibody or cx1547. Figure 38D Show representative images of remaining adherent tumor cells after 6 days of treatment without antibody (left panel), with a common FRα antibody (middle panel), or cx1547 (right panel).

[0153] Embodiments

[0154] The present invention provides restricted T cell engaging fusion proteins in the form of multi - specific polypeptide constructs that bind at least CD3 and a second antigen. The multi - specific polypeptide constructs provided herein include at least a first component comprising one or more copies of an antigen - binding domain that binds an antigen operably linked to an immunoglobulin Fc region; a second component comprising one or more copies of at least one CD3 - binding domain (referred to herein as an anti - CD3 binding domain or CD3 - binding region, terms that are used interchangeably herein); and a linker, such as a polypeptide linker, that couples the first component to the second component. In some embodiments, the antigen is a tumor - associated antigen (TAA). In some embodiments, the linker is a cleavable linker.

[0155] The multi - specific polypeptide constructs provided include a configuration in which the first component containing the Fc region is at the N - terminus of the second component containing the CD3 - binding region. In this embodiment, the first and second components are coupled via a C - terminal linker at the Fc - region end. In some embodiments, the antigen - binding domain is located on the amino - terminal (N - terminal) region of the multi - specific polypeptide construct. In some embodiments, the antigen - binding domain is located on the carboxyl - terminal (C - terminal) region of the multi - specific polypeptide construct. In some embodiments, the antigen - binding domain is located on both the N - and C - terminal regions of the multi - specific polypeptide construct. Various configurations of the multi - specific polypeptide constructs provided herein are shown in Figure 1 .

[0156] The multi - specific polypeptide constructs provided exhibit restricted T cell engaging activity, because once the antigen binds via the antigen - binding domain, such constructs only substantially bind to CD3. This is illustrated in the embodiments and figures provided herein, which show that the restricted CD3 - engaging protein effectively binds TAA - positive cells while having little ability to bind to non - T - cell - binding cells. This unique property allows the restricted CD3 - engaging protein to distribute to sites where TAA is present without binding to peripheral T cells. This form differs from other CD3 - engaging multi - specific constructs in that it does not permit or eliminates constitutive CD3 binding, thus providing the significant benefit of avoiding peripheral T cell binding and allowing preferential distribution to sites of antigens recognized by the antigen - binding domain. For example, as shown in the embodiments, the restricted CD3 - engaging form can have similar efficacy to the DART - Fc form (e.g., PCT published invention No. WO2017 / 030926) but with significantly reduced binding to peripheral T cells. Additionally, other CD3 - engaging constructs mediate antigen - dependent T cell activation, whereas the multi - specific polypeptide constructs provided herein mediate antigen - dependent T cell binding and activation.

[0157] In some aspects, the restricted T cell engaging activity of the provided multispecific polypeptide construct is due to the Fc region being located at the N-terminus of the CD3 binding region. In some embodiments, this localization reduces, attenuates, inhibits, 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 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, such as enhancing T cell activity, including cytolytic (or cytotoxic) T cell activity. In some aspects, modulation of the immune response can treat a disease or condition of a subject.

[0158] In some embodiments, one or more antigen binding domains bind antigens on tumor cells or cells of the tumor microenvironment. In some aspects, the provided multispecific polypeptide constructs can be used to increase an immune response against a tumor or cancer, such as T cell activity, such as cytotoxic activity. In some embodiments, the provided multispecific polypeptide constructs can be used to treat a tumor or cancer in a subject.

[0159] The multispecific polypeptide constructs of the present disclosure ensure via CD3-unbound T cells in peripheral blood, as the CD3 binding region of such constructs is restricted or otherwise blocked and / or inhibited due to the presence of the Fc region. Accordingly, the multispecific polypeptide constructs of the present disclosure provide multiple advantages. In some aspects, such constructs limit the bystander effect of binding to all T cells. In some aspects, such constructs reduce systemic toxicity.

[0160] In some embodiments, the provided multispecific polypeptide constructs of the present disclosure permit controlled biodistribution to a desired site in a subject, such as a site of tumor-associated antigen (TAA) expression. Sites of TAA expression include, for example, tumors and the surrounding tumor microenvironment.

[0161] 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 construct can contain more than one antigen binding domain capable of binding one or more TAAs, such as 2, 3, or 4 antigen binding domains, see, for example, Figure 1。In some embodiments, one or more antigen-binding domains bind the same antigen. In some embodiments, the multispecific polypeptide construct comprises more than one antigen-binding domain that binds different epitopes of the same antigen. In some embodiments, the multispecific polypeptide construct comprises more than one antigen-binding domain that binds one or more different antigens. In some embodiments, the multispecific polypeptide construct comprises more than one antigen-binding domain that binds different epitopes on the same antigen, and further comprises additional antigen-binding domains that bind to one or more different antigens. In some aspects, the provided multispecific polypeptide construct is a bispecific polypeptide construct such that it is capable of binding to CD3 and another antigen (e.g., TAA) via the binding of the antigen-binding domains of the multispecific polypeptide construct. In some embodiments, the provided multispecific polypeptide construct is a bispecific polypeptide construct that provides a tetravalent engagement of one or more TAAs via 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 Figure 1 and 2 shown.

[0162] In some embodiments, the multispecific polypeptide constructs provided herein exist in two states with respect to the ability to bind CD3 and subsequently activate T cells: (1) an "inactive" state, i.e., an uncleaved state, occurs when there is no binding by any or all of the antigen-binding domains, such that CD3 binding is restricted and T cell interaction is precluded, and (2) an "active" state occurs when there is antigen binding by any or all of the antigen-binding domains, such that the CD3-binding region is able to bind CD3 and permit T cell interaction.

[0163] In some embodiments, the Fc region is linked to the CD3-binding domain via one or more linkers. In some embodiments, the Fc region is linked to the CD3-binding region via one or more non-cleavable linkers. In some embodiments, the Fc region is linked to the CD3-binding region via a cleavable linker or otherwise labile linker.

[0164] In some embodiments, the Fc region and the CD3-binding region are linked by a cleavable linker, wherein in some aspects, enhanced CD3 binding occurs after cleavage of the cleavable linker. In some such aspects, the "active" state can be further amplified via several mechanisms including cleavage of the linker that couples the CD3-binding region to the Fc region. In some embodiments, the cleavable linker is a linker that contains a protease substrate recognition site. 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.

[0165] In some aspects, the multispecific polypeptide constructs of the present disclosure permit therapeutic efficacy in the absence of proteolysis.

[0166] In some embodiments, the Fc region is a homodimeric Fc region. In some embodiments, the Fc region is a heterodimeric Fc region. In some embodiments, the Fc region is a monomeric Fc region. In some embodiments, the Fc region of the multispecific polypeptide construct can interact with FcγR and mediate innate immune effector functions, such as antibody-dependent cell cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP). In some embodiments, the Fc region of the multispecific polypeptide construct can interact with complement proteins (i.e., C1q) and mediate complement-dependent cytotoxicity. Thus, in some aspects, the multispecific polypeptide constructs of the present disclosure permit multiple immune effector mechanisms, including innate immune effectors and T cells.

[0167] In some embodiments, wherein 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 split the multispecific polypeptide construct into first and second components. Depending on the composition of the multispecific polypeptide construct, the first and second components can have different functionalities. In some embodiments, the Fc region is a region that exhibits one or more effector functions (e.g., ADCC, CDC, or ADCP functions). In such examples, the multispecific polypeptide constructs of the present disclosure can be used to generate 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 in the Fc region causes the release of granzyme B, which can extracellular proteolyze and cleave the linker between the first and second components of the multispecific polypeptide construct.

[0168] In some embodiments, the linker is a cleavable linker. The multispecific polypeptide construct provides a two-in-one therapeutic moiety with dual effector functions, wherein proteolytic activation of the multispecific polypeptide construct generates two biologically active components each. The multispecific polypeptide constructs of the present disclosure can provide only Fc-mediated effector functions, such as ADCC (e.g., release of granzyme B by NK cells), ADCP, and / or CDC.

[0169] The subject limited CD3 engaging constructs are contemplated to be suitable for use with any TAA binding domain, thereby allowing for improved therapeutic exposure in the tumor or tumor microenvironment by avoiding interaction with peripheral T cells and mediating potent TAA-dependent T cell cytotoxicity. Incorporating a protease-cleavable linker between the components of the Fc and CD3 binding domains allows for full exposure of the CD3 binding domain, enabling amplification of the T cell activation capacity. Depending on the particular linker included, the amplification step can be mediated by tumor-associated proteases or granzyme released after antigen-dependent T cell activation. If a tumor protease-cleavable linker is included, amplification is mediated by the tumor or tumor microenvironment. Whereas if a granzyme B-cleavable linker is included, amplification can be self-mediated by the T cells after antigen-dependent activation. Additionally, in cases where an effector-enabled Fc is included in the construct, amplification can be mediated by granzyme released from NK cells, which occurs via the ADCC mechanism.

[0170] In some embodiments, the protease is a protease that is produced in the tumor microenvironment and / or induced by the initial binding of the CD3 binding region to CD3 in the tumor microenvironment upon binding of the antigen binding domain to the TAA upon T cell activation. In some embodiments, the protease is granzyme B. In some aspects, the multispecific polypeptide constructs of the present disclosure affect the ability of proteases and / or granzyme B within the tumor microenvironment to cleave a linker within the multispecific polypeptide construct at a position below the Fc immunoglobulin polypeptide, thereby generating two therapeutically active proteins that in some cases have different effector cell engagement. In some aspects, upon cleavage of the cleavable linker, the first portion or component that is cleaved retains the Fc-effector function and bivalent targeting of the first antigen (e.g., TAA) via the first antigen binding domain, and the second portion or component retains the ability to engage T cells, as separation of the CD3 binding region from the Fc region allows for CD3 binding. The second portion or component that is cleaved also retains the ability to bind to the TAA in some cases, which can be bivalent binding via the second antigen binding domain.

[0171] In some embodiments, the second portion or component contains a CD3 binding region that is monovalent with respect to CD3, such that T cells will not be activated unless the TAA is present. In some aspects, if the multivalent polypeptide construct contains a cleavable linker, the second portion or component that is cleaved allows for TAA-dependent T cell-mediated cytotoxicity. In some cases, the second portion or component that is cleaved ensures no FcRn interaction. Additionally, the size of the second portion or component that is cleaved will be small enough, e.g., only about 50 kDa, to ensure rapid release if for any reason the second portion or component that is cleaved distributes outside of the tumor site and / or if it is aberrantly cleaved outside of the tumor site.

[0172] In some embodiments, the multispecific polypeptide constructs of the present disclosure permit simultaneous T cell- and NK cell-mediated cytotoxicity. In some cases, this activity can occur in a multispecific polypeptide construct comprising 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 on a given TAA.

[0173] In some aspects, the multispecific polypeptide constructs of the present disclosure offer a number of advantages over current bispecific therapeutic agents. The multispecific polypeptide constructs of the present disclosure are smaller than conventional therapeutic antibodies, e.g., 150 kDa versus 125 kDa, which will permit better target (e.g., tumor) penetration. First, the size of the entire multispecific polypeptide construct provides a long half-life for the uncleaved construct, and when the construct is cleaved, the cleaved second moiety or component will be small enough to ensure a short half-life. In some aspects, the multispecific polypeptide constructs of the present disclosure exhibit reduced systemic toxicity or reduced toxicity in any area outside the tumor and / or tumor microenvironment because, prior to CD3 engagement, CD3 binding by the CD3-binding region depends on TAA engagement. In some cases, the incorporation of a cleavable linker that is specific for proteases in the tumor environment reduces CD3 binding by the multispecific construct until proteolytic activation and TAA engagement, thereby amplifying CD3 engagement.

[0174] The multispecific polypeptide constructs of the present disclosure are designed to ensure that the protease that cleaves the cleavable linker does not need to be tumor-biased (e.g., does not need to be differentially expressed only at tumor sites and / or in the tumor environment). Instead, such multispecific polypeptide constructs only require the protease to be present at the same location as the TAA. The valency of such constructs will drive biodistribution and retention in the tumor and / or tumor microenvironment.

[0175] All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. The citation of publications and patent documents is not intended to admit any kind of related prior art, nor is it intended to constitute any admission as to the content or date of such publications and patent documents. The invention has now been described in writing, and those skilled in the art will recognize that the invention can be practiced in various embodiments and that the following description and embodiments are for illustrative purposes and do not limit the following claims.

[0176] I. Definitions

[0177] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The term "an" entity or "a" entity refers to one or more of that entity. For example, a compound refers to one or more compounds. Thus, the terms "an", "a", "one or more" and "at least one" are used interchangeably. In addition, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature and techniques utilized in connection with the cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those that are well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications, or as commonly practiced in the art, or as described herein. Generally, the above techniques and procedures are performed according to commonly known methods in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclature and laboratory procedures and techniques utilized in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those that are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, medical preparation, formulation and delivery, and patient treatment.

[0178] As used in accordance with the present invention, the following terms shall be understood to have the following meanings unless otherwise indicated:

[0179] As used herein, the term "antibody" refers to an immunoglobulin molecule and the antigen-binding portion of an immunoglobulin (Ig) molecule, i.e., a molecule that contains an antigen-binding site that specifically binds to an antigen (immunoreacts with the antigen). "Specifically binds" or "immunoreacts with" or "immunologically specifically binds" means that an antibody reacts with one or more antigenic determinants of a desired antigen and does not bind to other polypeptides or binds with a far lower affinity (K d > 10 -6 ) binding. Antibodies include (but are not limited to) polyclonal, monoclonal, chimeric, fully human, domain antibodies, single-chain, Fab, and F(ab′)2 fragments, Fv, scFv, and Fab expression libraries.

[0180] It is known that the basic antibody structural unit comprises a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50 - 70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that are primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region that is primarily responsible for effector functions. Generally, antibody molecules obtained from humans refer to any one of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other by the nature of the heavy chains present in the molecule. Certain classes also have subclasses, such as IgG1, IgG2, IgG3, IgG4, and others. In addition, in humans, the light chain can be a κ chain or a λ chain.

[0181] As used herein, the term "monoclonal antibody" (mAb) or "monoclonal antibody composition" refers to a population of antibody molecules that contains only one molecular species, which antibody molecules are composed 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. A MAb contains an antigen-binding site that is capable of immunoreacting with a specific epitope of an antigen and is characterized by a unique binding affinity for the antigen.

[0182] The term "antigen-binding site" or "binding portion" refers to the part of an immunoglobulin molecule that participates in antigen binding. The antigen-binding site is formed by the amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches of sequences (termed "hypervariable regions") within the V regions of the heavy and light chains are inserted between more conserved flanking stretches termed "framework regions" or "FRs". Thus, the term "FR" refers to the amino acid sequences that are naturally found between and near the hypervariable regions in an immunoglobulin. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions" or "CDRs". The assignment of amino acids in each domain is according to the following definitions: Sequences of Proteins of Immunological Interest (Kabat Sequences of Proteins of Immunological Interest) (National Institutes of Health, Bethesda, Maryland (1987 and 1991)), or Chothia and Lesk J. Mol. Biol. 196: 901 - 917 (1987), Chothia et al., Nature 342: 878 - 883 (1989).

[0183] As used herein, the term "epitope" includes any 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. Epitope determinants are generally composed of the chemical active surface moieties of a molecule (e.g., amino acid or sugar side chains), and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, antibodies can be raised against the N-terminal, central, or C-terminal peptides of a polypeptide. Additionally, antibodies can be raised against linear or discontinuous epitopes of a polypeptide. An antibody is said to specifically bind an antigen when the dissociation constant is ≤1 μM, e.g., ≤100 nM in some embodiments and ≤10 nM in some embodiments, and the antibody does not exhibit binding to other proteins that are closely related or different.

[0184] As used herein, the terms "specifically bind", "immunologically bind", and "immunological binding properties" refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction can be represented by the dissociation constant (K d ) of the interaction, where a smaller K d represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, where those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates equally in both directions. Thus, the "association rate constant" (K on ) and the "dissociation rate constant" (K off ) can be determined by calculating the concentrations and the actual association and dissociation rates. (See Nature 361:186-87 (1993)). The ratio of K off / K on cancels out all parameters that are not related to affinity and is equal to the dissociation constant K d . (Generally see Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody of the invention is said to specifically bind to EGFR when the binding constant (K d ) measured by an assay such as a radioligand binding assay or a similar assay known to those skilled in the art is ≤1 μM, e.g., ≤100 nM in some embodiments, ≤10 nM in some embodiments, and ≤100 pM to about 1 pM in some embodiments.

