Targeted activation of endogenous cell death

Abstract

Disclosed herein are multi-specific binding polypeptide molecules (e.g., multi-specific antibodies) that can interact with TNF-related apoptosis-inducing ligand (TRAIL) receptors and cell-specific antigens. In some embodiments, also described herein are methods for treating one or more diseases or conditions (e.g., cancers) using the multi-specific binding polypeptides (e.g., multi-specific antibodies).

Description

TARGETED ACTIVATION OF ENDOGENOUS CELL DEATHCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Patent Application No. 63 / 540,237, filed on September 25, 2023, the contents of which are incorporated herein by reference in their entirety.BACKGROUND OF THE INVENTION

[0002] TNF-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) family of ligands. Targeting the TRAIL receptors is a useful approach in developing cancer and autoimmune disease therapies because activating the TRAIL-receptor can induce apoptosis in cancer and immune cells. There is a need to develop therapeutically efficacious, but safe, cell-specific biological molecules which can function as TRAIL-receptor agonist molecules, and induce apoptosis in cancer and immune cells.SUMMARY OF THE DISCLOSURE

[0003] Described herein are multi-specific binding polypeptides useful for the treatment of cancers, immune disorders, autoimmunity, longevity, etc. by selective elimination of target cells or disease cells. The multi-specific binding polypeptides comprise at least two distinct binding moieties with one of the binding moieties specific for a cell-specific antigen, and the other specific for TNF-related apoptosis-inducing ligand receptors: TRAIL-R2 (DR5) and TRAIL-R1 (DR4). In certain embodiments, the cell-specific antigen is an antigen in lipid raft of cell membrane, lipid rich microdomains of cell membrane and on the cell membrane. In certain embodiments, the cell-specific antigen is Glycosylphosphatidylinositol (GPI)-anchored protein. In certain embodiments, the cell-specific antigen is a tumor specific antigen. In certain embodiments, the target cell or disease cells is cancer cells, immune cells, hyperimmune cells, T-cells, immunosuppressive cells, senescent cells, etc.

[0004] In certain aspects, described herein is a multi-specific binding polypeptide comprising an antigen binding moiety that specifically binds to a cell-specific antigen and a binding moiety that specifically binds to TNF-related apoptosis-inducing ligand receptors: TRAIL-R2 (DR5) and TRAIL-R1 (DR4). In some embodiments, the multi-specific binding polypeptide is a multi-specific antibody. In some embodiments, the multi-specific binding polypeptide comprises a cell-specific antigen binding moiety that specifically binds to a cellspecific antigen and a binding moiety that specifically binds to TNF-related apoptosis-inducing ligand receptors: TRAIL-R2 (DR5) and TRAIL-R1 (DR4).

[0005] In certain embodiments, described herein is a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a multi-specific binding polypeptide (e.g., a multi-specific antibody), a pharmaceutical composition comprising, consisting essentially of, or consisting of the multispecific binding polypeptide (e.g., the multi-specific antibody), or a nucleic acid encoding the multi-specific binding polypeptide (e.g., the multi-specific antibody). In some aspects, also described herein is a method of treating an individual with a cancer, the method comprising administering to the individual with cancer the multi-specific binding polypeptide (e.g., the multi-specific antibody), the pharmaceutical composition comprising, consisting essentially of, or consisting of the multi-specific binding polypeptide (e.g., the multi-specific antibody), or a nucleic acid encoding the multi-specific binding polypeptide (e.g., the multi-specific antibody). In certain embodiments, the individual has previously been treated with an immune checkpoint inhibitor treatment.

[0006] In certain embodiments, described herein is a method of inducing cell killing effect in a target cell population, the method comprises contacting a cell-specific antigen on the cell membrane and a TNF-related apoptosis-inducing ligand receptors: TRAIL-R2 (DR5) and TRAIL-R1 (DR4) with a multi-specific binding polypeptide (e.g., a multi-specific antibody), a pharmaceutical composition comprising, consisting essentially of, or consisting of the multispecific binding polypeptide (e.g., the multi-specific antibody), or a nucleic acid encoding the multi-specific binding polypeptide (e.g., the multi-specific antibody) for a time sufficient to induce cell kill effect.

[0007] In certain embodiments, additionally described herein is a method for making a treatment comprising contacting the nucleic acid encoding the multi-specific binding polypeptide to a suitable cell line to establish a transfected cell line, culturing the transfected cell line under conditions that promote secretion of the multi-specific binding polypeptide, and harvesting the multi-specific binding polypeptide from the supernatant of the transfected cell line. In certain embodiments, the method further comprises purifying the multi-specific polypeptide from the supernatant of the transfected cell line. In certain embodiments, the transfected cell line is stably transfected. In certain embodiments, described herein is a method for making a cancer treatment comprising, consisting essentially of, or consisting of admixing the multi-specific binding polypeptide and a pharmaceutically acceptable excipient, carrier, or diluent.

[0008] In certain aspects, described herein is a multi-specific binding polypeptide comprising a TNF-related apoptosis-inducing ligand (TRAIL) receptor binding moiety that specifically binds to a TRAIL receptor and a cell-specific antigen binding moiety that specifically binds to a cell-specific antigen, wherein the off-rate (kd ( 1 / s)) of the TRAIL receptor binding moiety is higher than the off-rate (kd (1 / s)) of the cell-specific antigen binding moiety.