[0185] As used herein, the term "isolated polynucleotide" shall mean a polynucleotide of genomic, cDNA or synthetic origin, or some combination thereof, which by virtue of its origin, the "isolated polynucleotide" (1) is not associated with all or a portion of a polynucleotide where the "isolated polynucleotide" is found in nature, (2) is operably linked to a polynucleotide where it is not linked to the polynucleotide in nature, or (3) does not occur in nature as part of a larger sequence. Polynucleotides according to the present disclosure include nucleic acid molecules encoding heavy chain immunoglobulin molecules as shown herein and nucleic acid molecules encoding light chain immunoglobulin molecules as shown herein.

[0186] The term "isolated protein" as referred to herein means a protein of cDNA, recombinant RNA or synthetic origin, or some combination thereof, which by virtue of its origin or the source from which it is derived, the "isolated protein" (1) is not associated with a protein found in nature, (2) does not contain other proteins from the same source, such as does not contain murine proteins, (3) is expressed by cells from different species, or (4) does not occur in nature.

[0187] The term "polypeptide" is used herein as a general term to refer to natural proteins, fragments of polypeptide sequences or analogs. Thus, natural protein fragments and analogs are species within the genus of polypeptides. Polypeptides according to the present disclosure include heavy chain immunoglobulin molecules as shown herein and light chain immunoglobulin molecules as shown herein, as well as antibody molecules in the form of a combination of a heavy chain immunoglobulin molecule and a light chain immunoglobulin molecule (such as a κ light chain immunoglobulin molecule) and vice versa, and fragments and analogs thereof.

[0188] As used herein with respect to an object, the term "natural" refers to the fact that the object can be found in nature. By way of example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a source in nature and has not been intentionally modified or otherwise modified by a person in the laboratory is natural.

[0189] As used herein, the term "operably linked" means that the components so described are positioned in a relationship that permits them to function in their intended manner. A control sequence is "operably linked" to a coding sequence in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence.

[0190] As used herein, the term "control sequence" refers to a polynucleotide sequence necessary to effect the expression and processing of an encoding sequence to which it is ligated. The nature of such control sequences differs depending upon the host organism in prokaryotes and typically includes a promoter, ribosome binding site, and transcription termination sequence in eukaryotes. Generally, such control sequences include a promoter and a transcription termination sequence. The term "control sequence" is intended to include at least all components whose presence is essential for expression and processing and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. As used herein, the term "polynucleotide" means a nucleotide, ribonucleotide, or deoxynucleotide of at least 10 bases in length, or a modified form of any type of nucleotide. The term includes DNA in both single-stranded and double-stranded form.

[0191] As used herein, the term oligonucleotide includes natural and modified nucleotides linked together by natural and non-natural oligonucleotide linkages. Oligonucleotides generally comprise a subgroup of polynucleotides of 200 bases in length or less. In some embodiments, the oligonucleotide is 10 to 60 bases in length, e.g., in some embodiments, 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are generally single-stranded, e.g., for use as probes, but oligonucleotides can be double-stranded, e.g., for use in constructing gene mutants. The oligonucleotides of the present disclosure are sense or antisense oligonucleotides.

[0192] As used herein, the term "natural nucleotide" includes deoxyribonucleotides and ribonucleotides. As used herein, the term "modified nucleotide" includes nucleotides having modified or substituted sugar groups, and the like. As used herein, the term "oligonucleotide linkage" includes oligonucleotide linkages such as phosphorothioates, dithiophosphates, selenophosphates, diselenophosphates, phosphoroanilothioates, phoshoraniladates, phosphoramidates, and the like. See, e.g., LaPlanche et al., Nucl. Acids Res. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984); Stein et al., Nucl. Acids Res. 16:3209 (1988); Zon et al., Anti Cancer Drug Design 6:539 (1991); Zon et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein ed., Oxford University Press, Oxford, England (1991)); Stec et al., U.S. Patent No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990). Optionally, an oligonucleotide may include a label for detection.

[0193] As used herein, the 20 conventional amino acids and their abbreviations follow common usage. See Immunology - A Synthesis (2nd Edition, edited by E.S. Golub and D.R. Gren, Sinauer Associates, Sunderland, Mass. (1991)). Stereoisomers of the 20 conventional amino acids, unnatural amino acids (such as α,α - disubstituted amino acids, N - alkyl amino acids, lactic acid, and other non - conventional amino acids) (such as D - amino acids) can also be components suitable for the polypeptides of the present invention. Examples of non - conventional amino acids include: 4 - hydroxyproline, γ - carboxyglutamate, 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 (such as 4 - hydroxyproline). In the polypeptide notation used herein, according to standard usage and convention, the left - hand direction is the amino - terminal direction, and the right - hand direction is the carboxyl - terminal direction.

[0194] Similarly, unless otherwise stated, the left - hand end of a single - stranded polynucleotide sequence is the 5′ end in the left - hand direction of a double - stranded polynucleotide sequence, referred to as the 5′ direction. The 5′ to 3′ addition direction of a nascent RNA transcript is called the transcription direction; the sequence region on a DNA strand that has the same sequence as the RNA and is the 5′ to 5′ end of the RNA transcript is called the "upstream sequence", and the sequence region on a DNA strand that has the same sequence as the RNA and is the 3′ to 3′ end of the RNA transcript is called the "downstream sequence".

[0195] When applied to polypeptides, the term "substantial identity" means that when two peptide sequences are optimally aligned, for example, using the programs GAP or BESTFIT with default gap weights, they share at least 80% sequence identity, for example, at least 90% sequence identity in some embodiments, at least 95% sequence identity in some embodiments, and at least 99% sequence identity in some embodiments.

[0196] In some embodiments, the different residue positions differ by conservative amino acid substitutions.

[0197] As discussed herein, minor changes in the amino acid sequence of an antibody or immunoglobulin molecule are contemplated, as included by the present invention, provided that the changes in the amino acid sequence maintain at least 75%, such as 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 are within the families of related amino acids in their side chains. Genetically encoded amino acids are generally divided into the following families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartate, glutamate, glutamine, histidine, lysine, serine and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide-containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan and tyrosine, which are the aromatic family. For example, it is reasonable to expect that an isolated substitution of leucine with isoleucine or valine, substitution of aspartate with glutamate, substitution of threonine with serine or substitution of an amino acid with a structurally related amino acid analog would not have a major effect on the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid within the framework site. Whether an amino acid change results in a functional peptide can be readily determined by analyzing the specific activity of the polypeptide derivative. Analysis is elaborated herein. Fragments or analogs of an antibody or immunoglobulin molecule can be readily prepared by those skilled in the art. In some embodiments, the amino- and carboxyl-termini of the fragment or analog occur near the boundaries of the functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data with public or patent sequence databases. Computerized comparison methods are used to identify sequence motifs or predicted protein configuration domains that occur in other proteins of known structure and / or function. Methods are known for identifying protein sequences that fold into known three-dimensional structures. Bowie et al., Science 253:164 (1991). Thus, the above embodiments demonstrate that those skilled in the art can identify sequence motifs and structural configurations that can be used to define the structural and functional domains according to the present disclosure.

[0198] In some embodiments, the amino acid substitutions are those that: (1) reduce sensitivity to proteolysis, (2) reduce sensitivity to oxidation, (3) alter binding affinity to form protein complexes, (4) alter binding affinity, and (4) confer or modify other physicochemical or functional properties of such analogs. The analogs can include various mutant proteins with sequences in addition to the native peptide sequence. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in the native sequence (e.g., in the polypeptide portion outside the structural domain that forms intermolecular contacts). Conservative amino acid substitutions should not significantly alter the structural characteristics of the parent sequence (e.g., the substituted amino acid should not tend to break helices that occur in the parent sequence or disrupt other types of secondary structure characteristic of the parent sequence). Examples of polypeptide secondary and tertiary structures recognized in the art are set forth in the following: Proteins, Structures and Molecular Principles (edited by Creighton, W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (edited by C. Branden and J. Tooze, Garland Publishing, New York, N.Y. (1991)); and Thornton et al., Nature 354:105 (1991).

[0199] As used herein, the term "polypeptide fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion and / or one or more internal deletions, but wherein the remaining amino acid sequence is the same as the corresponding position in the deduced native sequence from, for example, a full-length cDNA sequence. Fragments are typically at least 5, 6, 8, or 10 amino acids in length, e.g., at least 14 amino acids in length in some embodiments, at least 20 amino acids in length in some embodiments, typically at least 50 amino acids in length, and at least 70 amino acids in length in some embodiments. As used herein, the term "analog" refers to a polypeptide comprising a segment of at least 25 amino acids such that such amino acids have significant identity to a portion of the deduced amino acid sequence and specifically bind to EGFR under suitable binding conditions. Typically, relative to the native sequence, the polypeptide analog comprises conservative amino acid substitutions (or additions or deletions). Analogs are typically at least 20 amino acids in length, e.g., at least 50 amino acids in length or longer in some embodiments, and can typically be as long as the full-length native polypeptide.

[0200] The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from a biological material.

[0201] As used herein, the term "labeled" or "labeled" refers to incorporating a detectable label, such as by incorporating a radiolabeled amino acid or attaching a polypeptide to a biotinyl moiety that can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent label or an enzyme activity detectable by optical or colorimetric methods). In some cases, the label or label may also be therapeutic. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labeling for polypeptides include (but are not limited to) the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., fluorophores, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotinyl groups, predetermined polypeptide epitopes recognized by a second reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the label is attached by a spacer arm of various lengths to reduce potential steric hindrance. As used herein, the term "pharmaceutical agent or drug" refers to a chemical compound or composition that can induce a desired therapeutic effect when appropriately administered to a patient.

[0202] As used herein, "substantially pure" means that the target substance is the major substance present (i.e., it is more abundant than any other individual substance in the composition on a molar concentration basis), and the substantially purified fraction is a composition in which the target substance comprises at least about 50% (on a molar concentration basis) of all macromolecular substances present.

[0203] Typically, a substantially pure composition will comprise more than about 80% of all macromolecular substances present in the composition, such as in some embodiments about 85%, 90%, 95% and more than 99%. In some embodiments, the target substance is purified to substantial homogeneity (no contaminating substances can be detected in the composition by common detection methods), where the composition consists essentially of a single macromolecular substance.

[0204] The term patient includes human and domestic animal subjects.

[0205] Other chemical terms in this text are used according to industry usage, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (edited by Parker, S., McGraw-Hill, San Francisco (1985)).

[0206] II. Multispecific Polypeptide Constructs

[0207] Provided herein are multispecific polypeptide constructs 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, wherein the Fc region is located at the N-terminus of the CD3-binding region; and one or both of the first and second components comprise an antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0208] In some embodiments, the multispecific polypeptide construct sequentially comprises, from the N-terminus to the C-terminus: an immunoglobulin Fc region; a linker; a CD3-binding region that binds CD3 (CD3ε); and an antigen-binding domain that binds to a tumor-associated antigen (TAA). In some embodiments, the multispecific polypeptide construct sequentially comprises, from the N-terminus to the C-terminus: an antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker; and a CD3-binding region that binds CD3 (CD3ε). In some embodiments, the multispecific polypeptide construct comprises 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 sequentially comprises, from the N-terminus to the C-terminus: a first antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker; a CD3-binding region that binds CD3 (CD3ε); and a second antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0209] Each component of the multispecific polypeptide constructs of the present disclosure is described in more detail below.

[0210] 1. Anti-CD3 Binding Domain:

[0211] The multispecific polypeptide constructs of the present disclosure include one or more copies of an anti-CD3 binding domain. The anti-CD3 binding domain of the present disclosure activates T cells via engagement of CD3ε on T cells. The anti-CD3 binding domain of the present disclosure agonizes, stimulates, activates, and / or otherwise enhances CD3-mediated T cell activation. The biological activities of CD3 include, for example, T cell activation and other signal transduction via the interaction between CD3 and the antigen-binding subunits of the T cell receptor (TCR). For example, the anti-CD3 binding domain of the present disclosure fully or partially activates T cells by regulating, e.g., agonizing, stimulating, activating, or otherwise enhancing CD3-mediated T cell activation via engagement of CD3ε on T cells.

[0212] In a preferred embodiment, 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 via 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, and humanized monoclonal antibodies and chimeric antibodies, as well as antigen-binding fragments thereof. In some embodiments, the anti-CD3ε binding domain includes one or more copies of an antibody or an antigen-binding fragment thereof.

[0213] In some embodiments, the anti-CD3ε binding domain includes one or more copies of an antibody or an antigen-binding fragment thereof selected from the group consisting of: Fab fragments, F(ab′)2 fragments, Fv fragments, scFv, scAb, dAb, single domain heavy chain antibodies, and single domain light chain antibodies. In some embodiments, the anti-CD3 binding domain includes an Fv antibody fragment that binds 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 binds CD3 monovalently.

[0214] In some embodiments, its 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, its 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, its 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, its 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, its 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 consisting of SEQ ID NOs: 32-81. In some embodiments, its anti-CD3ε binding domain comprises a combination of a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62 and a light chain variable region amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81.

[0215] In some embodiments, its anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity 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 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, its anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence of SEQ ID NO: 14 and a variable light chain (Lv) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence of SEQ ID NO: 15.

[0216] In some embodiments, its anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity 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 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, its anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence of SEQ ID NO: 44 and a variable light chain (Lv) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence of SEQ ID NO: 72.

[0217] In some embodiments, the anti-CD3ε Fv antibody fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81. In some embodiments, the anti-CD3ε Fv antibody fragment comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62 and an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81.

[0218] In some embodiments, the anti-CD3ε binding domain comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence is selected from VH CDR1 sequences comprising at least the amino acid sequence TYAMN (SEQ ID NO: 16); VH CD2 sequences comprising at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 17); and VH CDR3 sequences comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18).

[0219] 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 selected from VL CDR1 sequences comprising at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 19); VL CDR2 sequences comprising at least the amino acid sequence GTNKRAP (SEQ ID NO: 20); and VLCDR3 sequences comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 21).

[0220] 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); a VHCD2 sequence comprising at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 17); a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1 sequence comprising at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 19); a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO: 20); and a VLCDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 21).

[0221] In some embodiments, the anti-CD3ε binding domain comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence is selected from VH CDR1 sequences comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence TYAMN (SEQ ID NO: 16); VH CD2 sequences comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 17); and VH CDR3 sequences comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18).

[0222] 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 selected from VL CDR1 sequences comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 19); VL CDR2 sequences comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence GTNKRAP (SEQ ID NO: 20); and VL CDR3 sequences comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence ALWYSNLWV (SEQ ID NO: 21).

[0223] In some embodiments, the anti-CD3ε binding domain comprises a VH CDR1 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence TYAMN (SEQ ID NO: 16); a VH CDR2 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 17); a VH CDR3 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 19); a VL CDR2 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence GTNKRAP (SEQ ID NO: 20); and a VL CDR3 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence ALWYSNLWV (SEQ ID NO: 21).

[0224] In some embodiments, its 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, its 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, wherein the heavy chain variable amino acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81. In some embodiments, its 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, wherein the heavy chain variable amino acid sequence is selected from the group consisting of SEQ ID NOs: 32-81. In some embodiments, its anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62 and a light chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81. In some embodiments, its anti-CD3ε binding domain is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62 and a light chain variable amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81.

[0225] 2. Immunoglobulin Fc polypeptide:

[0226] The first component of the multispecific polypeptide construct of the present disclosure comprises an immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is an IgG isotype selected from the group consisting of IgG1 isotype, IgG2 isotype, IgG3 isotype, and IgG4 subclass. In some embodiments, the Fc region is a human Fc. In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6. In some embodiments, the immunoglobulin Fc region contains an Fc chain that is an immunologically active fragment of any one of SEQ ID NOs: 1-6. In some embodiments, the immunoglobulin Fc region contains an Fc polypeptide chain or an immunologically active fragment thereof having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of any one of SEQ ID NOs: 1-6.

[0227] In some embodiments, the multispecific polypeptide construct is a dimer formed from polypeptides each containing an Fc. In some specific embodiments, the same or substantially the same polypeptides will dimerize to produce a homodimer. In some embodiments, the dimer is a homodimer in which the two polypeptides of the multispecific polypeptide construct are the same. In other cases, the Fc region is formed from Fc domains that are mutated or modified to promote heterodimerization, where different polypeptides can dimerize to produce 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 known to promote heterodimerization include any of those described below.

[0228] Generally speaking, in addition to antigen-binding ability, which is the primary function of immunoglobulins, the Fc region is also responsible for effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell cytotoxicity (ADCC). Additionally, the FcRn sequence present in the Fc region plays a role in regulating IgG levels by extending the in vivo half-life through coupling to the FcRn receptor in the body. In some embodiments, when using the Fc region of the provided multispecific polypeptide construct, such functions can be altered, for example, reduced or enhanced.

[0229] In some embodiments, the Fc region of the provided multispecific polypeptide construct exhibits one or more effector functions. In some cases, the Fc region can provide Fc-mediated effector functions such as 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 biologically active components: a CD3-binding region capable of binding and engaging CD3 on T cells, and an Fc region linked to a TAA-antigen-binding domain capable of exhibiting target-specific effector functions.

[0230] In some embodiments, the Fc region includes an Fc polypeptide that is mutated or modified to alter one or more effector functions. Various embodiments of mutating an Fc polypeptide to alter (e.g., reduce) effector functions are known and include any of those described below. In some embodiments, when referring to amino acid substitutions in the Fc region, unless otherwise specified with respect to a particular SEQ ID NO, the EU numbering of Kabat (also known as Kabat numbering) is used. The EU numbering is known and is based on the most recently updated IMGT Scientific Chart ( the international ImMunoGeneTics information http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (Created on May 17, 2001, last updated on Jan 10, 2013) and the EU index as reported in: Kabat, E.A. et al., "Sequences of Proteins of Immunological interest". 5th Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 (1991).

[0231] In some embodiments, the provided multispecific polypeptide constructs containing an Fc region that exhibits reduced effector function may be desirable candidates for applications where limited CD3 binding is desired but certain effector functions (e.g., CDC and ADCC) are non-essential or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / abrogation of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the multispecific polypeptide construct and / or its cleaved components do not bind FcγR (and thus likely have no ADCC activity), but retain the ability to bind FcRn. Primary cells (NK cells) that mediate ADCC only express FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Non-limiting embodiments of in vitro assays for assessing the ADCC activity of molecules of interest are described in: U.S. Patent No. 5,500,362 (e.g., see Hellstrom, I. et al., Proc. Nat′l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat′l Acad. Sci. USA 82:1499-1502 (1985); U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be employed (e.g., see ACTI for flow cytometry TM Non-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, Calif.) and CvtoTox Non-radioactive cytotoxicity assays (Promega, Madison, Wis.). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be evaluated in vivo (e.g., in animal models such as those shown in Clynes et al., Proc. Nat’l Acad. Sci. USA 95:652-656 (1998)). A C1q binding assay can also be performed to confirm that the multispecific polypeptide construct and / or its cleaved components do not bind C1q and thus have no CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO2006 / 029879 and WO 2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int′l. Immunol. 18(12):1759-1769 (2006)).

[0232] In some embodiments, the immunoglobulin Fc region or an immunologically active fragment thereof is of the IgG isotype. By way of example, the immunoglobulin Fc region of the fusion protein is of the human IgG1 isotype and has the following amino acid sequence:

[0233]

[0234] In some embodiments, the immunoglobulin Fc region or an immunologically active fragment thereof comprises a human IgG1 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:1.

[0235] In some embodiments, the human IgG1 Fc region is modified to alter antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), e.g., amino acid modifications as described in the following: Natsume et al., 2008 Cancer Res, 68(10):3863-72; Idusogie et al., 2001 J Immunol, 166(4):2571-5; Moore et al., 2010 mAbs, 2(2):181-189; Lazar et al., 2006 PNAS, 103(11):4005-4010, Shields et al., 2001 JBC, 276(9):6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18):8882-8890; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48:152-164; Alegre et al., 1992 J Immunol, 148:3461-3468; see Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11, the entire content of which is incorporated herein by reference.

[0236] In some embodiments, the Fc region (e.g., human IgG1 Fc region) is modified to enhance ADCC activity or CDC activity. Embodiments of mutations that enhance ADCC include modifications of Ser239 and Ile332, e.g., Ser239Asp and Ile332Glu (S239D, I332E). Examples of mutations that enhance CDC include modifications of Lys326 and Glu333. In some embodiments, the Fc region is modified at one or both of such positions using the Kabat numbering system, e.g., Lys326Ala and / or Glu333Ala (K326A and E333A).

[0237] In some embodiments, the human IgG1 Fc region fusion protein of the present invention has no or reduced fucose attached to N-linked glycan chains at N297. There are multiple ways to prevent fucosylation, including (but not limited to) production in FUT8-deficient cell lines; addition of inhibitors, e.g., castanospermine, to mammalian cell culture media; and metabolic engineering of the cell line. In some embodiments, the human IgG1 Fc region is modified at amino acid Asn297 (boxed, Kabat numbering) to prevent glycosylation of the fusion protein, e.g., Asn297Ala (N297A) or Asn297Asp (N297D).

[0238] 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: Leu 234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325), or Ala327 (A327). For example, Leu 234Ala (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 (boxed in SEQ ID NO: 1 above, Kabat numbering) to alter Fc receptor interaction, such as Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (boxed in SEQ ID NO: 1 above, Kabat numbering) to alter Fc receptor interaction, such as Leu234Ala (L234A). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234 and 235, such as Leu234Ala and Leu235Ala (L234A / L235A) or Leu234Val and Leu235Ala (L234V / L235A). In a preferred embodiment, the modification within the Fc region reduces binding to the Fc-receptor-γ receptor while having a minimal impact on binding to the neonatal Fc receptor (FcRn).

[0239] In some embodiments, the human IgG Fc region is modified to enhance FcRn binding. Embodiments of Fc mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E respectively) (Kabat numbering, Dall’Acqua et al., 2006, J. Biol Chem Vol.281(33)23514 - 23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al., 2010 Nature Biotech, Vol.28(2), 157 - 159) (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest). In some embodiments, the mutated 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).

[0240] 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 such embodiments, deletion of these three amino acids in the Fc reduces complement protein C1q binding.

[0241]

[0242] In some embodiments, the Fc region of the fusion protein is altered at Gly236 (boxed in SEQ ID NO:1 above) to reduce Fc receptor binding. For example, Gly236 is deleted from the fusion protein. In some embodiments, the human IgG1 Fc region is modified at amino acid Gly236 to enhance interaction with CD32A, such as Gly236Ala (G236A).

[0243] In some embodiments, the human IgG1 Fc region lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).

[0244] In some embodiments, the fusion or its immunologically active fragment comprises a human IgG2 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2.

[0245] In some embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG2 isotype and has the following amino acid sequence:

[0246]

[0247] In some embodiments, the fusion or its immunologically active fragment comprises a human IgG2 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 3.

[0248] In some embodiments, the human IgG2 Fc region is modified at amino acid Asn297 (boxed) to prevent glycosylation of the antibody, such as Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human IgG2 Fc region lacks Lys447 (EU index of Kabat et al. 1991 Sequences of Proteins of Immunological Interest).

[0249] In some embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG3 isotype and has the following amino acid sequence:

[0250]

[0251] In some embodiments, the antibody or its immunologically active fragment comprises a human IgG3 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 4.

[0252] In some embodiments, the human IgG3 Fc region is modified at amino acid Asn297 (boxed, Kabat numbering) to prevent glycosylation of the antibody, such as Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human IgG3 Fc region is modified at amino acid 435 to extend the half-life, such as Arg435His (R435H). In some embodiments, the human IgG3 Fc region lacks Lys447 (EU index of Kabat et al. 1991 Sequences of Proteins of Immunological Interest).

[0253] In some embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG4 isotype and has the following amino acid sequence:

[0254]

[0255] In some embodiments, the antibody or its immunologically active fragment comprises a human IgG4 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 5.

[0256] In some embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG4 isotype and has the following amino acid sequence:

[0257]

[0258] In some embodiments, the antibody or its immunologically active fragment comprises a human IgG4 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6.

[0259] In other embodiments, the human IgG4 Fc region is modified at amino acid 235 to alter Fc receptor interaction, such as Leu235Glu (L235E). In some embodiments, the human IgG4 Fc region is modified at amino acid Asn297 (boxed, Kabat numbering) to prevent glycosylation of the antibody, such as Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human IgG4 Fc region lacks Lys447 (EU index of Kabat et al. 1991 Sequences of Proteins of Immunological Interest).

[0260] In some embodiments, the human IgG Fc region is modified to stabilize homodimerization by introducing two disulfide bonds at the CH3:CH3 interface by changing Ser354 to Cys (S354C) and Tyr349 to Cys (Y349C) (S354C / Y349C).

[0261] In some embodiments, the human IgG Fc region is modified to induce heterodimerization. A variety of methods are known for promoting the heterodimerization of complementary Fc polypeptides, see, e.g., Ridgway et al., Protein Eng. 9:617-621 (1996); Merchant et al., Nat. Biotechnol. 16(7):677-81 (1998); Moore et al. (2011) MAbs, 3:546-57; Von Kreudenstein et al. MAbs, (2013) 5:646-54; Gunasekaran et al. (2010) J. Biol. Chem., 285:19637-46; Leaver-Fay et al. (2016) Structure, 24:641-51; Ha et al. (2016) Frontiers in Immunology, 7:1; Davis et al. (2010) Protein Eng Des Sel, 23:195-202; published international PCT inventions WO 1998 / 050431, WO 2009 / 089004, WO 2011143545, WO 2014 / 067011, WO 2012 / 058768, WO 2018027025; published U.S. patent inventions US20140363426, US20150307628, US20180016354, US20150239991; and U.S. patents US5731168, US7183076, US9701759, US9605084, and US9650446. Methods for promoting the heterodimerization of Fc chains include mutating the Fc region, e.g., by including a set of "knobs-into-holes" mutations or by including mutations to effect electrostatic manipulation of the Fc to favor attractive interactions between different polypeptide chains. For example, in some embodiments, the Fc polypeptides of the heterodimer include mutations to alter the charge polarity across the surface of the Fc dimer such that co-expression of electrostatically complementary Fc chains supports favorable attractive interactions, thereby promoting the formation of the desired Fc heterodimer, while unfavorable repulsive charge interactions inhibit the formation of undesired Fc homodimers (Gunasekaran et al. (2010) JBC, 285:19637-19646). When co-expressed in cells, association between the chains is possible, but due to charge repulsion, such chains do not substantially self-associate. Other strategies for generating heterodimeric Fc include mixing human IgG and IgA CH3 domain segments to produce complementary CH3 heterodimers, which are referred to as SEED Fc.

[0262] In some embodiments, to promote heterodimerization, both polypeptides of the Fc heterodimer contain paired or complementary amino acid modifications. Exemplary paired amino acid modifications of the polypeptides of the Fc fusion are described in Table 1.

[0263]

[0264] In some embodiments, the modification comprises introducing a protrusion (knob) into a first Fc polypeptide and introducing a cavity (hole) into a second Fc polypeptide such that the protrusion can be positioned in the cavity to facilitate complexing of the first and second Fc-containing polypeptides. The amino acids targeted for replacement and / or modification to create a protrusion or cavity in a polypeptide are typically surface amino acids that interact or contact one or more amino acids in the surface of the second polypeptide.

[0265] In some embodiments, the first Fc polypeptide modified to contain a protrusion (hole) amino acid comprises replacing a natural or original amino acid with an amino acid having at least one side chain that protrudes from the surface of the first Fc polypeptide and can therefore be positioned in a complementary cavity (hole) in the adjacent surface of the second polypeptide. Most often, the replacement amino acid is one with a side chain volume larger than the original amino residue. Those skilled in the art know how to determine and / or evaluate the properties of amino acid residues to identify those that are ideal replacement amino acids to produce protrusions. In some embodiments, the replacement residues used to form the protrusion are natural amino acid residues and include, for example, arginine (R), phenylalanine (F), tyrosine (Y) or tryptophan (W). In some embodiments, the original residue identified for replacement is an amino acid residue with a small side chain, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine.

[0266] In some embodiments, the second Fc polypeptide modified to contain a cavity (hole) comprises replacing a natural or original amino acid with an amino acid having at least one side chain that is recessed from the surface of the second polypeptide and is thus able to accommodate a corresponding protrusion from the surface of the first polypeptide. Most often, the replacement amino acid is one that has a side chain volume that is smaller than the original amino residue. Those skilled in the art know how to determine and / or evaluate the properties of amino acid residues to identify those that are ideal replacement amino acids for forming a cavity. Typically, replacement residues for forming a cavity are natural amino acid residues and include, for example, alanine (A), serine (S), threonine (T) and valine (V). In some embodiments, the original amino acid identified for replacement is an amino acid with a large side chain, such as tyrosine, arginine, phenylalanine or tryptophan.

[0267] The CH3 surface of human IgG1, for example, includes 16 residues on each domain, which are located on four antiparallel β3 strands and are buried 1090 from each surface. (See, e.g., Deisenhofer et al. (1981) Biochemistry, 20:2361-2370; Miller et al., (1990) J Mol. Biol., 216, 965-973; Ridgway et al., (1996) Prot. Engin., 9:617-621; U.S. Patent No. 5,731,168). Modifications of the CH3 domain for generating protrusions or cavities are described, e.g., in U.S. Patent No. 5,731,168; International Patent Invention WO98 / 50431 and WO2005 / 063816; and Ridgway et al., (1996) Prot. Engin., 9:617-621. In some embodiments, modifications of the CH3 domain for generating protrusions or cavities generally target residues located on two central antiparallel β-strands. The aim is to minimize the risk that the generated protrusions can be accommodated by extending into the surrounding solvent rather than by complementary cavities in the partner CH3 domain.)

[0268] For example, in some embodiments, the heterodimeric Fc comprises a polypeptide having an amino acid modification at Thr366 within the CH3 domain, which, when replaced with a larger amino acid (e.g., Try (T366W)), preferentially pairs with a second CH3 domain having amino acid modifications (e.g., Ser, Ala, and Val (T366S / L368A / Y407V) respectively) at positions Thr366, Leu368, and Tyr407 with smaller amino acids. Heterodimerization via CH3 modification can be further stabilized by introducing disulfide bonds, e.g., by changing Ser354 to Cys (S354C) and Tyr349 to Cys (Y349C) on the opposing CH3 domains (see Carter, 2001 Journal of Immunological Methods, 248:7-15).

[0269] The resulting multispecific polypeptide construct can be purified by any suitable method, e.g., by affinity chromatography on a Protein A or Protein G column. If two nucleic acid molecules encoding different polypeptides are transformed into a cell, homodimers and heterodimers will form. The conditions of expression can be adjusted to make heterodimer formation more favorable than homodimer formation.)

[0270] Techniques are known for the differential affinity purification of heterodimers from homodimers using heterodimer pairs as affinity reagents. In some aspects, such techniques involve engineering 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 for Protein A reagent in one of the polypeptides of the Fc heterodimer, see for example WO2017134440, WO2010151792, Jendeberg et al. (Jendeberg et al., (1997) J. Immunol. Methods, 201(1):25-34). In some of such embodiments, the Fc region may be modified at the Protein-A binding site on one member of the heterodimer to prevent Protein-A binding and thereby more effectively purify the heterodimer fusion protein. An exemplary modification within this binding site is Ile253, such as Ile253Arg (I253R). In some embodiments, the modification may be H435R or H435R / Y436F. In some embodiments, the Fc polypeptide of the Fc heterodimer may contain modifications to enable it to bind Protein A rather than Protein G (pA+ / pG-). Exemplary pA+ / pG- amino acid modifications include an Fc containing serine at position 428, serine at position 434 and optionally histidine at position 436, with reference to human IgG1 or containing such residues at the corresponding positions in human IgG 2, 3, or 4. In some aspects, such amino acid modifications in one IgG Fc polypeptide at positions 428, 434 and optionally 436 reduce or prevent binding of Protein G, thereby enhancing purification of the protein.

[0271] In some embodiments, any such modification conferring differential affinity to the affinity reagent may be combined with any one or more of the other amino acid modifications described above. For example, the I253R modification may be combined with the T366S / L368A / Y407V modification or the T366W modification. The Fc with the T366S / L368A / Y407V modification is able to form homodimers as there is no steric occlusion of the dimerization surface as in the case of the Fc with the T336W modification. Thus, in some embodiments, the I253R modification is combined with the Fc with the T366S / L368A / Y407V modification to not allow purification of any homodimeric Fc that may form. Similar modifications can be utilized by combining T366S / L368A / Y407V and H453R.

[0272] In some embodiments, the Fc region of the heterodimer molecule may additionally contain one or more other Fc mutations, such as any of those described above. In some embodiments, the heterodimer molecule contains an Fc region with mutations that reduce effector function.

[0273] In some embodiments, one Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NO: 82, 86, 94 or 96, and the other Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NO: 83, 87, 90, 92, 98 or 100. In some embodiments, one Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NO: 84, 88, 95 or 97 and the other Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NO: 85, 89, 93, 99 or 101.

[0274] In some embodiments, the human IgG Fc region is modified to prevent dimerization. In such embodiments, the fusion proteins of the invention are monomeric. 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.

[0275] 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: Leu 234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325) or Ala327 (A327). For example, Leu 234Ala (L234A), Leu235Ala (L235A), Asp265Asn (D265N), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Asn325Glu (N325E) or Ala327Ser (A327S). In a preferred embodiment, the modification within the Fc region reduces binding to the Fc-receptor - gamma receptor while having a minimal impact on binding to the neonatal Fc receptor (FcRn).