[0009] In certain embodiments, the off-rate (kd (1 / s)) of the TRAIL receptor binding moiety is at least 20-fold higher than the off-rate (kd (1 / s)) of cell-specific antigen binding moiety. In certain embodiments, a first binding affinity between the TRAIL receptor binding moiety and the TRAIL receptor is less than a second binding affinity between the cell-specific antigen binding moiety and the cell-specific antigen. In certain embodiments, the first binding affinity is less than the second binding affinity by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, or higher.

[0010] In certain embodiments, the TRAIL receptor binding moiety has a KD for TRAIL receptor that is lower than the KD of the cell-specific antigen binding moiety for the antigen expressed on the cell. In certain embodiments, the cell killing potency of the polypeptide decreases with decreasing binding affinity to the tumor antigen and increasing rate of dissociation from the tumor antigen.[OH] In certain embodiments, the cell-specific antigen comprises CD33, HSP90, FOLR1, HER2, GPC3, GD2, FLT3, CD20, CEA, CD38, CD22, PDL1, or TROP2. In certain embodiments, the TRAIL receptor comprises TRAIL-R2 (DR5) or TRAIL-R1 (DR4).

[0012] In certain embodiments, the multi-specific binding polypeptide comprises an Fc portion. In certain embodiments, the multi-specific binding polypeptide comprises an Fc region that has been modified to reduce the affinity for human neonatal Fc receptor (FcRn).

[0013] In certain embodiments, the multi-specific binding polypeptide comprises an Fc region including a modification to reduce antibody-dependent cellular cytotoxicity (ADCC), wherein the modification optionally comprises L234, L235, P238, or P331, or a combination thereof, wherein L234, L235, P238, and P331 correspond to positions 234, 235, 238, and 331 of a wild-type IgGl, according to the EU numbering convention. In certain embodiments, the Fc region comprises an L234A, L235A, P238S, or P331 S modification, or a combination thereof.

[0014] In certain embodiments, the multi-specific polypeptide comprises an Fc region that has been modified to reduce neutropenia. In certain embodiments, the multi-specific binding polypeptide comprises an Fc region including a modification at L234, S239, S442, or a combination thereof, wherein L234, S239, and S442 correspond to positions 234, 239, 442 of a wild-type IgGl, according to the EU numbering convention. In certain embodiments, the Fc region comprises an L234F, S239C, or S442C modification, or a combination thereof. In certain embodiments, the multi-specific binding polypeptide comprises an Fc region including an L234A modification and an L235A modification.

[0015] In certain embodiments, the multi-specific polypeptide comprises a modification to ahinge region. In certain embodiments, the multi-specific polypeptide comprises a hinge region having a modification at S228, wherein S228 correspond to position 228 of a wild-type IgG4, according to the EU numbering convention. In certain embodiments, the multi-specific polypeptide comprises a hinge region having an S228P modification.

[0016] In certain embodiments, the multi-specific polypeptide is a full-length antibody, an appended antibody, chimeric antibody, a bispecific fusion protein, a bispecific antibody conjugate, or an antigen binding fragment thereof. In certain embodiments, the multi-specific binding polypeptide comprises an Fc region that has been modified to enhance antibodydependent cellular cytotoxicity (ADCC). In certain embodiments, the Fc region comprises a modification at S239, A330, 1332, or a combination thereof, wherein S239, A330, and 1332 correspond to positions 239, 330, and 332 of a wild-type IgGl, according to the EU numbering convention.

[0017] In certain embodiments, the multi-specific polypeptide comprises a nanobody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KH4, Fab-scFv, scFv-CH-CL-scFv, F(ab’)2, F(ab’)2-scFv2. scFv- KIH, Fab-scFv-Fc, tetravalent HC Ab, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, or intrabody.

[0018] In certain embodiments, the multi-specific binding polypeptide comprises an IgG framework, optionally an IgGl, IgG2, or IgG4 framework, further optionally an IgGl or IgG4 framework. In certain embodiments, the multi-specific binding polypeptide is a humanized antibody.

[0019] In certain embodiments, the multi-specific binding polypeptide can induce apoptosis in a target cell that expresses the cell-specific antigen. In certain embodiments, the cell-specific antigen is expressed in a cancer cell, immune cell, hyperimmune cell, T-cell, immunosuppressive cell, and / or senescent cell.

[0020] In certain embodiments, the cell-specific antigen is located in a lipid raft of a cell membrane, in lipid rich microdomains of the cell membrane, and / or on the cell membrane. In certain embodiments, the cell-specific antigen is Glycosylphosphatidylinositol (GPI)-anchored protein.

[0021] In certain embodiments, the cell-specific antigen is a tumor-specific antigen and / or tumor associated antigen. In certain embodiments, the cell-specific antigen is an immune cellspecific antigen and / or immune cell associated antigen, a hyperimmune cell-specific antigen and / or hyperimmune cell associated antigen, T-cell-specific antigen and / or T-cell associated antigen, or an immunosuppressive cell-specific antigen or and / or immunosuppressive cell associated antigen.

[0022] In certain embodiments, the TRAIL receptor binding moiety comprises an IgG antibody framework, optionally an IgGl or IgG4 framework. In certain embodiments, the TRAIL receptor binding moiety comprises a full-length antibody or an antigen binding fragment thereof. In certain embodiments, the TRAIL receptor binding moiety comprises a Fab, F(ab)2, singledomain antibody, a single chain variable fragment (scFv), or a nanobody. In certain embodiments, the TRAIL receptor binding moiety is a humanized antibody. In certain embodiments the TRAIL receptor binding moiety is a chimeric antibody.

[0023] In certain embodiments, the TRAIL receptor binding moiety comprises:

[0024] an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 84, HCDR2 comprising SEQ ID NO: 86, and a HCDR3 comprising SEQ ID NO: 87, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 88, a LCDR2 comprising SEQ ID NO: 89, and a LCDR3 comprising SEQ ID NO: 90; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 87; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 91.

[0025] In certain embodiments, the TRAIL receptor binding moiety comprises:

[0026] an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 131, HCDR2 comprising SEQ ID NO: 132, and a HCDR3 comprising SEQ ID NO: 133, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 135, a LCDR2 comprising SEQ ID NO: 136, and a LCDR3 comprising SEQ ID NO: 137; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 134; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 138.

[0027] In certain embodiments, the cell-specific antigen binding moiety comprises an IgG antibody framework, optionally an IgGl or IgG4 framework. In certain embodiments, the cellspecific antigen binding moiety comprises a full-length antibody or an antigen binding fragment thereof. In certain embodiments, the cell-specific antigen binding moiety comprises a Fab, F(ab)2, single-domain antibody, a single chain variable fragment (scFv), or a nanobody. In certain embodiments, the cell-specific antigen binding moiety is a humanized antibody. In certain embodiments, the cell-specific antigen binding moiety is a chimeric antibody.

[0028] In certain embodiments, the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ IDNOs: 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 118, or 122; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 126, or 130.

[0029] In certain embodiments, the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 4, a HCDR2 comprising SEQ ID NO: 5, and a HCDR3 comprising SEQ ID NO: 6, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 44, a LCDR2 comprising SEQ ID NO: 45, and a LCDR3 comprising SEQ ID NO: 46; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 47.

[0030] In certain embodiments, the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 8, a HCDR2 comprising SEQ ID NO: 9, and a HCDR3 comprising SEQ ID NO: 10, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 48, a LCDR2 comprising SEQ ID NO: 49, and a LCDR3 comprising SEQ ID NO: 50; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 11; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 51.

[0031] In certain embodiments, the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 12, a HCDR2 comprising SEQ ID NO: 13, and a HCDR3 comprising SEQ ID NO: 14, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 52, a LCDR2 comprising SEQ ID NO: 53, and a LCDR3 comprising SEQ ID NO: 54; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 15; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 55.

[0032] In certain embodiments, the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 16, a HCDR2 comprising SEQ ID NO: 17, and a HCDR3 comprising SEQ ID NO: 18, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 56, a LCDR2 comprising SEQ ID NO: 57, and a LCDR3 comprising SEQ ID NO: 58; or an immunoglobulin heavy chain variableregion comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 19; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 59.

[0033] In certain embodiments, the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 20, a HCDR2 comprising SEQ ID NO: 21, and a HCDR3 comprising SEQ ID NO: 22, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 60, a LCDR2 comprising SEQ ID NO: 61, and a LCDR3 comprising SEQ ID NO: 62; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 23; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 63.

[0034] In certain embodiments, the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 24, a HCDR2 comprising SEQ ID NO: 25, and a HCDR3 comprising SEQ ID NO: 26, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 64, a LCDR2 comprising SEQ ID NO: 65, and a LCDR3 comprising SEQ ID NO: 66; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 27; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 67.

[0035] In certain embodiments, the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 28, a HCDR2 comprising SEQ ID NO: 29, and a HCDR3 comprising SEQ ID NO: 30, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 68, a LCDR2 comprising SEQ ID NO: 69, and a LCDR3 comprising SEQ ID NO: 70; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 31; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 71.

[0036] In certain embodiments, the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 32, a HCDR2 comprising SEQ ID NO: 33, and a HCDR3 comprising SEQ ID NO: 34, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 72, a LCDR2 comprising SEQ ID NO:73, and a LCDR3 comprising SEQ ID NO: 74; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 35; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 75.

[0037] In certain embodiments, the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 36, a HCDR2 comprising SEQ ID NO: 37, and a HCDR3 comprising SEQ ID NO: 38, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 76, a LCDR2 comprising SEQ ID NO: 77, and a LCDR3 comprising SEQ ID NO: 78; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 39; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 79.

[0038] In certain embodiments, the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 40, a HCDR2 comprising SEQ ID NO: 41, and a HCDR3 comprising SEQ ID NO: 42, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 80, a LCDR2 comprising SEQ ID NO: 81, and a LCDR3 comprising SEQ ID NO: 82; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 43; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 83.

[0039] In certain embodiments, the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 115, a HCDR2 comprising SEQ ID NO: 116, and a HCDR3 comprising SEQ ID NO: 117, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 123, a LCDR2 comprising SEQ ID NO: 124, and a LCDR3 comprising SEQ ID NO: 125; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 118; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 126.

[0040] In certain embodiments, the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 119, a HCDR2 comprising SEQ ID NO: 120, and a HCDR3 comprising SEQ ID NO: 121, and aVL domain comprising a LCDR1 comprising SEQ ID NO: 127, a LCDR2 comprising SEQ ID NO: 128, and a LCDR3 comprising SEQ ID NO: 129; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 122; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 130.