[0276] 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), where the cysteine at Cys220 that forms a disulfide bond with the C-terminal cysteine of the light chain is mutated to serine, such as Cys220Ser (C220S). In other embodiments, the fusion protein contains a truncated hinge having the sequence DKTHTCPPC (SEQ ID NO: 8).

[0277] In some embodiments, the fusion protein has a modified hinge from IgG4 that is modified to prevent or reduce chain exchange, such as 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 a hinge polypeptide.

[0278] 3. Linker:

[0279] The provided multispecific polypeptide construct contains a linker that couples or conjugates a first component containing an immunoglobulin Fc region and a second component containing a CD3-binding region. In some embodiments, the linker is located 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. Since the provided multispecific polypeptide construct is a multimer (e.g., a dimer), the provided construct includes a linker that couples the first Fc polypeptide to the CD3-binding region of the first polypeptide (e.g., VH) and the second Fc polypeptide to the CD3-binding region of the second polypeptide (e.g., VL). Generally, the linkers present in the first and second polypeptides of the multispecific polypeptide construct are the same. Thus, in some embodiments, each domain of the CD3-binding domain is linked via a linker (e.g., the same linker) to the opposite polypeptide of the Fc (e.g., heterodimeric Fc).

[0280] A variety of polypeptide linkers are known for use in fusion proteins (e.g., see 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).

[0281] In some embodiments, the linker is selected such that when the CD3 binding region is linked to the Fc region of the multispecific polypeptide conjugate, the CD3 binding region is constrained and, upon contact of the multispecific polypeptide construct with a cell, is unable or substantially unable to bind or engage CD3 on the surface of the cell (e.g., a T cell). A variety of assays can be employed to evaluate the binding or engagement of CD3 by the multispecific polypeptide construct, including assays to evaluate T cell binding, NFAT activation using a reporter gene system, cytolytic T cell activity, cytokine production, and / or expression of T cell activation markers. Exemplary assays are shown in the provided examples. Generally, the linker also ensures proper folding of the polypeptide construct, does not exhibit a charge that would be inconsistent with the activity or function of the linked polypeptides, and does not form bonds or other interactions with amino acid residues in one or more domains that could impede or alter the activity of the linked polypeptides. In some embodiments, the linker is a polypeptide linker. The polypeptide linker can be a flexible linker, a rigid 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, the linker is up to 25 amino acids in length. In some embodiments, the linker is at least or at least about 2 amino acids in length. In some aspects, a suitable length is (e.g.) at least one and generally less than about 40 amino acid residues (e.g., 2-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, 8-12 amino acids) in length. In some embodiments, the linker is about 2 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.

[0282] In some aspects, the longer the linker length, the stronger the CD3 binding when the multispecific polypeptide conjugate binds to its antigen (e.g., TAA). Thus, in some aspects, the linker has a length greater than 12 amino acids, such as a length greater than 13, 14, 15, 16, 17, or 18 amino acids. In some embodiments, the linker has a length of 12 to 40 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 12 to 15 amino acids, 15 to 40 amino acids, 15 to 30 amino acids, 15 to 24 amino acids, 15 to 18 amino acids, 18 to 40 amino acids, 18 to 30 amino acids, 18 to 24 amino acids, 24 to 40 amino acids, 24 to 30 amino acids, or 30 to 40 amino acids.

[0283] The linker can be natural, synthetic, or a combination of both. Particularly suitable linker polypeptides mainly comprise amino acid residues selected from glycine (Gly), serine (Ser), alanine (Ala), and threonine (Thr). For example, the linker can contain at least 75% (calculated based on the total number of residues present in the peptide linker), such as at least 80%, at least 85%, or at least 90% amino acid residues selected from Gly, Ser, Ala, and Thr. The linker can also consist solely 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, such as at least 75% glycine residues. In some embodiments, the peptide linker consists solely of glycine residues. In some embodiments, the peptide linker consists solely of glycine and serine residues.

[0284] In some embodiments, such linkers are mainly composed of the amino acids glycine and serine, herein referred to as GS-linkers. In some embodiments, the linker contains (GGS)n, where n is 1 to 10, such as 1 to 5, such as 1 to 3, such as GGS(GGS)n (SEQ ID NO: 171), where n is 0 to 10. In certain embodiments, the linker contains the sequence (GGGGGS)n (SEQ ID NO: 173), where n is 1 to 10 or n is 1 to 5, such as 1 to 3. In other embodiments, the linker contains (GGGGGS)n (SEQ ID NO: 172), where n is 1 to 4, such as 1 to 3. The linker can include a combination of any of the above, such as a combination of 2, 3, 4, or 5 repeats of GS, GGS, GGGGGS, and / or GGGGGGGS linkers. In some embodiments, the linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids in length.

[0285] In some embodiments, the linker is (in single-letter amino acid code): GGS, GGGGS (SEQ ID NO: 149), or GGGGGGGS (SEQ ID NO: 135). In some embodiments, the GS-linker comprises the following amino acid sequences: GGSGGS, i.e., (GGS)2 (SEQ ID NO: 10); GGSGGSGGS, i.e., (GGS)3 (SEQ ID NO: 11); GGSGGSGGSGGS, i.e., (GGS)4 (SEQ ID NO: 12); GGSGGSGGSGGSGGS, i.e., (GGS)5 (SEQ ID NO: 13); GGGGGGSGGGGGSGGGGGS, i.e., (G5S)3 (SEQ ID NO: 119); GGSGGGGSGGGGSGGGGS (SEQ ID NO: 147); and GGGGSGGGGSGGGGS (SEQ ID NO: 170). In some embodiments, the linker is GGGG (SEQ ID NO: 103). In some of any of the above embodiments, serine may be replaced by alanine (e.g., (Gly4Ala) or (Gly3Ala)).

[0286] In some embodiments, the linker comprises a peptide linker having the amino acid sequence Gly x Xaa-Gly y -Xaa-Gly z (SEQ ID NO: 174), where 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), aspartate (Asp), and glutamate (Glu), and where x, y, and z are each integers in the range of 1-5. In some embodiments, each Xaa is independently selected from the group consisting of Ser, Ala, and Thr. In a specific variant, each of x, y, and z is equal to 3 (resulting in 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).

[0287] In some embodiments, the linker is a serine-rich linker based on repeats of the (SSSSG)y (SEQ ID NO: 185) motif, where y is at least 1, but y can be 2, 3, 4, 5, 6, 7, 8, and 9.

[0288] In some cases, it may be desirable to provide some rigidity to the peptide linker. This can be accomplished by including proline residues in the amino acid sequence of the peptide linker. Thus, in some embodiments, the linker comprises at least one proline residue in the amino acid sequence of the peptide linker. For example, the peptide linker can have an amino acid sequence in which at least 25% (such as at least 50% or at least 75%) of the amino acid residues are proline residues. In a particular embodiment, the peptide linker consists only of proline residues.

[0289] In some aspects, the peptide linker comprises at least one cysteine residue, such as one cysteine residue. For example, in some embodiments, the linker comprises at least one cysteine residue and amino acid residues selected from the group consisting of Gly, Ser, Ala, and Thr. In some such embodiments, the linker comprises glycine residues and cysteine residues, such as only glycine residues and cysteine residues. Typically, each peptide linker will include only one cysteine residue. An example of a specific linker that contains a cysteine residue includes having an amino acid sequence Gly m -Cys-Gly n where n and m are each integers from 1-12, such as 3-9, 4-8, or 4-7. In a specific variation, the peptide linker has the amino acid sequence GGGGG-C-GGGGG (SEQ ID NO: 177).

[0290] In some embodiments, the linker of the fusion protein is a structured or constrained linker. In certain embodiments, the structured linker contains the sequence (AP)n or (EAAAK)n (SEQ ID NO: 178), where n is from 2 to 20, preferably from 4 to 10, including but not limited to AS-(AP)n-GT (SEQ ID NO: 179) or AS-(EAAAK)n-GT (SEQ ID NO: 180), where n is from 2 to 20, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In other embodiments, the linker contains the sequence (GGGGA)n (SEQ ID NO: 181), (PGGGS)n (SEQ ID NO: 182), (AGGGS)n (SEQ ID NO: 183) or GGS-(EGKSSGSGSESKST)n-GGS (SEQ ID NO: 184), where n is from 2 to 20. In some embodiments, the linker is SSSASASSA (SEQ ID NO: 186), GSPGSPG (SEQ ID NO: 187) or ATTTGSSPGPT (SEQ ID NO: 176). In some embodiments, such linkers may be more resistant to proteolytic degradation due to their structure, thereby providing advantages upon in vivo injection.

[0291] In some embodiments, the linker is not a cleavable linker, 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, non-cleavable linkers include those that do not contain at least one peptide bond, which is preferably within the cleavable peptide sequence or recognition site of a protease. In certain embodiments, the non-cleavable linker is not a target substrate of a protease such that it is not preferentially or specifically cleaved by a protease compared to a linker containing the substrate recognition site of the same protease.

[0292] In some embodiments, the linker is a cleavable linker. In some aspects, a cleavable linker is a linker that includes a sequence that is a substrate of a protease due to the presence of at least one bond that can be broken under physiological conditions. In some cases, a cleavable linker is prone to cleavage or sensitive to cleavage under specific conditions present in vivo, such as upon exposure to extracellular proteases (including those present in vivo in the cellular environment). In some cases, proteases may be present in a specific physiological microenvironment, such as the tumor microenvironment, thereby restricting the site where cleavage may occur.

[0293] Compared to another non-target substrate, a protease generally exhibits specificity or preference for cleavage of a specific target substrate. The degree of this specificity can be determined based on the cleavage rate constant of a sequence (e.g., a linker), which is a measure of the protease's preference for its substrate and the enzyme's efficiency. Any method for determining the rate of increase in cleavage over time in the presence of different concentrations of substrate can be used to calculate the specificity constant. For example, the substrate is linked to a fluorescent probe moiety that is released upon cleavage by the protease. By measuring the cleavage rate at different protease concentrations, the cleavage specificity constant (k cat / K m ) of a specific protease for a specific linker can be determined. In some embodiments, the cleavable linker is a linker that can be specifically cleaved by a protease at a rate of about at least 1×10 4 M -1 S -1 , or at least 5×10 4 M -1 S, at least 10×10 4 M -1 S, at least 10×10 5 M -1 S or greater.

[0294] Cleavable linker

[0295] In some embodiments, the multispecific polypeptide construct of the present disclosure includes a cleavable linker that joins a first and a second component. In some embodiments, the cleavable linker includes an amino acid sequence that can serve as a substrate for a protease, typically an extracellular protease. For example, the cleavable linker can contain a cleavage sequence that contains at least one peptide bond within the cleavable peptide sequence of the protease. Suitable proteases include, for example, matrix metalloproteinases (MMPs), cysteine proteases, serine proteases, and plasminogen activators, which are formed or activated in an enhanced manner in diseases such as rheumatoid arthritis or cancer, leading to excessive tissue degradation, inflammation, and metastasis. In certain embodiments, the protease is a protease produced by a tumor, an activated immune effector cell (e.g., a T cell or an NK cell), or a cell in the tumor microenvironment. In some embodiments, the protease is granzyme B, stromelysin, or an MMP (e.g., MMP-2).

[0296] The cleavable linker can be selected based on a protease produced by a tumor that is proximal to the cell expressing the target or produced by a tumor that is co-localized in the tissue with the desired target of the multispecific polypeptide construct. It has been reported in the literature that the levels of proteases with known substrates are increased in many cancers, such as solid tumors. See, for example, La Rocca et al., (2004) British J. of Cancer 90(7): 1414-1421.

[0297] In some embodiments, the cleavable linker that joins the first and second components of the multispecific polypeptide construct can be cleaved by proteases produced by immune effector cells that are activated by one of such components. For example, a multispecific polypeptide construct that encompasses an effector-enabled or enhanced IgG Fc region can elicit ADCC when engaged with a target antigen. Critical to ADCC is the release of granzyme B and perforin from effector cells, namely NK cells and cytotoxic T cells. Upon release, granzyme B enters the target cell in a perforin-dependent manner, where it mediates apoptosis. Importantly, granzyme B is active within the extracellular synapse between the effector cell and the target cell. In some embodiments, the cleavable linker that joins the first and second components of the multispecific polypeptide construct can be 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, upon binding of the TAA by the multispecific polypeptide construct, T cell activation via CD3 engagement can release such proteases, which can then cleave the specific cleavable linker, thereby enhancing or increasing the activity of the CD3-binding molecule in engaging CD3. In some embodiments, cleavage can amplify or increase the activity achieved by the multispecific construct when bound to the TAA in the uncleaved state.

[0298] Exemplary substrates include, but are not limited to, substrates cleavable by one or more of the following enzymes or proteases: ADAMS, ADAMTS, such as ADAM8; ADAM9; ADAM10; ADAM12; ADAM15; ADAM17 / TACE; ADAMDEC1; ADAMTS1; ADAMTS4; ADAMTS5; aspartate proteases, such as BACE or renin; asparagine endopeptidases, such as endolysin D or endolysin E; caspases, such as caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10 or caspase 14; cysteine endopeptidases, such as endolysin B, endolysin C, endolysin K, endolysin L, endolysin S, endolysin V / L2, endolysin X / Z / P; cysteine proteases, such as cathepsin K; asparagine endopeptidase; Otubain-2; KLK, such as KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 or KLK14; metalloproteases, such as Meprin; neprilysin; PSMA; BMP-1; MMP, such as MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26 or MMP27; serine proteases, such as activated protein C, endolysin A, endolysin G, chymotrypsin, blood coagulation factor proteases (such as FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, tosyl-L-arginine methyl esterase, HtrA1, human neutrophil elastase, lactoferrin, Marapsin, NS3 / 4A, PACE4, plasminogen, PSA, tPA, thrombin, plasmin, uPA; type II transmembrane serine proteases (TTSP), such as DESC1, DPP-4, FAP, Hepsin, matriptase-2, matriptase, TMPRSS2, TMPRSS3, or TMPRSS4; and any combination thereof.

[0299] In some embodiments, the cleavable linker may be cleaved by multiple proteases, such as 2 or more proteases, 3 or more proteases, 4 or more proteases, etc.

[0300] In some embodiments, the cleavable linker is selected to use a specific protease, such as a protease known to be produced by a tumor in a cell proximal to the target of expression and / or by a tumor co-localized with the target of the multispecific polypeptide construct.

[0301] In some embodiments, the cleavable linker contains a substrate recognition site or cleavage site for a specific protease, which is a sequence recognized by the active site of the protease that cleaves the protease. Generally, for example, for serine proteases, the cleavage sequence consists of P1-P4 and P1'-P4' amino acids in the substrate, where cleavage occurs after the P1 position. Generally, the cleavage sequence of serine proteases is 6 residues long to match the expanded substrate specificity of many proteases, but can be longer or shorter depending on the protease. Generally, the cleavable linker includes a P1-P1' labile bond sequence recognized by the protease. In some aspects, the cleavable linker is engineered 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.

[0302] In some embodiments, the cleavable linker includes 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 contains 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), where P4 is the amino acid I, L, Y, M, F, V, or A; P3 is the amino acid A, G, S, V, E, D, Q, N, or Y; P2 is the amino acid H, P, A, V, G, S, or T; P1 is the amino acid D or E; and P1' is the amino acid I, L, Y, M, F, V, T, S, G, or A. In some embodiments, the granzyme B cleavable linker contains an amino acid sequence having the general formula P4 P3 P2 P1 ↓ P1' (SEQ ID NO: 151), where P4 is the amino acid I or L; P3 is the amino acid E; P2 is the amino acid P or A; P1 is the amino acid D; and P1' is the amino acid I, V, T, S, or G.

[0303] In some embodiments, the substrate of 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 contains 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).

[0304] In some embodiments, the cleavable linker comprises an amino acid that is a substrate of a stromelysin. In some embodiments, the cleavable linker comprises the sequence P4QAR↓(A / V) (SEQ ID NO: 154), where P4 is any amino acid. In some embodiments, the cleavable linker comprises the sequence RQAR(A / V) (SEQ ID NO: 155). In some embodiments, the substrate of the stromelysin 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).

[0305] In some embodiments, the cleavable linker comprises an amino acid that is a substrate of one or more matrix metalloproteinases (MMPs). In some embodiments, the MMP is MMP-2. In some embodiments, the cleavable linker has the general formula P3P2P1↓P1’ (SEQ ID NO: 157), where P3 is P, V, or A; P2 is Q or D; P1 is A or N; and P1’ is L, I, or M. In some embodiments, the cleavable linker has the general formula P3P2P1↓P1’ (SEQ ID NO: 158), where P3 is P; P2 is Q or D; P1 is A or N; and P1’ is L or I. In some embodiments, the substrate of the MMP comprises the amino acid sequence PAGL (SEQ ID NO: 24).