[0041] In certain embodiments, the TRAIL receptor binding moiety comprises an IgG antibody framework, optionally a full-length IgG antibody framework, the cell-specific antigen binding moiety is an scFv, and the cell-specific antigen binding moiety is coupled to the C-terminus of the TRAIL receptor binding moiety, optionally recombinantly fused to the C-terminus of the TRAIL receptor binding moiety.

[0042] In certain embodiments, the TRAIL receptor binding moiety comprises an IgG antibody framework, optionally a full-length IgG antibody framework, the cell-specific antigen binding moiety is an scFv, and the cell-specific antigen binding moiety is coupled to the N-terminus of the TRAIL receptor binding moiety, optionally recombinantly fused to the N-terminus of the TRAIL receptor binding moiety.

[0043] In certain embodiments, the cell-specific antigen binding moiety comprises an IgG antibody framework, optionally a full-length IgG antibody framework, the TRAIL receptor binding moiety is an scFv, and the TRAIL receptor binding moiety is coupled to the C-terminus of the cell-specific antigen binding moiety, optionally recombinantly fused to the C-terminus of the cell-specific antigen binding moiety.

[0044] In certain embodiments, the cell-specific antigen binding moiety comprises an IgG antibody framework, optionally a full-length IgG antibody framework, the TRAIL receptor binding moiety is an scFv, and the TRAIL receptor binding moiety is coupled to the N-terminus of the cell-specific antigen binding moiety, optionally recombinantly fused to the N-terminus of the cell-specific antigen binding moiety.

[0045] In certain embodiments, the cell-specific antigen binding moiety is coupled to the TRAIL receptor binding moiety by a polypeptide linker. In certain embodiments, the polypeptide linker comprises (Gly4Ser)n, wherein n is an integer from 1 to 10, optionally from 1 to 6, 1 to 4, or 1 to 2, further optionally 1, 2, 3, or 4.

[0046] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in Table 5; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in Table 5.

[0047] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 92; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 93.

[0048] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 94; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 95.

[0049] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 96; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 97.

[0050] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 98; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 99.

[0051] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 100; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 101.

[0052] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 102; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 103.

[0053] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 104; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 105.

[0054] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 106; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 107.

[0055] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 108; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 109.

[0056] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 110; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 111.

[0057] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 139; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 140.

[0058] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 141; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 142.

[0059] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 143; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 93.

[0060] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 144; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 103.

[0061] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 145; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 101.

[0062] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 146; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 140.

[0063] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 147; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 142.

[0064] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 148; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 111.

[0065] In certain embodiments, the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 149; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 99.

[0066] In certain embodiments, the multi-specific binding polypeptide further comprises at least one payload and / or cytotoxic moiety. In certain embodiments, the payload and / or cytotoxic moiety comprises an auristatin, an auristatin derivative, maytansine, a maytansinoid, a taxane, a calicheamicin, cemadotin, a duocarmycin, a pyrrolobenzodiazepine (PBD), tubulysin, dexamethasone, or dasatinib. In certain embodiments, the auristatin derivative is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). In certain embodiments, the maytansinoid is DM1, DM2, or DM4. In certain embodiments, the pyrrolobenzodiazepine is a pyrrolobenzodiazepine dimer.

[0067] In certain embodiments, the at least one payload and / or cytotoxic moiety is attached to the multi-specific binding polypeptide via a linker, optionally a cleavable linker or a non-cleavable linker. In certain embodiments, the linker comprises a pH-sensitive linker, a proteasesensitive linker, a self-immolative linker, or a combination thereof. In certain embodiments, the linker comprises a zero-length linker, a homobifunctional linker, a heterobifunctional linker, a di-peptide linker, a spacer, a maleimide-based conjugating moiety, or a combination thereof. In certain embodiments, the linker comprises a polymer, a linear or branched polyethylene glycol, a peptide, or a combination thereof. In certain embodiments, the linker is a peptidomimetic linker. In certain embodiments, a ratio of the payload and / or cytotoxic moiety to the multispecific binding polypeptide is about 1 : 1, about 2:1, about 3: 1, about 4: 1, about 5: 1, about 6:1, about 7: 1, about 8: 1, about 10: 1, or about 12: 1.

[0068] In certain aspects, described herein is a pharmaceutical composition comprising a multi-specific binding polypeptide of any of the embodiments described herein, and a pharmaceutically acceptable excipient, carrier, or diluent. In certain embodiments, the pharmaceutical composition further comprises an additional therapeutic agent selected from the group consisting of Mylotarg, Venetoclax, Daratumumab, Cisplatin, Doxorubicin, Paclitaxel, or a combination thereof. In certain embodiments, the pharmaceutical composition is formulated for parenteral administration, optionally for subcutaneous, intramuscular, or intravenous administration.

[0069] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 92; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 93.

[0070] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 94; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 95.

[0071] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 96; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 97.

[0072] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 98; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 99.

[0073] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 100; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 101.

[0074] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 102; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 103.

[0075] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 104; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 105.

[0076] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 106; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 107.

[0077] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 108; and optionally an immunoglobulin light chain at leastabout 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 109.

[0078] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 110; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 111.

[0079] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 139; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 140.

[0080] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 141; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 142.

[0081] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 143; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 93.

[0082] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 144; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 103.

[0083] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acidsequence set forth in SEQ ID NO: 145; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 101.

[0084] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 146; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 140.

[0085] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 147; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 142.

[0086] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 148; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 111.

[0087] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 149; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 99.

[0088] In certain aspects, described herein is a nucleic acid encoding a multi-specific binding polypeptide according to any of the embodiments described herein.

[0089] In certain aspects, described herein is a vector comprising a nucleic acid of any of the embodiments described herein.

[0090] In certain aspects, described herein is a cell comprising the nucleic acid or the vector of any of the embodiments described herein.

[0091] In certain aspects, described herein is a method of inducing a cell killing effect in a target cell population, comprising contacting the target cell population with a multi-specific bindingpolypeptide, a pharmaceutical composition, or a nucleic acid of any of the embodiments described herein for a time sufficient to induce cell kill effect, thereby killing the at least one cell in the target cell population.

[0092] In certain aspects, described herein is a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a multi-specific binding polypeptide, pharmaceutical composition, or a nucleic acid of any of the embodiments described herein. In certain embodiments, the disease or condition is cancer. In certain embodiments, the disease or condition is bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, hematologic malignancy, kidney cancer, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, testicular cancer, or thyroid cancer.

[0093] In certain embodiments, the breast cancer is luminal A breast cancer, luminal B breast cancer, triple-negative breast cancer, HER2-enriched breast cancer, normal-like breast cancer, ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC) such as tubular carcinoma of the breast, medullary carcinoma of the breast, papillary carcinoma of the breast, or cribriform carcinoma of the breast, invasive lobular carcinoma (ILC), inflammatory breast cancer, lubular carcinoma in situ (LCIS), male breast cancer, Paget’s disease of the Nipple, or phyllodes tumors of the breast.

[0094] In certain embodiments, the ovarian cancer is epithelial carcinoma, serous carcinoma, small-cell carcinoma, primary peritoneal carcinoma, clear-cell carcinoma, clear-cell adenocarcinoma, endometrioid, malignant mixed mullerian tumor, mucinous, mucinous adenocarcinoma, pseudomyxoma peritonei, undifferentiated epithelial, malignant Brener tumor, transitional cell carcinoma, sex cord-stromal tumor, granulosa cell tumor, adult granulosa cell tumor, juvenile granulosa cell tumor, Sertoli-Leydig cell tumor, sclerosing stromal tumors, germ cell tumor, dysgerminoma, choriocarcinoma, immature (solid) teratoma, mature teratoma (dermoid cyst), yolk sac tumor (endodermal sinus tumor), embryonal carcinoma, polyembryoma, squamous cell carcinoma, mixed tumors, or low malignant potential tumors.

[0095] In certain embodiments, the lung cancer is small cell lung cancer (SCLC) or nonsmall cell lung cancer (NSCLC), such as adenocarcinoma, squamous cell cancer, large cell carcinoma, or undifferentiated non-small cell lung cancer.

[0096] In certain embodiments, the brain cancer is glioblastoma.

[0097] In certain embodiments, the hematologic malignancy is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom’s macroglobulinemia,multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt’s lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, or acute myeloid leukemia.

[0098] In certain embodiments, the liver cancer is hepatocellular carcinoma (HCC), cholangiocarcinoma, liver angiosarcoma, or hepatoblastoma.

[0099] In certain embodiments, the prostate cancer is acinar adenocarcinoma, ductal adenocarcinoma, transitional cell (or urothelial) cancer, squamous cell cancer, small cell prostate cancer, carcinoid in the prostate, or sarcoma in the prostate.

[0100] In certain embodiments, the cancer is a metastatic cancer. In certain embodiments, the cancer relapsed or refractory cancer.

[0101] In certain embodiments, the disease or condition is an autoimmune disease. In certain embodiments, the autoimmune disease is rheumatoid arthritis, multiple sclerosis (MS), type 1 diabetes, systemic lupus erythematosus (SLE), Graves Disease, or Hashimoto’s thyroiditis.

[0102] In certain embodiments, the disease or condition is a liver disease or condition. In certain embodiments, the liver disease or condition is non-alcoholic fatty liver disease (NASH) or alcoholic steatohepatitis.

[0103] In certain embodiments, the method further comprises administering an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent, radiation, or a combination thereof. In certain embodiments, the additional therapeutic agent is Mylotarg, Venetoclax, Daratumumab, Cisplatin, Doxorubicin, Paclitaxel, or a combination thereof. In certain embodiments, the multi-specific binding polypeptide and the additional therapeutic agent are administered simultaneously or sequentially. In certain embodiments, the multi-specific binding polypeptide is administered to the subject prior to administering the additional therapeutic agent. In certain embodiments, the additional therapeutic agent is administered to the subject prior to administering the multispecific binding polypeptide. In certain embodiments, the multi-specific binding polypeptide and the additional therapeutic agent are administered as a combination. In certain embodiments, the multi-specific binding polypeptide and the additional therapeutic agent are administered as separate dosage forms.

[0104] In certain embodiments, the subject has previously been treated with an immune checkpoint inhibitor treatment. In certain embodiments, the subject is insensitive to treatmentwith an immune checkpoint inhibitor, has failed to respond to treatment with an immune checkpoint inhibitor, or who expresses low level of or does not express an immune checkpoint protein. In certain embodiments, the subject has undergone surgery.

[0105] In certain embodiments, the multi-specific binding polypeptide, the pharmaceutical composition, or the nucleic acid decreases tumor cells, optionally tumor cell proliferation, in the target cell population by about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more. In certain embodiments, the multi-specific binding polypeptide, the pharmaceutical composition, or the nucleic acid decreases immunosuppressive cells, optionally immunosuppressive cell proliferation in the target cell population by about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more.