[0306] In some embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate of granzyme B and an amino acid sequence that is a substrate of a stromelysin. 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).

[0307] 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 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).

[0308] In some embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for a stromelysin and an amino acid sequence that is a substrate for MMP. In 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).

[0309] 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 a stromelysin, and an amino acid sequence that is a substrate for 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 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).

[0310] The cleavable linker can include any known linker. Examples of cleavable linkers are set forth in the following: Be’liveau et al. (2009) FEBS Journal, 276; U.S. Published Inventor No. US20160194399; No. US20150079088; No. US20170204139; No. US20160289324; No. US20160122425; No. US20150087810; No. US20170081397; U.S. Patent No. US9644016.

[0311] In some embodiments, the cleavable linker comprises an amino acid sequence selected from the group consisting of: TGLEADGSPAGLGRQARVG (SEQ ID NO: 25); TGLEADGSRQARVGPAGLG (SEQ ID NO: 26); TGSPAGLEADGSRQARVGS (SEQ ID NO: 27); TGPAGLGLEADGSRQARVG (SEQ ID NO: 28); TGRQARVGLEADGSPAGLG (SEQ ID NO: 29); TGSRQARVGPAGLEADGS (SEQ ID NO: 30); and TGPAGLGSRQARVGLEADGS (SEQ ID NO: 31); GPAGLGLEPDGSRQARVG (SEQ ID NO: 104); GGSGGGGIEPDIGGSGGS (SEQ ID NO: 105); GGSGGGGLEADTGGSGGS (SEQ ID NO: 106); GSIEPDIGS (SEQ ID NO: 107); GSLEADTGS (SEQ ID NO: 108); GGSGGGGIEPDGGGSGGS (SEQ ID NO: 109); GGSGGGGIEPDVGGSGGS (SEQ ID NO: 110); GGSGGGGIEPDSGGSGGS (SEQ ID NO: 111); GGSGGGGIEPDTGGSGGS (SEQ ID NO: 112); GGGSLEPDGSGS (SEQ ID NO: 113); and GPAGLGLEADGSRQARVG (SEQ ID NO: 114), GGEGGGGSGGSGGGS (SEQ ID NO: 115); GSSAGSEAGGSGQAGVGS (SEQ ID NO: 116); GGSGGGGLEAEGSGGGGS (SEQ ID NO: 117); GGSGGGGIEPDPGGSGGS (SEQ ID NO: 118); TGGSGGGGIEPDIGGSGGS (SEQ ID NO: 148).

[0312] 4. Antigen-binding domain:

[0313] The multispecific polypeptide construct of the present invention comprises at least one antigen-binding domain, such as at least a first antigen-binding domain and a second antigen-binding domain. In some aspects, the antigen-binding domain, or each antigen-binding domain independently, is selected from antibodies or antigen-binding fragments, natural cognate binding partners, Anticalins (engineered lipocalins), Darpins, Fynom ers, Centyrins (engineered fibronectin type III domains), cysteine-stub domains, Affilins, Affibodvs or engineered CH3 domains. In some embodiments, the natural cognate binding partner comprises the extracellular domain of the natural cognate binding partner of the TAA or a binding fragment thereof, or a variant thereof that exhibits binding activity to the TAA.

[0314] In some embodiments, the antigen-binding domain, or each antigen-binding domain independently, such as the first antigen-binding domain and the second antigen-binding domain, comprises one or more copies of an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding domain, or each antigen-binding domain independently, such as the first antigen-binding domain and the second antigen-binding domain, comprises one or more copies of an antibody or an antigen-binding fragment thereof selected from the group consisting of: Fab fragments, F(ab′)2 fragments, Fv fragments, scFv, scAb, dAb, single-domain heavy-chain antibodies and single-domain light-chain antibodies. In some embodiments, the antigen-binding domain, or each antigen-binding domain independently, such as the first antigen-binding domain and the second antigen-binding domain, comprises one or more single-domain antibody (sdAb) fragments, such as V H H, V NAR , engineered V H or V K domains. V H H can be generated from natural camelid heavy-chain only antibodies, genetically modified rodents that produce heavy-chain only antibodies, or naive / synthetic camelid or humanized camelid single-domain antibody libraries. V NAR can be generated from chondrichthyan heavy-chain only antibodies. Various methods have been implemented to generate monomeric sdAbs from common heterodimeric V H and V K domains, including surface engineering and selection of specific germline families. In some embodiments, the antigen-binding domain of the multispecific polypeptide construct, or each antigen-binding domain independently, such as the first antigen-binding domain and / or the second antigen-binding domain, contains VH and VL sequences assembled into a FAB or scFv. In some embodiments, the antigen-binding domain of the multispecific polypeptide construct, or each antigen-binding domain independently, such as the first antigen-binding domain and / or the second antigen-binding domain, contains a binding domain as a single-domain antibody (sdAb).

[0315] In some embodiments, the antigen-binding domain or, independently, each antigen-binding domain is or comprises the extracellular domain of a natural cognate binding partner of a TAA or a binding fragment thereof, or a variant thereof that exhibits binding activity to the TAA.

[0316] In some embodiments, the antigen-binding domain, or independently each antigen-binding domain, such as the first antigen-binding domain and the second antigen-binding domain, binds the same antigen. In some embodiments, the antigen-binding domain, or independently each antigen-binding domain, such as the first antigen-binding domain and the second antigen-binding domain, binds different antigens. In some embodiments, the antigen-binding domain, or independently each antigen-binding domain, such as the first antigen-binding domain and the second antigen-binding domain, binds the same tumor-associated antigen (TAA). In some embodiments, the antigen-binding domain, or independently each antigen-binding domain, such as the first antigen-binding domain and the second antigen-binding domain, binds different TAAs. In some embodiments, the antigen-binding domain, or independently each antigen-binding domain, such as the first antigen-binding domain and the second antigen-binding domain, binds different epitopes on the same TAA. In some embodiments, the antigen-binding domain, or independently each antigen-binding domain, such as the first antigen-binding domain and the second antigen-binding domain, binds the same epitope on the same TAA.

[0317] In some embodiments, the antigen-binding domain that binds a TAA or, independently, each antigen-binding domain binds to the TAA with monovalent, divalent, trivalent, or tetravalent binding.

[0318] In some embodiments, the TAA is selected from the group consisting of: 1-92-LFA-3, 5T4, α-4 integrin, α-V integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis-Y, Ephrin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9 (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), Claudin-3, Claudin-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG 1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor α (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, 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, IL23, IL23R, IL27 / IL27R (wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged1, Jagged2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LvPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, Nakastrin protein, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine-1-phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFB, 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.

[0319] 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 comprises a binding domain of an sdAb that binds to FRα. Exemplary FRα-binding sdAbs are set forth in SEQ ID NOs: 120, 121, and 122.

[0320] In some embodiments, at least one antigen-binding domain, or each antigen-binding domain independently, binds to the tumor-associated antigen (TAA) cMET. For example, the antigen-binding domain comprises a binding domain of an sdAb that binds cMET. Exemplary cMET-binding sdAbs are set forth in SEQ ID NO: 123 (U.S. Patent No. 9,346,884).

[0321] 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 comprises a binding domain of an scFv that binds B7H3. Exemplary B7H3-binding scFvs are set forth in SEQ ID NO: 124. In some embodiments, the antigen-binding domain is or comprises a Fab antibody fragment comprising VH-CH1 (Fd) and LC. Exemplary B7H3 Fd is set forth in SEQ ID NO: 127 and exemplary B7H3 LC is set forth in SEQ ID NO: 128 (PCT Publication No. WO2017 / 030926).

[0322] In some embodiments, the antigen-binding domain binds to the tumor-associated antigen (TAA) CD20. For example, the antigen-binding domain comprises a binding domain of an scFv that binds CD20. Exemplary CD20-binding scFvs are set forth in SEQ ID NO: 125, 189, and 190 (U.S. Publication No. US 2005 / 0123546).

[0323] In some embodiments, the antigen-binding domain binds to the tumor-associated antigen (TAA) DLL3. For example, the antigen-binding domain comprises a binding domain of an scFv that binds DLL3. Exemplary DLL3-binding scFvs are set forth in SEQ ID NO: 126 and 189 (U.S. Publication No. US 2017 / 0037130). In some embodiments, the antigen-binding domain is or comprises a Fab antibody fragment comprising an Fd and LC that bind DLL3. Exemplary DLL3 Fd is set forth in SEQ ID NO: 133 and exemplary DLL3 LC is set forth in SEQ ID NO: 134 (U.S. Patent No. US 8,044,178).

[0324] In some embodiments, the antigen-binding domain binds to the tumor-associated antigen (TAA) 5T4. Exemplary 5T4 Fd is set forth in SEQ ID NO: 129 and exemplary 5T4 LC is set forth in SEQ ID NO: 130. In some embodiments, the antibody-binding domain comprises VH-CH1 (Fd) or VL-CL as shown in SEQ ID NOs: 167 and 168 (U.S. Patent No. US 8,044,178).

[0325] In some embodiments, the antigen-binding domain binds to the tumor-associated antigen (TAA) gpNMB. In some embodiments, the antigen-binding domain is or comprises a Fab fragment comprising an Fd and an LC chain. Exemplary gpNMB Fd is set forth in SEQ ID NO: 131 and exemplary gpNMB LC is set forth in SEQ ID NO: 132.

[0326] In some embodiments, the antigen-binding domain is directly or indirectly linked to the Fc region and / or the CD3-binding region via a linker. In some embodiments, the linkage is via a linker. In some embodiments, the linker is a linking peptide (LP), which may comprise any flexible or rigid linker as shown in Part II.3, but the peptide linking one or more antigen-binding domains is generally not a cleavable linker.

[0327] In some embodiments, the multispecific polypeptide construct comprises a first linking peptide (LP1) between the first antigen-binding domain and the Fc region. In some embodiments, the multispecific polypeptide construct comprises a second linking peptide (LP2) between the CD3-binding region and the second antigen-binding domain. In some embodiments, the multispecific polypeptide construct comprises a first linking peptide (LP1) between the first antigen-binding domain and the Fc region and a second linking peptide (LP2) between the CD3-binding region and the second antigen-binding domain. In some aspects, the multispecific polypeptide construct has the following structural configuration from the N-terminus to the C-terminus: first antigen-binding domain - LP1 - Fc region - linker - CD3-binding region - LP2 - second antigen-binding domain. In some embodiments, the two linking peptides are different from each other.

[0328] In some embodiments, LP1 or LP2 is independently a peptide about 1 to 20 amino acids in length. In some embodiments, LP1 or LP2 is independently a peptide that is or comprises any Gly-Ser linker as shown in SEQ ID NOs: 10-13, 119, 135, 147, 149 or GGS.

[0329] III. Pharmaceutical Compositions

[0330] The present disclosure provides a composition of any of the provided multispecific polypeptide constructs. It should be understood that administration of a therapeutic entity according to the present disclosure should be administered together with a suitable carrier, excipient, and other reagents incorporated into the formulation to provide improved transfer, delivery, tolerance, etc. A variety of suitable formulations can be found in the formulations known to all pharmaceutical chemists: Remington’s Pharmaceutical Sciences (15th Edition, Mack Publishing Company, Easton, Pennsylvania (1975)), specifically Chapter 87 therein, Blaug, Seymour. Such formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (e.g., Lipofectin TM ), DNA conjugates, anhydrous absorbent pastes, water-in-oil and oil-in-water emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowaxes. Any of the above mixtures can be suitable for the treatments and therapies according to the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. For additional information regarding formulations, excipients, and carriers known to pharmaceutical chemists, also see Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000), Charman WN “Lipids, lipophilic drugs, and oral drug delivery - some emerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) and the references therein.

[0331] In some embodiments, multispecific polypeptide constructs, conjugated multispecific polypeptide constructs, and compositions thereof (collectively referred to herein as therapeutic agents and derivatives, fragments, analogs, and homologs thereof) can be incorporated into pharmaceutical compositions suitable for administration. The principles and considerations involved in preparing such compositions and guidelines for the selection of components are provided in the following, for example: Remington’s Pharmaceutical Sciences: The Science And Practice Of Pharmacy, 19th Edition (edited by Alfonso R. Gennaro et al.), Mack Publishing Company, 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, Volume 4), 1991, M. Dekker, New York.

[0332] Such compositions generally comprise a multispecific polypeptide construct or conjugate thereof and a pharmaceutically acceptable carrier. If the multispecific polypeptide construct comprises a fragment of an antibody, the smallest fragment of the antibody that specifically binds to the target protein can be used. For example, based on the antibody variable region sequence, peptide molecules can be designed that retain the ability of the antibody to bind to the target protein sequence. Such peptides can be synthesized chemically and / or produced by recombinant DNA technology. (See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)).

[0333] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and similar agents compatible with the administration of pharmaceuticals. Suitable carriers are described in the most recent edition of Remington’s Pharmaceutical Sciences, a standard reference text in the art, which is incorporated herein by reference. Suitable embodiments 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 can also be used. The use of such media and agents for pharmaceutical active substances is well known in the art. The use thereof in therapeutic compositions is also contemplated, except for any conventional media or agents that are incompatible with the active compound.

[0334] Formulations for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0335] The pharmaceutical compositions of the present disclosure are formulated to be compatible with their intended route of administration. Embodiments of the route of administration include parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate, or phosphate, and agents for the adjustment of tonicity (e.g., sodium chloride or dextrose). The pH may be adjusted with an acid or a base (e.g., hydrochloric acid or sodium hydroxide). Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0336] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and its degree of fluidity should be such that it has easy injectability. It must be stable under the conditions of manufacture and storage, and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycols, etc.) and suitable mixtures thereof. The appropriate fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the desired particle size (in the case of a dispersion) and by using surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (such as, for example, p-hydroxybenzoic acid, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, isotonic agents (such as sugars, polyols (such as mannitol, sorbitol) or sodium chloride) are suitably incorporated into the composition. The long-acting absorption of an injectable composition can be achieved by incorporating a delaying absorbent (such as, for example, monostearate and gelatin) into the composition.

[0337] A sterile injectable solution can be prepared by incorporating the required amount of the active compound into a suitable solvent containing one or a combination (as required) of the ingredients listed above, followed by filtration sterilization. Generally, a dispersion is 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 the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which can produce a powder consisting of the active ingredient and any desired additional ingredients (from its previously sterile-filtered solution). If the composition is lyophilized, sterilization using this method can be carried out before or after lyophilization and reconstitution. Parenteral compositions can be stored in lyophilized form or in solution. Additionally, parenteral compositions are usually placed in a container with an infusion port, such as an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle.

[0338] In some embodiments, the pharmaceutical composition is administered to a subject by any route, including orally, transdermally, by inhalation, intravenously, intraarterially, intramuscularly, directly applied to a wound site, applied to a surgical site, intraperitoneally, by suppository, subcutaneously, intradermally, transdermally, by nebulization, intrapleurally, intracavitarily, intraarticularly, intravitreally or intraspinally.

[0339] Oral compositions generally include an inert diluent or an edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compounds can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compounds in the fluid carrier are orally administered and then rinsed and spat out or swallowed. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, carboxymethyl starch (Primogel), or corn starch; lubricants such as magnesium stearate or fully hydrogenated vegetable oil (Sterotes); glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0340] For administration by inhalation, the multispecific polypeptide construct is delivered in the form of an aerosol spray from a pressure vessel or dispenser or nebulizer containing a suitable propellant (such as a gas, e.g., carbon dioxide).

[0341] Systemic administration can also be effected by transmucosal or transdermal means. For transmucosal or transdermal administration, permeants suitable for the permeation barrier are used in the formulation. Such permeants are generally known in the art and, for example, for transmucosal administration, include detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished via the use of a nasal spray or a suppository. For transdermal administration, the active compounds are formulated as ointments, pastes, gels, or creams as are generally known in the art.

[0342] 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.

[0343] In one embodiment, a therapeutic agent is prepared with a carrier that can protect the compound from rapid elimination in the body, such as a sustained / controlled release formulation, including implant and microencapsulation delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The methods for preparing such formulations are obvious to those skilled in the art.

[0344] By way of example, the therapeutic agent can be incorporated into microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or coarse emulsion drops, prepared respectively by (e.g.) coacervation techniques or surface polymerization.

[0345] In some embodiments, the pharmaceutical composition comprises pharmaceutically acceptable excipients such as fillers, binders, coatings, preservatives, lubricants, flavoring agents, sweetening agents, coloring agents, solvents, buffering agents, chelating agents or stabilizers. Examples of pharmaceutically acceptable fillers include cellulose, calcium hydrogen phosphate, calcium carbonate, microcrystalline cellulose, sucrose, lactose, glucose, mannitol, sorbitol, maltitol, 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, 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 sweetening agents include sucrose, saccharin, aspartame or sorbitol. Examples of pharmaceutically acceptable buffering agents include carbonates, citrates, gluconates, acetates, phosphates or tartrates.