[0106] In certain embodiments, the multi-specific binding polypeptide, the pharmaceutical composition, or the nucleic acid enhances T-cell proliferation, optionally tumor-infiltrating lymphocyte (TIL) proliferation.

[0107] In certain embodiments, the target cell population is an in vivo target cell population. In certain embodiments, the target cell population is within a tumor microenvironment.

[0108] In certain aspects, described herein is a kit comprising the multi-specific binding polypeptide, the nucleic acid, the vector, or the cell of any of the embodiments described herein. In certain embodiments, the kit further comprises an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is Mylotarg, Venetoclax, Daratumumab, Cisplatin, Doxorubicin, Paclitaxel, or a combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0109] Various aspects of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0110] FIG. 1A illustrates a non-limiting embodiment of a multi-specific binding polypeptide of the current disclosure.

[0111] FIG. IB illustrates a non-limiting embodiment of a multi-specific binding polypeptide of the current disclosure with a cytotoxic moiety.

[0112] FIG. 2A is a schematic illustration of an exemplary multi-specific binding polypeptide that binds to a cell-specific antigen located in the lipid raft of a target cell which facilitates recruitment and oligomerization (e.g., dimerization) of the TRAIL-R2 within the lipidraft for subsequent activation of the apoptotic signaling pathway.

[0113] FIG. 2B is a schematic illustration of an exemplary multi-specific binding polypeptide that binds to a cell-specific antigen located outside of the lipid raft of a target cell. The exemplary multi-specific antibody, upon binding to the cell-specific antigen, does not activate the TRAIL-R2 mediated apoptosis within the target cell.

[0114] FIG. 3A is a schematic illustration of a conventional antibody, which generates nontumor toxicity due to leakage of inflammatory cytokines, granzymes, and-or perforins to nearby normal cells and tissues. As shown in this figure, recruiting an immune effector cell to a cancer cell and activation of the immune effector cell by the conventional antibody, the activated immune effector cells not only secrete soluble TRAILs that can lead to activation of apoptosis within the cancer cell via the TRAIL-R2 pathway, but can also release cytokines, granzymes, and / or perforins which leakage to nearby normal cells leads to non-tumor toxicity.

[0115] FIG. 3B is a schematic illustration of an exemplary multi-specific binding polypeptide which modulates TRAIL-R2 mediated apoptosis of the cancer cell and does not activate immune effector cells. As shown in this figure, the lack of immune effector cell activation minimizes or prevents the release of inflammatory cytokines, granzymes, and / or perforin, and as such, reduces or inhibits toxicity to nearby normal cells.

[0116] FIG. 4A illustrates a non-limiting embodiment of an aCD33 x aTRAIL-R2 multispecific binding polypeptide of the current disclosure.

[0117] FIG. 4B illustrates a solution profile analysis of protein A purified aCD33 x aTRAIL-R2 multi-specific binding polypeptide by Size-Exclusion Ultra Performance Liquid Chromatography (SE-UPLC). The major peak with retention time = 7.08 corresponds to the monomeric form of the intact multi-specific binding polypeptide construct and is 80% of all the species noted. The peak with a retention time of 6.0 is a higher molecular weight species of the multi-specific binding polypeptide construct and could be an artifact associated with the method employed.

[0118] FIG. 4C illustrates an SDS-PAGE analysis of protein A purified aCD33 x aTRAIL- R2 multi-specific antibody. The samples were analyzed under reducing (R) and non-reducing (NR) conditions. Two bands are observed under reducing conditions, top band corresponding to the molecular weight of the heavy chain and lower band corresponding to the molecular weight of the light chain. Under non-reducing conditions only one band was observed corresponding to the molecular weight of the intact bispecific antibody construct.

[0119] FIG. 5A illustrates a Biacore multi-cycle kinetics sensogram of aCD33 x aTRAIL- R2 multi-specific binding polypeptide binding to CD33.

[0120] FIG. 5B illustrates a Biacore multi-cycle kinetics sensogram of aCD33 x aTRAIL-R2 multi-specific binding polypeptide binding to TRAIL-R2.

[0121] FIG. 6A illustrates dose dependent TRAIL-R2 mediated apoptosis in U937 cells by aCD33 x aTRAIL-R2 multi-specific antibody. aCD33 x aTRAIL-R2 demonstrated 145 x more potent induction of apoptosis (ECso = 0.02 nM) than TRAIL-R2’s natural ligand, TRAIL (ECso = 2.9 nM).

[0122] FIG. 6B illustrates dose dependent TRAIL-R2 mediated cell death in U937 cells by aCD33 x aTRAIL-R2 multi-specific antibody. aCD33 x aTRAIL-R2 demonstrated 65.66x more potent cell killing (ICso = 0.009 nM) than TRAIL-R2’s natural ligand, TRAIL (ICso = 0.59 nM).

[0123] FIG .7A illustrates low levels of expression of CD33 on the surface of U937 cells determined by the low level of binding of anti- CD33 antibody measured by Fluorescent Activated Cell Sorting (FACS). The non-binding antibody isotype control did not bind on the surface of the U937 cells.

[0124] FIG. 7B illustrates low levels of expression of TRAIL-R2 on the surface of U937 determined by the low level of binding of anti- TRAIL-R2 antibody measured by Fluorescent Activated Cell Sorting (FACS). The non-binding antibody isotype control did not bind on the surface of the U937 cells.

[0125] FIG. 8 illustrates negligible TRAIL-R2 mediated apoptosis in CD33 -negative, COLO-205 cells by aCD33 x aTRAIL-R2 multi-specific antibody. aCD33 x aTRAIL-R2 induced apoptosis by 10% above background compared to TRAIL, which induced apoptosis to 40% above background.

[0126] FIG. 9 illustrates negligible TRAIL-R2 mediated apoptosis in TROP2-negative, U937 cells by aTROP2 x aTRAIL-R2 multi-specific antibody. TROP2 x aTRAIL-R2 induced apoptosis by 12% above background. Herceptin is shown as a negative control.

[0127] FIG. 10A illustrates dose dependent TRAIL-R2 mediated cell killing in U937 cells by aCD33 x aTRAIL-R2 multi-specific antibody. aCD33 x aTRAIL-R2 (IC50 = 0.0007 nM) demonstrated 3718x higher cell killing potency than Mylotarg (IC50 = 2.63 nM), an approved ADC drug.

[0128] FIG. 10B illustrates dose dependent TRAIL-R2 mediated cell killing in Karpas-422 cells by aCD33 x aTRAIL-R2 multi-specific antibody. aCD33 x aTRAIL-R2 (IC50 = 0.001 nM) demonstrated 1000x higher cell killing potency than Mylotarg (IC50 = 1 nM), an approved ADC drug.

[0129] FIG. 11 illustrates lack of dose dependent TRAIL-R2 mediated cell killing in Colo- 205 cells not expressing CD33 by aCD33 x aTRAIL-R2 multi-specific antibody. aCD33 x aTRAIL-R2 demonstrated no cell killing compared to TRAIL, a natural ligand of TRAIL-R2.

[0130] FIG. 12 illustrates dose dependent TRAIL-R2 mediated cell killing in U937 cells by aCD33 x aTRAIL-R2 multi-specific antibody. aCD33 x aTRAIL-R2 demonstrated higher cell killing of IC50 = 0.00036 nM compared to the standard of care, Venetoclax, tested at 100 nM, which showed no cell killing. Cell killing by aCD33 x aTRAIL-R2 is increased by combining with 100 nM Venetoclax, IC50 = 0.00008 nM.

[0131] FIG. 13 illustrates no dose dependent TRAIL-R2 mediated cell killing in Hematopoietic Stem Cells by aCD33 x aTRAIL-R2 multi-specific antibody. aCD33 x aTRAIL-R2, TRAIL, anti- TRAIL-R2 antibody showed no cell killing in Hematopoietic Stem Cells, whereas Mylotarg showed a full dose dependent cell killing.

[0132] FIG. 14 illustrates dose dependent single agent activity by TRAIL-R2 mediated cell killing in Karpas-422 xenograft model in mice by aCD33 x aTRAIL-R2 multi-specific antibody. aCD33 x aTRAIL-R2 exhibited single agent activity in stopping tumor growth at doses 5, 10, and 15 mg / kg.

[0133]

[0134] FIG. 15A illustrates a non-limiting embodiment of an aHER2 x aTRAIL-R2 multispecific binding polypeptide of the current disclosure.

[0135] FIG. 15B illustrates a solution profile analysis of protein A purified aHER2 x aTRAIL-R2 multi-specific binding polypeptide by Size-Exclusion Ultra Performance Liquid Chromatography (SE-UPLC). The major peak with retention time = 3.6 corresponds to the monomeric form of the intact multi-specific antibody construct.

[0136] FIG. 15C illustrates an SDS-PAGE analysis of protein A purified aHER2 x aTRAIL-R2 multi-specific antibody. The samples were analyzed under reducing (R) and nonreducing (NR) conditions. Two bands are observed under reducing conditions, top band corresponding to the molecular weight of the heavy chain and lower band corresponding to the molecular weight of the light chain. Under non-reducing conditions only one band was observed corresponding to the molecular weight of the intact bispecific antibody construct.

[0137] FIG. 16A illustrates a non-limiting embodiment of an aHSP90 x aTRAIL-R2 multispecific binding polypeptide of the current disclosure.

[0138] FIG. 16B illustrates a solution profile analysis of protein A purified aHSP90 x aTRAIL-R2 multi-specific antibody by Size-Exclusion Ultra Performance Liquid Chromatography (SE-UPLC). The major peak with retention time = 3.6 corresponds to the monomeric form of the intact multi-specific binding polypeptide construct.

[0139] FIG. 16C illustrates an SDS-PAGE analysis of protein A purified aHSP90 x aTRAIL-R2 multi-specific antibody. The samples were analyzed under reducing (R) and nonreducing (NR) conditions. Two bands are observed under reducing conditions, top bandcorresponding to the molecular weight of the heavy chain and lower band corresponding to the molecular weight of the light chain. Under non-reducing conditions only one band was observed corresponding to the molecular weight of the intact bispecific antibody construct.

[0140] FIG. 17A illustrates a Biacore multi-cycle kinetics sensogram of aHER2 x aTRAIL- R2 multi-specific binding polypeptide binding to HER2.

[0141] FIG. 17B illustrates a Biacore multi-cycle kinetics sensogram of aHER2 x aTRAIL- R2 multi-specific binding polypeptide binding to TRAIL-R2.

[0142] FIG. 18A illustrates a Biacore multi-cycle kinetics sensogram of aHSP90 x aTRAIL-R2 multi-specific binding polypeptide binding to HSP90.