[0346] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, such matrices being in the form of shaped articles such as membranes or microcapsules. In some embodiments, the pharmaceutical composition further comprises a reagent for controlling or sustaining the release of the product, such as injectable microspheres, biodegradable particles, polymeric compounds (polylactic acid, polyglycolic acid), beads or liposomes. Examples of sustained release matrices include polyesters, hydrogels (such as poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), poly(lactide) (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ-L-glutamyl ethyl ester, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)) and poly-D-(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable the release of molecules for more than 100 days, certain hydrogels release proteins for a shorter period of time.

[0347] The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions, including liposomes targeted to infected cells with monoclonal antibodies against viral antigens, can also be used as pharmaceutically acceptable carriers. Such materials can be prepared according to methods known to those skilled in the art, such as those described in U.S. Patent No. 4,522,811.

[0348] Particularly advantageously, oral or parenteral compositions are formulated in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suitable as unit doses for use by a subject to be treated; each unit contains a predetermined quantity of the active compound which is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of this disclosure depend on and are directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of a subject.

[0349] Kits are further provided that contain the pharmaceutical compositions (or articles) described herein. Such pharmaceutical compositions can be packaged in a container, package, or dispenser together with instructions for administration. The kits described herein can also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for practicing any of the methods described herein.

[0350] The formulation can also contain more than one multispecific polypeptide construct required for the particular indication being treated, such as those having complementary activities that do not adversely affect each other. In some embodiments, or additionally, the composition can contain reagents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors. Such molecules are adapted to be present in combination in an amount effective for the intended purpose.

[0351] In some embodiments, the dosage of the pharmaceutical composition is a single dose or a repeated dose. In some embodiments, the dosage is administered to a subject once a day, twice a day, three times a day, four times a day, or more times a day. In some embodiments, about 1 or more (e.g., about 2 or more, about 3 or more, about 4 or more, about 5 or more, about 6 or more, or about 7 or more) doses are administered within a week. In some embodiments, multiple doses are administered over the course of days, weeks, months, or years. In some embodiments, the treatment course is about 1 or more doses (e.g., about 2 or more doses, about 3 or more doses, about 4 or more doses, about 5 or more doses, about 7 or more doses, about 10 or more doses, about 15 or more doses, about 25 or more doses, about 40 or more doses, about 50 or more doses, or about 100 or more doses).

[0352] In some embodiments, a pharmaceutical composition is administered to a subject. Generally, the dosage and route of administration of the pharmaceutical composition are determined according to the size and condition of the subject and according to standard medical practice. By way of example, a therapeutically effective dose can initially be estimated in cell culture assays or in animal models (e.g., mice, rats, rabbits, dogs, pigs, or monkeys). Animal models can also be used to determine appropriate concentration ranges and routes of administration. This information can then be used to determine the available dosage and route of administration in humans. The precise dosage will be determined according to factors relevant to the subject to be treated. The dosage and administration are adjusted to provide an adequate amount of the active compound or to maintain the desired effect. Factors that can be considered include the severity of the disease state, the general health of the subject, the age, weight, and sex of the subject, the time and frequency of administration, drug combinations, response sensitivity, and response to the therapy. A person skilled in the medical arts can readily determine the optimal dosage and treatment regimen for a particular patient by monitoring the signs of the patient's disease and adjusting the treatment accordingly.

[0353] IV. Methods of Use and Therapeutic Administration

[0354] Also provided are methods of using the multispecific polypeptide constructs and their uses. Such methods and uses include therapeutic methods and uses, e.g., involving administering a molecule or a composition comprising the same to a subject suffering from a disease, disorder, or condition such as a tumor or cancer. In some embodiments, the molecule and / or composition is administered in an effective amount to effectively treat the disease or disorder. Uses include the use of the multispecific polypeptide constructs in such methods and treatments, and in the preparation of medicaments for 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 suffering from or suspected of suffering from a disease or disorder. In some embodiments, the method thereby treats the disease, disorder, or condition of the subject.

[0355] In one embodiment, the multispecific polypeptide constructs of the present disclosure can be used as therapeutic agents. Such agents will generally be used to diagnose, prognose, monitor, treat, palliate, and / or prevent diseases or conditions in a subject. A treatment regimen is implemented by identifying a subject (e.g., a human patient or other mammal) having a disorder (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 have an effect due to its binding to a target.

[0356] In some embodiments, provided herein are methods of 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 of modulating the immune response increases or enhances the immune response of the subject. For example, the increased or enhanced response can be an increase in cell-mediated immunity. In some embodiments, the method increases T cell activity, such as cytotoxic T lymphocyte (CTL) activity. In some embodiments, the modulated (e.g., increased) immune response is against a tumor or cancer.

[0357] Administration of the multispecific polypeptide construct can activate innate immune cells via engagement of the Fc-region of the multispecific polypeptide construct by FcγR. Administration of the multispecific polypeptide construct can agonize, stimulate, activate, and / or augment innate immune cell effector functions, including ADCC, cytokine release, degranulation, and / or ADCP. Once the linker connecting the first and second components is cleaved by a protease, thereby allowing the anti-CD3 binding moiety to bind CD3ε on T cells, administration of the multispecific polypeptide construct can activate T cells. Administration of the multispecific polypeptide construct can agonize, stimulate, activate, and / or augment CD3-mediated T cell activation, cytotoxicity, cytokine release, and / or proliferation.

[0358] In some embodiments, the provided methods are used to treat a disease or condition in a subject by administering a therapeutically effective amount of any of the provided multispecific conjugates or pharmaceutical compositions. In some embodiments, the disease or condition is a tumor or cancer. Generally, alleviating or treating a disease or disorder involves 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 the drug can accomplish one or a combination of the following: reducing the number of cancer cells; reducing tumor size; inhibiting (i.e., reducing to a certain extent / or stopping) cancer cell infiltration into peripheral organs; inhibiting tumor metastasis; inhibiting tumor growth to a certain extent; and / or alleviating one or more symptoms associated with cancer to a certain extent. 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 companion animal (e.g., a cat, dog, horse), a farm animal, a working animal, or a zoo animal). The terms subject and patient are used interchangeably herein.

[0359] In some embodiments, the pharmaceutical composition can be used to inhibit the growth of mammalian cancer cells (such as human cancer cells). The methods for treating cancer can include administering an effective amount of any one of the pharmaceutical compositions described herein to a subject having cancer. An effective amount of the pharmaceutical composition can be administered to inhibit, arrest, or reverse the progression of cancer. Human cancer cells can be treated in vivo or ex vivo. In the ex vivo treatment of a human patient, a tissue or fluid containing cancer cells is treated in vitro and then the tissue or fluid is reintroduced into the patient. In some embodiments, in some embodiments, cancer in a human patient is treated by administering a therapeutic composition to the patient.

[0360] Non-limiting embodiments of the disease include: all types of cancer (such as breast cancer, lung cancer, colorectal cancer, prostate cancer, melanoma, head and neck cancer, and pancreatic cancer, etc.), rheumatoid arthritis, Crohn's disease, SLE, cardiovascular injury, ischemia, etc. For example, the indications will include leukemia (including T-cell acute lymphoblastic leukemia (T-ALL)), lymphoblastic diseases (including multiple myeloma), and solid tumors (including lung tumors, colorectal tumors, prostate tumors, pancreatic tumors, and breast tumors (including triple-negative breast cancer)). For example, the indications include bone diseases or cancer metastases, which are independent of the primary tumor origin; breast cancer, including (by non-limiting embodiments) ER / PR+ breast cancer, Her2+ breast cancer, triple-negative breast cancer; colorectal cancer; endometrial cancer; gastric cancer; glioblastoma; head and neck cancer, such as esophageal cancer; lung cancer, such as by non-limiting embodiments, non-small cell lung cancer; multiple myeloma, ovarian cancer; pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; kidney cancer, such as by non-limiting embodiments, renal cell carcinoma; and / or skin cancer, such as by non-limiting embodiments, 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.

[0361] A therapeutically effective amount of the multispecific polypeptide construct of the present disclosure generally refers to the amount required to achieve a therapeutic goal. As described above, this can be the binding interaction between the multispecific polypeptide construct and its target antigen, which, in some cases, agonizes, stimulates, activates, and / or enhances FcγR-mediated innate immune cell activation or CD3-mediated T cell activation. The amount to be administered will further depend on the binding affinity of the multispecific polypeptide construct for its specific antigen and also on the rate at which the administered multispecific polypeptide construct is depleted from the free volume of the other subject to which it is administered. A common range for the therapeutically effective dose of the multispecific polypeptide construct can be (by non-limiting example) from about 0.01 μg / kg body weight to about 10 mg / kg body weight. In some embodiments, the therapeutically effective dose of the multispecific polypeptide construct of the present disclosure can be (by non-limiting example) from about 0.01 mg / kg body weight to about 5-10 mg / kg body weight. Common dosing frequencies can range, for example, from twice daily to once a week.

[0362] The effectiveness of the treatment is determined in combination with any known method for diagnosing or treating a particular disorder. Methods for screening multispecific polypeptide constructs for desired specificities include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immuno-mediated techniques known in the art. There are a variety of known ways to determine whether administration of the provided multispecific polypeptide construct is sufficient to modulate immune activity: inducing, generating, or turning on immune cells that mediate or are capable of mediating a protective immune response; altering the physical or functional properties of immune cells; or combinations of such effects. Examples of measurements of modulation of immune activity include, but are not limited to, examining the presence or absence of immune cell populations (using flow cytometry, immunohistochemistry, histology, electron microscopy, polymerase chain reaction (PCR)); measuring the functional capabilities of immune cells, including the ability to proliferate or divide in response to signals or resistance thereto (e.g., T-cell proliferation assays and peptide scanning assays based on 3H-thymidine incorporation after stimulation with anti-CD3 antibody, anti-T cell receptor antibody, anti-CD28 antibody, calcium ionophore, PMA (phorbol 12-myristate 13-acetate) antigen-presenting cells loaded with peptide or protein antigen; B-cell proliferation assays); measuring the ability to kill or lyse other cells (e.g., cytotoxic T cell assays); measuring cytokines, chemokines, cell surface molecules, antibodies, and other cell products (e.g., by flow cytometry, enzyme-linked immunosorbent assay, Western blot analysis, protein microarray analysis, immunoprecipitation analysis); measuring biochemical markers of activation of signal transduction pathways within immune cells or immune cells (e.g., Western blot and immunoprecipitation analysis of tyrosine, serine, or threonine phosphorylation, polypeptide cleavage and formation or dissociation of protein complexes; protein array analysis; DNA transcription, typing using DNA arrays or subtractive hybridization); measuring cell death by apoptosis, necrosis, or other mechanisms (e.g., annexin V staining, TUNEL assay, gel electrophoresis to measure DNA ladders, histology; fluorescence probe caspase assays, Western blot analysis of caspase substrates); measuring genes, proteins, and other molecules produced by immune cells (e.g., Northern blot analysis, polymerase chain reaction, DNA microarrays, protein microarrays, two-dimensional gel electrophoresis, Western blot analysis, enzyme-linked immunosorbent assay, flow cytometry); measuring clinical symptoms or outcomes, such as by measuring recurrence rates or disease severity, e.g., improvement in autoimmune, neurodegenerative, and other diseases involving self-proteins or self-polypeptides (clinical scoring, need for additional therapy, functional status, imaging studies).

[0363] Multispecific polypeptide constructs can also be used in a variety of diagnostic and prophylactic formulations. In one embodiment, a multispecific polypeptide construct is administered to a patient at risk of developing one or more of the aforementioned conditions. The predisposition of a patient or organ to one or more conditions can be determined using genotypic, serological, or biochemical markers.

[0364] In another embodiment of the invention, a multispecific polypeptide construct is administered to a human subject diagnosed with a clinical indication associated with one or more of the aforementioned conditions. At the time of diagnosis, the multispecific polypeptide construct is administered to mitigate or reverse the effects of the clinical indication.

[0365] Combination therapy

[0366] In some embodiments, a multispecific polypeptide construct, a conjugated multispecific polypeptide construct, and their compositions (collectively referred to herein as therapeutic agents) are administered in combination with one or more additional reagents or a combination of additional reagents. Suitable additional reagents include current medical and / or surgical therapies for the intended application. For example, the therapeutic agent can be used in combination with an additional chemotherapeutic agent or an anti-tumor agent. For example, the therapeutic agent and the additional reagent are formulated into a single therapeutic composition, and the therapeutic agent and the additional reagent are administered simultaneously. In some embodiments, the therapeutic agent and the additional reagent are separate from each other, e.g., each is formulated into a separate therapeutic composition, and the therapeutic agent and the additional reagent are administered simultaneously, or the therapeutic agent and the additional reagent are administered at different times during the treatment regimen. For example, the therapeutic agent is administered before the additional reagent, the therapeutic agent is administered after the additional reagent, or the therapeutic agent and the additional reagent are administered in an alternating manner. As described herein, the therapeutic agent and the additional reagent are administered in a single dose or multiple doses. In some embodiments, the additional reagent is conjugated or otherwise attached to the therapeutic agent. Suitable additional reagents are selected according to the purpose of the intended application (i.e., killing, preventing cell proliferation, hormone therapy, or gene therapy). Such reagents can include, but are not limited to, for example, pharmaceutical agents, toxins, fragments of toxins, alkylating agents, enzymes, antibiotics, antimetabolites, anti-proliferative agents, hormones, neurotransmitters, DNA, RNA, siRNA, oligonucleotides, antisense RNA, aptamers, diagnostic agents, radiopaque dyes, radioisotopes, fluorescent probe compounds, magnetic labels, nanoparticles, labeled compounds, lectins, compounds that alter cell membrane permeability, photochemical compounds, small molecules, liposomes, micelles, gene therapy vectors, viral vectors, etc. Finally, combinations of reagents or combinations of different classes of reagents can be used.

[0367] In one embodiment, the multispecific polypeptide construct is administered in a combination therapy, i.e., in combination with other agents (e.g., therapeutic agents) that can be used to treat a pathological condition or disorder (e.g., autoimmune disorders and inflammatory diseases). The term "combination" in this context means that such agents are administered substantially simultaneously, simultaneously, or sequentially. If administered sequentially, at the start of the administration of the second compound, the first of the two compounds can still be detected at an effective concentration at the treatment site.

[0368] For example, the combination therapy can include co-formulating and / or co-administering one or more multispecific polypeptide constructs of the present disclosure with one or more additional therapeutic agents (e.g., one or more cytokine and growth factor inhibitors, immunosuppressive agents, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxic or cell growth inhibitors, as elaborated in more detail below). In addition, one or more multispecific polypeptide constructs described herein can be used in combination with two or more therapeutic agents described herein. Such combination therapies can advantageously utilize lower doses of the administered therapeutic agents, thereby avoiding possible toxicities or complications associated with various single therapies.

[0369] In other embodiments, one or more multispecific polypeptide constructs of the present disclosure are co-formulated and / or co-administered with one or more anti-inflammatory agents, immunosuppressive agents, or metabolic or enzyme inhibitors. Non-limiting embodiments of drugs or inhibitors that can be used in combination with the antibodies described herein include one or more of the following: non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, tenidap, naproxen, meloxicam, piroxicam, diclofenac, and indomethacin; sulfasalazine; corticosteroids, such as prednisolone; cytokine inhibitory anti-inflammatory drugs (CSAIDs); inhibitors of nucleotide biosynthesis, e.g., inhibitors of purine biosynthesis, folate antagonists (e.g., methotrexate (N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid)); and inhibitors of pyrimidine biosynthesis, such as 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).

[0370] Examples of additional inhibitors include one or more of the following: corticosteroids (oral, inhaled, and locally injected); immunosuppressive agents such as cyclosporin, tacrolimus (FK-506); and mTOR inhibitors such as sirolimus (rapamycin - RAPAMUNE TM or rapamycin derivatives such as soluble rapamycin derivatives (e.g., ester rapamycin derivatives such as CCI-779); agents that interfere with pro-inflammatory cytokine signaling such as TNFα or IL-1 (e.g., IRAK, NIK, IKK, p38, or MAP kinase inhibitors); COX2 inhibitors such as celecoxib, rofecoxib, and their variants; phosphodiesterase inhibitors such as R973401 (phosphodiesterase type IV inhibitor); phospholipase inhibitors such as inhibitors of cytosolic phospholipase 2 (cPLA2) (e.g., trifluoromethyl ketone analogs); inhibitors of vascular endothelial growth factor or growth factor receptors such as VEGF inhibitors and / or VEGF-R inhibitors; and inhibitors of angiogenesis. Therapeutic agents suitable for use in combination with the antibodies of the present disclosure are immunosuppressive agents such as cyclosporin, tacrolimus (FK-506); mTOR inhibitors such as sirolimus (rapamycin) or rapamycin derivatives such as soluble rapamycin derivatives (e.g., ester rapamycin derivatives such as CCI-779); COX2 inhibitors such as celecoxib and its variants; and phospholipase inhibitors such as inhibitors of cytosolic phospholipase 2 (cPLA2), such as trifluoromethyl ketone analogs. Additional examples of therapeutic agents that can be combined with the multispecific polypeptide constructs include one or more of the following: 6-mercaptopurine (6-MP); azathioprine sulfasalazine; mesalazine; olsalazine; chloroquine / hydroxychloroquine penicillamine; gold thiomalate (intramuscular and oral); azathioprine; colchicine; β-2 adrenergic receptor agonists (salbutamol, terbutaline, salmeteral); xanthines (theophylline, aminophylline); cromolynate; nedocromil; ketotifen; ipratropium and oxitropium; mycophenolate mofetil; adenosine agonists; antithrombotic agents; complement inhibitors; and adrenergic agents.

[0371] V. Exemplary Embodiments

[0372] The embodiments provided are:

[0373] 1. A multispecific polypeptide construct, the multispecific polypeptide construct comprising a first component containing an immunoglobulin Fc region and a second component containing a CD3 binding region, wherein:

[0374] the first component and the second component are coupled by a linker, wherein the Fc region is located at the N-terminus of the CD3 binding region; and

[0375] one or both of the first component and the second component comprise an antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0376] 2. The multispecific polypeptide construct according to embodiment 1, wherein the CD3 binding region binds to CD3 (CD3ε).

[0377] 3. The multispecific construct according to embodiment 1 or embodiment 2, wherein the antigen-binding domain is located at the amino terminus relative to the Fc region of the multispecific polypeptide construct and / or at the carboxyl terminus relative to the CD3 binding region.

[0378] 4. The multispecific polypeptide construct according to any one of embodiments 1 to 3, wherein the first component comprises a first antigen-binding domain and the second component comprises a second antigen-binding domain, wherein each of such antigen-binding domains binds to a tumor-associated antigen (TAA).

[0379] 5. The multispecific polypeptide construct according to embodiment 4, wherein the first antigen-binding domain is located at the amino terminus relative to the Fc region of the multispecific construct and the second antigen-binding domain is located at the carboxyl terminus relative to the CD3 binding region of the multispecific construct.

[0380] 6. A multispecific polypeptide construct, wherein the multispecific construct sequentially comprises from the N-terminus to the C-terminus:

[0381] a first antigen-binding domain that binds to a tumor-associated antigen (TAA);

[0382] an immunoglobulin Fc region;

[0383] a linker;

[0384] a CD3 binding region that binds to CD3 (CD3ε); and

[0385] a second antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0386] 7. A multispecific polypeptide construct, wherein the multispecific construct sequentially comprises from the N-terminus to the C-terminus:

[0387] an immunoglobulin Fc region;

[0388] Connector;

[0389] A CD3-binding region that binds to CD3 (CD3ε); and

[0390] An antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0391] 8. A multispecific polypeptide construct, wherein the multispecific construct sequentially comprises, from the N-terminus to the C-terminus:

[0392] An antigen-binding domain that binds to a tumor-associated antigen (TAA);

[0393] An immunoglobulin Fc region;

[0394] A connector; and

[0395] A CD3-binding region that binds to CD3 (CD3ε).

[0396] 9. The multispecific polypeptide construct according to any one of embodiments 1 to 8, wherein the Fc region is a homodimeric Fc region.

[0397] 10. The multispecific polypeptide construct according to any one of embodiments 1 to 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.

[0398] 11. The multispecific polypeptide construct according to any one of embodiments 1 to 10, wherein the Fc region comprises a polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 1.

[0399] 12. The multispecific polypeptide construct according to any one of embodiments 1 to 10, wherein the Fc region comprises a polypeptide containing the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 2;

[0400] The Fc region comprises a polypeptide containing the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 4; or

[0401] The Fc region comprises a polypeptide containing the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 5.

[0402] 13. The multispecific polypeptide construct according to any one of embodiments 1 to 6, 9 and 12, wherein the Fc region is a heterodimeric Fc region.

[0403] 14. The multispecific polypeptide construct according to embodiment 13, wherein compared to the polypeptide of the homodimeric Fc region, optionally compared to the Fc polypeptide shown in SEQ ID NO: 1 or an immunologically active fragment thereof, one or both of the Fc polypeptides of the heterodimeric Fc region comprise at least one modification to induce heterodimerization.

[0404] 15. The multispecific polypeptide construct according to embodiment 14, wherein each of such Fc polypeptides of the heterodimeric Fc independently comprises at least one amino acid modification.

[0405] 16. The multispecific polypeptide construct according to embodiment 15, wherein each of such Fc polypeptides of the heterodimeric Fc comprises a knob-into-hole structure modification or comprises a charge mutation to increase the electrostatic complementarity of such polypeptides.

[0406] 17. The multispecific polypeptide construct according to embodiment 16, wherein the amino acid modification is a knob-into-hole structure modification.

[0407] 18. The multispecific fusion polypeptide according to any one of embodiments 13 to 17, wherein the first Fc polypeptide of the heterodimeric Fc comprises a modification selected from Thr366Ser, Leu368Ala, Tyr407Val and combinations thereof and the second Fc polypeptide of the heterodimeric Fc comprises the modification T366W.

[0408] 19. The multispecific fusion polypeptide according to embodiment 18, wherein the first Fc polypeptide and the second Fc polypeptide further comprise a modification from 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.

[0409] 20. The multispecific polypeptide construct according to embodiment 16, wherein the amino acid modification is a charge mutation to increase the electrostatic complementarity of such polypeptides.

[0410] 21. A multispecific polypeptide construct according to any one of embodiments 13 to 16 and 20, wherein the first Fc polypeptide and / or the second Fc polypeptide, or each of the first Fc polypeptide and the second Fc polypeptide, comprises a modification at a complementary position, wherein the modification is a replacement of an amino acid with an amino acid having a charge opposite to that of the complementary amino acid of the other polypeptide.

[0411] 22. A multispecific polypeptide construct according to any one of embodiments 14 to 21, wherein one of the first Fc polypeptide or the second Fc polypeptide of the heterodimeric Fc further comprises a modification of residue Ile253.

[0412] 23. The multispecific polypeptide construct according to embodiment 22, wherein the modification is Ile253Arg.

[0413] 24. A multispecific polypeptide construct according to any one of embodiments 14 to 23, wherein one of the first Fc polypeptide or the second Fc polypeptide of the heterodimeric Fc further comprises a modification of residue His435.

[0414] 25. The multispecific polypeptide construct according to embodiment 24, wherein the modification is His435Arg.

[0415] 26. A multispecific polypeptide construct according to any one of embodiments 1 to 25, wherein the Fc region comprises a polypeptide without Lys447.

[0416] 27. A multispecific polypeptide construct according to any one of embodiments 1 to 26, wherein the Fc region comprises a polypeptide containing at least one modification to enhance FcRn binding.

[0417] 28. The multispecific fusion polypeptide according to embodiment 27, wherein the modification is at a position selected from the group consisting of: Met252, Ser254, Thr256, Met428, Asn434, and combinations thereof.

[0418] 29. The multispecific fusion polypeptide according to embodiment 28, wherein the modification is at a position selected from the group consisting of: Met252Y, Ser254T, Thr256E, Met428L, Met428V, Asn434S, and combinations thereof.

[0419] 30. The multispecific fusion polypeptide according to embodiment 28, wherein the modification is at position Met252 and position Met428.

[0420] 31. The multispecific fusion polypeptide according to embodiment 30, wherein the modification is Met252Y and Met428L.

[0421] 32. The multispecific fusion polypeptide of embodiment 30, wherein the modification is Met252Y and Met428V.

[0422] 33. The multispecific polypeptide construct of any one of embodiments 13 to 32, wherein the first polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NO: 82, 86, 94 or 96, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NO: 83, 87, 90, 92, 98 or 100.

[0423] 34. The multispecific polypeptide construct of any one of embodiments 1 to 33, wherein the Fc region comprises a polypeptide containing at least one amino acid modification that reduces effector function and / or reduces binding to an effector molecule selected from Fcγ receptors or C1q.

[0424] 35. The multispecific polypeptide construct of embodiment 34, wherein the one or more amino acid modifications are deletion of one or more of Glu233, Leu234 or Leu235.

[0425] 36. The multispecific polypeptide construct of any one of embodiments 13 to 32, 34 and 35, wherein the first polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NO: 84, 88, 95 or 97 and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NO: 85, 89, 91, 93, 99 or 101.

[0426] 37. The multispecific polypeptide construct of any one of embodiments 1 to 32, wherein the Fc region comprises a polypeptide containing at least one modification to enhance FcγR binding.

[0427] 38. The multispecific polypeptide construct of embodiment 37, wherein the modification is a modification of Ser239 or Ile332.

[0428] 39. The multispecific polypeptide construct of any one of embodiments 1 to 32 and 37, wherein the glycosylation of the Fc region is modified to enhance FcγR binding as compared to an unmodified Fc region.

[0429] 40. The multispecific polypeptide construct of embodiment 39, wherein the Fc region has no or reduced fucose content.

[0430] 41. The multispecific polypeptide construct of any one of embodiments 1 to 40, wherein the CD3 binding region is an anti-CD3 antibody or antigen-binding fragment.

[0431] 42. The multispecific polypeptide construct of embodiment 41, wherein the anti-CD3 antibody or antigen-binding fragment comprises a variable heavy chain region (VH) and a variable light chain region (VL).

[0432] 43. The multispecific polypeptide construct of any one of embodiments 1 to 42, wherein the CD3-binding region is monovalent.

[0433] 44. The multispecific polypeptide construct of any one of embodiments 41 to 43, wherein the anti-CD3 antibody or antigen-binding fragment is not a single-chain antibody, optionally not a single-chain variable fragment (scFv).

[0434] 45. The multispecific polypeptide construct of embodiment 42 or embodiment 44, wherein the Fc is a heterodimeric Fc, and the VH and the VL constituting the anti-CD3 antibody or antigen-binding fragment are linked to opposite polypeptides of the heterodimeric Fc.

[0435] 46. The multispecific polypeptide construct of any one of embodiments 1 to 45, wherein the CD3-binding region cannot or substantially cannot bind or engage CD3 unless at least one of the antigen-binding domains binds to its TAA.

[0436] 47. The multispecific polypeptide construct of any one of embodiments 1 to 46, wherein the CD3-binding region cannot or substantially cannot bind or engage CD3 unless at least two of the antigen-binding domains bind to its TAA.

[0437] 48. The multispecific polypeptide construct of any one of embodiments 1 to 47, wherein the linker is a polypeptide linker.

[0438] 49. The multispecific polypeptide construct of embodiment 48, wherein the linker is a polypeptide that is at most 25 amino acids in length.

[0439] 50. A multispecific polypeptide construct according to embodiment 48 or embodiment 49, wherein the linker is or 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 length.

[0440] 51. A multispecific polypeptide construct according to any one of embodiments 48 to 50, wherein the linker is a polypeptide that is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length.

[0441] 52. A multispecific polypeptide construct according to any one of embodiments 1 to 51, wherein the linker is a cleavable linker.

[0442] 53. A multispecific polypeptide construct comprising a first component containing a heterodimeric Fc region and a second component comprising an anti-CD3 antibody or antigen-binding fragment containing a variable heavy chain region (VH) and a variable light chain region (VL), wherein:

[0443] The VH and the VL constituting the anti-CD3 antibody or antigen-binding fragment are linked to opposite polypeptides of the heterodimeric Fc;

[0444] The first component and the second component are coupled by a cleavable linker, wherein the heterodimeric Fc region is located at the N-terminus of the anti-CD3 antibody; and

[0445] One or both of the first component and the second component comprise an antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0446] 54. A multispecific polypeptide construct according to embodiment 52 or embodiment 53, wherein when the multispecific polypeptide construct is in an uncleaved state compared to a cleaved state, the binding of the CD3-binding region to CD3 is significantly reduced.

[0447] 55. A multispecific polypeptide according to any one of embodiments 52 to 54, wherein in the cleaved state, the first component and the second component are not linked.

[0448] 56. A multispecific polypeptide construct according to any one of embodiments 52 to 55, wherein the cleavable linker is a polypeptide that serves as a substrate for a protease.

[0449] 57. The multispecific polypeptide construct according to embodiment 56, wherein the protease is produced by immune effector cells, by a tumor, or by cells present in the tumor microenvironment.

[0450] 58. The multispecific polypeptide construct according to embodiment 57, wherein the protease is produced by immune effector cells, and the immune effector cells are activated T cells, natural killer (NK) cells, or NK T cells.

[0451] 59. The multispecific polypeptide construct according to any one of embodiments 56 to 58, wherein the protease is selected from interstitial proteases, matrix metalloproteinases (MMPs), granzyme B, and combinations thereof.

[0452] 60. The multispecific polypeptide construct according to embodiment 59, wherein the protease is granzyme B.

[0453] 61. The multispecific polypeptide construct according to any one of embodiments 52 to 60, wherein the cleavable linker comprises an amino acid sequence of the general formula P4 P3 P2 P1 ↓ P1’ (SEQ ID NO: 150), wherein P4 is 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.

[0454] 62. The multispecific polypeptide construct according to any one of embodiments 52 to 61, wherein the cleavable linker comprises an amino acid sequence of the general formula P4 P3 P2 P1 ↓ P1’ (SEQ ID NO: 151), wherein P4 is 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.

[0455] 63. A multispecific polypeptide construct according to any one of embodiments 52 to 62, 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).

[0456] 64. A multispecific polypeptide construct according to any one of embodiments 52 to 63, wherein the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 105 - 112, 136 - 141, 148, 150 - 153.

[0457] 65. A multispecific polypeptide construct according to embodiment 59, wherein the protease is a stromelysin.

[0458] 66. A multispecific polypeptide construct according to any one of embodiments 52 to 65, wherein:

[0459] the cleavable linker comprises the sequence P1QAR ↓(A / V) (SEQ ID NO: 154), where P1 is any amino acid; or

[0460] the cleavable linker comprises the sequence RQAR(A / V) (SEQ ID NO: 155).

[0461] 67. A multispecific polypeptide construct according to any one of embodiments 52 to 66, wherein the cleavable linker comprises the sequence RQARV (SEQ ID NO: 156).

[0462] 68. A multispecific polypeptide construct according to any one of embodiments 52 to 67, wherein the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 23, 154 - 156.

[0463] 69. A multispecific polypeptide construct according to embodiment 59, wherein the protease is MMP.

[0464] 70. A multispecific polypeptide construct according to embodiment 69, wherein the MMP is MMP - 2.

[0465] 71. A multispecific polypeptide construct according to any one of embodiments 52 to 70, wherein the cleavable linker comprises the general formula P3 P2 P1 ↓ P1’ (SEQ ID NO: 157), where P3 is P, V or A; P2 is Q or D; P1 is A or N; and P1’ is L, I or M.

[0466] 72. A multispecific polypeptide construct according to any one of embodiments 52 to 71, wherein the cleavable linker comprises the general formula P3 P2 P1 ↓ P1’ (SEQ ID NO: 158), where P3 is P; P2 is Q or D; P1 is A or N; and P1’ is L or I.

[0467] 73. A multispecific polypeptide construct according to any one of embodiments 52 to 72, wherein the cleavable linker comprises the sequence PAGL (SEQ ID NO: 24).

[0468] 74. A multispecific polypeptide construct according to any one of embodiments 52 to 73, 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.

[0469] 75. A multispecific polypeptide construct according to any one of embodiments 45 to 74, wherein the multispecific polypeptide construct comprises at least (i) a first polypeptide comprising the first Fc polypeptide of the heterodimeric Fc region, the linker, and the VH domain of the anti-CD3 antibody or antigen-binding fragment; and (ii) a second polypeptide comprising the second Fc polypeptide of the heterodimeric Fc region, the linker, and the VL domain of the anti-CD3 antibody or antigen-binding fragment, wherein one or both of the first polypeptide and the second polypeptide comprise at least one antigen-binding domain that binds to a tumor-associated antigen (TAA).

[0470] 76. A multispecific polypeptide construct according to any one of embodiments 1 to 75, wherein one or more antigen-binding domains that bind to the TAA bind to the TAA monovalently, divalently, trivalently, or tetravalently.

[0471] 77. The multispecific polypeptide construct according to embodiment 75, wherein only one of the first polypeptide or the second polypeptide comprises the at least one antigen-binding domain that binds to the TAA.

[0472] 78. A multispecific polypeptide construct according to embodiment 75 or embodiment 77, wherein the at least one antigen-binding domain is located at the amino terminus of one of the first polypeptide or the second polypeptide of the multispecific polypeptide construct relative to the Fc region and / or at the carboxyl terminus relative to the CD3-binding region.

[0473] 79. A multispecific polypeptide construct according to embodiment 75 or embodiment 77, wherein the at least one antigen-binding domain is located at the amino terminus relative to the Fc region of the multispecific construct and the second antigen-binding domain is located at the carboxyl terminus relative to the CD3-binding region of the multispecific construct.

[0474] 80. A multispecific polypeptide construct according to any one of embodiments 1 to 79, wherein the antigen-binding domain or each of such antigen-binding domains independently comprises the extracellular domain or a binding fragment thereof of the natural cognate binding partner of the TAA or a variant thereof that exhibits binding activity to the TAA.

[0475] 81. A multispecific polypeptide construct according to any one of embodiments 1 to 79, wherein the antigen-binding domain or each of such antigen-binding domains independently is an antibody or an antigen-binding fragment thereof selected from the group consisting of: Fab fragment, F(ab′)2 fragment, Fv fragment, scFv, scAb, dAb, single domain heavy chain antibody, and single domain light chain antibody.

[0476] 82. A multispecific polypeptide construct according to embodiment 81, wherein the antibody or antigen-binding fragment thereof is Fv, scFv, Fab, single domain antibody (sdAb), V NAR or V H H.

[0477] 83. A multispecific polypeptide construct according to embodiment 81 or embodiment 82, wherein the antibody or antigen-binding fragment is sdAb.

[0478] 84. A multispecific polypeptide construct according to embodiment 83, wherein the sdAb is a human or humanized sdAb.

[0479] 85. A multispecific polypeptide construct according to embodiment 83 or embodiment 84, wherein the sdAb is V H H, V NAR , a modified VH domain or a modified VK domain.

[0480] 86. A multispecific polypeptide construct according to embodiment 81 or embodiment 82, wherein the antibody or antigen-binding fragment thereof is scFv.

[0481] 87. A multispecific polypeptide construct according to embodiment 81 or embodiment 82, wherein the antibody or antigen-binding fragment thereof is a Fab.

[0482] 88. A multispecific polypeptide construct according to embodiment 87, wherein the multispecific polypeptide construct comprises:

[0483] (i) A first polypeptide comprising the first Fc polypeptide of the heterodimeric Fc region, the linker, and the VH domain of the anti-CD3 antibody or antigen-binding fragment;

[0484] (ii) A second polypeptide comprising the second Fc polypeptide of the heterodimeric Fc region, the linker, and the VL domain of the anti-CD3 antibody or antigen-binding fragment, and

[0485] (iii) A third polypeptide comprising the VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen, wherein the first polypeptide and / or the second polypeptide further comprises the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment.

[0486] 89. A multispecific polypeptide construct according to embodiment 88, wherein only one of the first polypeptide or the second polypeptide comprises the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment.

[0487] 90. A multispecific polypeptide construct according to embodiment 89, wherein both the first polypeptide and the second polypeptide comprise the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment.

[0488] 91. A multispecific polypeptide construct according to embodiment 89 or embodiment 90, wherein the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment is located at the amino terminus of one of the first polypeptide or the second polypeptide of the multispecific polypeptide construct relative to the Fc region and / or at the carboxyl terminus relative to the CD3-binding region.

[0489] 92. A multispecific polypeptide construct according to any one of embodiments 89 to 91, wherein the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment is located at the amino terminus relative to the Fc region of the first polypeptide or the second polypeptide and at the carboxyl terminus relative to the CD3-binding region of the other of the first polypeptide or the second polypeptide.

[0490] 93. A multispecific polypeptide construct according to any one of embodiments 1 to 92, wherein the antigen-binding domain or each of such antigen-binding domains independently binds to a tumor antigen selected from the following: 1-92-LFA-3, 5T4, α-4 integrin, α-V integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis-Y, Ephrin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9 (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), Claudin-3, Claudin-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor α (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, 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, 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, Nakasteroin protein, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidyl-serine, 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.

[0491] 94. A multispecific polypeptide construct according to any one of embodiments 1 to 93, wherein the multispecific antigen-binding domain comprises at least a first antigen-binding domain and a second antigen-binding domain, and wherein the first antigen-binding domain and the second antigen-binding domain bind to the same TAA.

[0492] 95. The multispecific polypeptide construct of embodiment 94, wherein the first antigen-binding domain and the second antigen-binding domain bind to different epitopes on the same TAA.

[0493] 96. The multispecific polypeptide construct of embodiment 94, wherein the first antigen-binding domain and the second antigen-binding domain bind to the same epitope on the same TAA.

[0494] 97. The multispecific polypeptide construct of any one of embodiments 1 to 96, wherein the multispecific antigen-binding domain comprises at least a first antigen-binding domain and a second antigen-binding domain, and wherein the first antigen-binding domain binds to a different TAA than the second antigen-binding domain.

[0495] 98. The multispecific polypeptide construct of any one of embodiments 5 to 97, wherein the multispecific polypeptide construct comprises a first linker peptide (LP1) between the first antigen-binding domain and the Fc region.

[0496] 99. The multispecific polypeptide construct of any one of embodiments 5 to 98, wherein the multispecific polypeptide construct comprises a second linker peptide (LP2) between the CD3-binding region and the second antigen-binding domain.

[0497] 100. The multispecific polypeptide construct of any one of embodiments 5 to 99, wherein the multispecific polypeptide construct comprises a first linker peptide (LP1) between the first antigen-binding domain and the Fc region and a second linker peptide (LP2) between the CD3-binding region and the second antigen-binding domain, and wherein the multispecific polypeptide construct has the following structural configuration from the N-terminus to the C-terminus: first antigen-binding domain - LP1 - Fc region - linker - CD3-binding region - LP2 - second antigen-binding domain.

[0498] 101. The multispecific polypeptide construct of embodiment 100, wherein the linker is a cleavable linker.

[0499] 102. The multispecific polypeptide construct of embodiments 100 and 101, wherein the two linker peptides are different from each other.

[0500] 103. The multispecific polypeptide construct of any one of embodiments 98 to 102, wherein LP1 or LP2 is independently a peptide about 1 to 20 amino acids in length.

[0501] 104. The multispecific polypeptide of embodiment 103, wherein LP1 or LP2 independently comprises a peptide that is or comprises any Gly-Ser linker as shown in SEQ ID NOs: 10-13, 119, 135, 147, 149 or GGS.

[0502] 105. The multispecific polypeptide construct of any one of embodiments 41 to 104, wherein the anti-CD3 antibody or antigen-binding fragment is an Fv antibody fragment.

[0503] 106. The multispecific polypeptide construct of embodiment 105, wherein the Fv antibody fragment comprises a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv).

[0504] 107. The multispecific polypeptide construct of any one of embodiments 41 to 106, wherein the anti-CD3 antibody or antigen-binding fragment comprises VH CDR1, which comprises the amino acid sequence TYAMN (SEQ ID NO: 16); VH CD2, which comprises the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 17); VH CDR3, which comprises the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18); VL CDR1, which comprises the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 19); VL CDR2, which comprises the amino acid sequence GTNKRAP (SEQ ID NO: 20); and VL CDR3, which comprises the amino acid sequence ALWYSNLWV (SEQ ID NO: 21).

[0505] 108. The multispecific polypeptide construct of embodiment 106 or embodiment 107, wherein the anti-CD3 dsFv comprises:

[0506] VH, which has the amino acid sequence of any one of SEQ ID NOs: 14 and 32-62 or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 14 and 32-62; and

[0507] VL, which has the amino acid sequence of any one of SEQ ID NOs: 15 and 63-81 or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 14 and 32-62.

[0508] 109. The multispecific polypeptide construct according to any one of embodiments 106 to 108, 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.

[0509] 110. The multispecific polypeptide construct according to any one of embodiments 102 to 104, wherein the anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO: 44 and the amino acid sequence of SEQ ID NO: 72.

[0510] 111. The multispecific polypeptide construct according to any one of embodiments 1 to 109, wherein the multispecific polypeptide construct is conjugated to a reagent.

[0511] 112. The multispecific polypeptide construct according to embodiment 111, wherein the reagent is a therapeutic agent, an anti-tumor agent, a toxin or a fragment thereof, a detectable moiety, or a diagnostic agent.

[0512] 113. The multispecific polypeptide construct according to embodiment 112, wherein the reagent is conjugated to the multispecific polypeptide construct via a linker.

[0513] 114. A polynucleotide encoding the multispecific polypeptide construct according to any one of embodiments 1 to 113.

[0514] 115. A polynucleotide encoding a polypeptide chain of any one of the multispecific polypeptide constructs according to any one of embodiments 1 to 113...

Claims

1. A multispecific polypeptide construct, the multispecific polypeptide construct comprising a first component containing a heterodimeric immunoglobulin Fc region and a second component containing a CD3 binding region, wherein: the first component and the second component are coupled by a first linker, wherein the heterodimeric Fc region is located at the amino terminus of the CD3 binding region; the CD3 binding region is a disulfide-stabilized anti-CD3 Fv antibody fragment (dsFv) comprising a variable heavy chain region (VH) and a variable light chain region (VL), wherein the amino acid sequence of the VH is SEQ ID NO: 44, and the amino acid sequence of the VL is SEQ ID NO: 72, wherein the VH of the dsFv is linked to a first polypeptide of the heterodimeric immunoglobulin Fc region, and the VL of the dsFv is linked to a second polypeptide of the heterodimeric immunoglobulin Fc region; the amino acid sequence of the first polypeptide of the heterodimeric immunoglobulin Fc region is SEQ ID NO: 82 and the amino acid sequence of the second polypeptide of the heterodimeric immunoglobulin Fc region is SEQ ID NO: 83; the first component comprises one or two first antigen-binding domains that bind to a tumor-associated antigen (TAA), wherein the first antigen-binding domain is a single-domain antibody (sdAb), wherein each antigen-binding domain of the first component is linked to the amino terminus of the Fc region by a second linker, and the second component further comprises one or two second antigen-binding domains that bind to a TAA, wherein the second antigen-binding domain is an sdAb, wherein each antigen-binding domain of the second component is linked to the carboxyl terminus of the CD3 binding region by a third linker, the third linker being a polypeptide of 2-6 amino acids in length that comprises at least 50% glycine residues.

2. The multispecific polypeptide construct according to claim 1, wherein the CD3 binding region binds to CD3ε.

3. The multispecific polypeptide construct according to claim 1, wherein the first component comprises a first antigen-binding domain and the second component comprises a second antigen-binding domain, wherein each of such antigen-binding domains binds to a tumor-associated antigen (TAA).

4. The multispecific polypeptide construct according to claim 3, wherein the first antigen-binding domain is located at the amino terminus relative to the Fc region of the multispecific polypeptide construct, and the second antigen-binding domain is located at the carboxyl terminus relative to the CD3 binding region of the multispecific polypeptide construct.

5. The multispecific polypeptide construct according to claim 1, wherein the first polypeptide of the heterodimeric immunoglobulin Fc region and the second polypeptide of the heterodimeric immunoglobulin Fc region further comprise non-cysteine residues modified to cysteine residues.

6. The multispecific polypeptide construct according to any one of claims 1 to 5, wherein the Fc region comprises a polypeptide containing at least one modification to enhance FcRn binding.

7. The multispecific polypeptide construct according to any one of claims 1 to 5, wherein the Fc region comprises a polypeptide containing at least one amino acid modification that reduces effector function and / or reduces binding to an effector molecule selected from Fcγ receptors or C1q.

8. The multispecific polypeptide construct according to any one of claims 1 to 5, wherein the CD3-binding region is monovalent.

9. The multispecific polypeptide construct according to any one of claims 1 to 5, wherein the first linker is a cleavable linker.

10. The multispecific polypeptide construct according to claim 9, wherein the cleavable linker is a polypeptide that functions as a substrate for a protease.

11. The multispecific polypeptide construct according to claim 10, wherein the protease is produced by immune effector cells, by a tumor, or by cells present in the tumor microenvironment.

12. The multispecific polypeptide construct according to claim 10, wherein the protease is selected from stromelysin, matrix metalloproteinase (MMP), granzyme B, and combinations thereof.

13. The multispecific polypeptide construct according to claim 9, wherein the cleavable linker comprises an amino acid sequence of the general formula P4 P3P2 P1 ↓ P1’, where P4 is the amino acid I, L, Y, M, F, V, or A; P3 is the amino acid A, G, S, V, E, D, Q, N, or Y; P2 is the amino acid H, P, A, V, G, S, or T; P1 is the amino acid D or E; and P1’ is the amino acid I, L, Y, M, F, V, T, S, G, or A.

14. The multispecific polypeptide construct according to claim 9, wherein the cleavable linker comprises an amino acid sequence of the general formula P4 P3P2 P1 ↓ P1’, where P4 is the amino acid I or L; P3 is the amino acid E; P2 is the amino acid P or A; P1 is the amino acid D; and P1’ is the amino acid I, V, T, S, or G.

15. The multispecific polypeptide construct according to claim 9, 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).

16. The multispecific polypeptide construct according to claim 9, wherein the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:22, 105 - 112, 136 - 141, 150 - 153.

17. The multispecific polypeptide construct according to claim 12, wherein the protease is stromelysin.

18. The multispecific polypeptide construct according to claim 9, wherein: The cleavable linker comprises the sequence RQARA or the sequence RQARV (SEQ ID NO: 156).

19. The multispecific polypeptide construct according to claim 12, wherein the protease is MMP.

20. The multispecific polypeptide construct according to claim 9, wherein the cleavable linker comprises the general formula P3 P2P1 ↓ P1’, where P3 is P, V or A; P2 is Q or D; P1 is A or N; and P1’ is L, I or M.

21. The multispecific polypeptide construct according to claim 9, wherein the cleavable linker comprises the general formula P3 P2P1 ↓ P1’, where P3 is P; P2 is Q or D; P1 is A or N; and P1’ is L or I.

22. The multispecific polypeptide construct according to claim 9, wherein the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-24, 105-113, 117-118, 136-144, 148, 150-158.

23. The multispecific polypeptide construct according to any one of claims 1 to 5, wherein the antigen-binding domain binds to the TAA to form a monovalent, divalent, trivalent or tetravalent binding.

24. The multispecific polypeptide construct according to any one of claims 1 to 5, wherein the antigen-binding domain binds to a tumor-associated antigen (TAA) selected from the following: 1-92-LFA-3, 5T4, α-4 integrin, α-V integrin, α4β1 integrin, α4β7 integrin, AGR2, Lewis-Y, ephrin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9 (Lewis a), CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, IL-2RG, CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5, CEACAM6 (NCA-90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor α (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GP IIb receptor, GPIIIa receptor, Gp130, GPIIB, GPIIIA, GPNMB, GRP78, HER2, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27, IL27R, IL29, IL-31R, IL31, IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16), Na / K ATPase, NGF, nakasterone protein, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidyl-serine, 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-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2 and WISP-3.

25. The multispecific polypeptide construct according to any one of claims 1 to 5, wherein the antigen-binding domain binds to EGFRviii.

26. The multispecific polypeptide construct according to any one of claims 1 to 5, wherein the multispecific polypeptide construct 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; or the multispecific polypeptide construct 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.

27. The multispecific polypeptide construct according to any one of claims 1 to 5, wherein the multispecific polypeptide construct comprises a second linker between the first antigen-binding domain and the Fc region and a third linker between the CD3-binding region and the second antigen-binding domain, and wherein the multispecific polypeptide construct has the following structural configuration from the N-terminus to the C-terminus: first antigen-binding domain - second linker - Fc region – first linker - CD3-binding region - third linker - second antigen-binding domain.

28. A polynucleotide encoding the multispecific polypeptide construct according to any one of claims 1 to 27.

29. A vector comprising the polynucleotide according to claim 28.

30. A cell comprising one or more polynucleotides according to claim 28.

31. A cell comprising the vector according to claim 29.

32. A method for producing a multispecific polypeptide construct, the method comprising introducing one or more polynucleotides according to claim 28 into a cell and culturing the cell under conditions for producing the multispecific polypeptide construct.

33. A method for generating a multispecific polypeptide construct, the method comprising introducing one or more vectors as claimed in claim 29 into a cell and culturing the cell under conditions for generating the multispecific polypeptide construct.

34. A method for generating a multispecific polypeptide construct, the method comprising culturing the cell under conditions suitable for the cell as claimed in claim 30 or 31 to generate the multispecific polypeptide construct.

35. A pharmaceutical composition comprising a multispecific polypeptide construct as claimed in any one of claims 1 to 27 and a pharmaceutically acceptable carrier.

36. Use of a multispecific polypeptide construct as claimed in any one of claims 1 to 27 in the preparation of a medicament for treating cancer in a subject.

37. Use of the pharmaceutical composition as claimed in claim 35 in the preparation of a medicament for treating cancer in a subject.

38. Use as claimed in any one of claims 36 to 37, wherein the subject is a human.

Images