[0143] FIG. 18B illustrates a Biacore multi-cycle kinetics sensogram of aHSP90 x aTRAIL-R2 multi-specific binding polypeptide binding to TRAIL-R2.

[0144] FIG. 19A illustrates a non-limiting embodiment of an aCD22 x aTRAIL-R2 multispecific binding polypeptide of the current disclosure.

[0145] FIG. 19B illustrates a solution profile analysis of protein A purified aCD22 x aTRAIL-R2 multi-specific binding polypeptide by Size-Exclusion Ultra Performance Liquid Chromatography (SE-UPLC). The major peak with a retention time of 6.25 corresponds to the monomeric form of the intact multi-specific binding polypeptide construct and is 95.2% of all the species noted. The peak with a retention time of 5.25 could be an artifact associated with the method employed.

[0146] FIG. 19C illustrates an SDS-PAGE analysis of protein A purified aCD22 x aTRAIL-R2 multi-specific antibody. The samples were analyzed under reducing (R) and nonreducing (NR) conditions. Two bands are observed under reducing conditions, top band corresponding to the molecular weight of the heavy chain and lower band corresponding to the molecular weight of the light chain. Under non-reducing conditions only one band was observed corresponding to the molecular weight of the intact bispecific antibody construct.

[0147] FIG. 20A illustrates dose dependent TRAIL-R2 mediated cell killing in Karpas-422 cells by aCD22 x aTRAIL-R2 multi-specific antibody. aCD22 x aTRAIL-R2 (IC50 = 0.04 nM) demonstrated 9.75 x higher cell killing potency than TRAIL-R2’s natural ligand, TRAIL (IC50 = 0.39 nM).

[0148] FIG. 20B illustrates lack of dose dependent TRAIL-R2 mediated cell killing in Colo- 205 cells not expressing CD22 by aCD22 x aTRAIL-R2 multi-specific antibody. aCD22 x aTRAIL-R2 demonstrated no cell killing compared to TRAIL, a natural ligand of TRAIL-R2.

[0149] FIG. 21 A illustrates a non-limiting embodiment of an aCD38 x aTRAIL-R2 multispecific binding polypeptide of the current disclosure.

[0150] FIG. 21B illustrates a solution profile analysis of protein A purified aCD38 xaTRAIL-R2 multi-specific binding polypeptide by Size-Exclusion Ultra Performance Liquid Chromatography (SE-UPLC). The major peak with a retention time of 4.1 corresponds to the monomeric form of the intact multi-specific binding polypeptide construct and is 91.2% of all the species noted.

[0151] FIG. 21C illustrates an SDS-PAGE analysis of protein A purified aCD38 x aTRAIL-R2 multi-specific antibody. The samples were analyzed under reducing (R) and nonreducing (NR) conditions. Two bands are observed under reducing conditions, top band corresponding to the molecular weight of the heavy chain and lower band corresponding to the molecular weight of the light chain. Under non-reducing conditions only one band was observed corresponding to the molecular weight of the intact bispecific antibody construct.

[0152] FIG. 22 illustrates dose dependent TRAIL-R2 mediated cell killing in Jeko-1 cells by aCD38 x aTRAIL-R2 multi-specific antibody. aCD38 x aTRAIL-R2 (IC50 = 1.314 nM) demonstrated higher cell killing potency than Daratumumab, an approved drug to treat Multiple Myeloma.

[0153] FIG. 23A illustrates a non-limiting embodiment of an aFOLRl x aTRAIL-R2 multispecific binding polypeptide of the current disclosure.

[0154] FIG. 23B illustrates a solution profile analysis of protein A purified aFOLRl x aTRAIL-R2 multi-specific binding polypeptide by Size-Exclusion Ultra Performance Liquid Chromatography (SE-UPLC). The major peak with a retention time of 6.25 corresponds to the monomeric form of the intact multi-specific binding polypeptide construct and is 100% of all the species noted.

[0155] FIG. 23C illustrates an SDS-PAGE analysis of protein A purified aFOLRl x aTRAIL-R2 multi-specific antibody. The samples were analyzed under reducing (R) and nonreducing (NR) conditions. Two bands are observed under reducing conditions, top band corresponding to the molecular weight of the heavy chain and lower band corresponding to the molecular weight of the light chain. Under non-reducing conditions only one band was observed corresponding to the molecular weight of the intact bispecific antibody construct.

[0156] FIG. 24A illustrates dose dependent TRAIL-R2 mediated cell killing in OVCAR-3 cells by aFOLRl x aTRAIL-R2 multi-specific antibody. aFOLRl x aTRAIL-R2 (IC50 = 0.011 nM) demonstrated 167x higher cell killing potency than Elahere an approved ADC drug for Ovarian Cancer (IC50 = 1.84), and 13.6x higher cell killing potency than TRAIL (IC50 = 0.15 nM), the natural ligand of TRAIL.

[0157] FIG. 24B illustrates dose dependent killing of SKOV3 cells by aFOLRl x aTRAIL- R2 in combination with standard of care drugs for ovarian cancer, Cisplatin, Doxorubicin, and Paclitaxel. Standard of care drugs Cisplatin, Doxorubicin, and Paclitaxel exhibited no cell killingactivity by themselves. Cell killing by aFOLRl x aTRAIL-R2 is further enhanced when combined with 30 pM Cisplatin, 10 pM Doxorubicin, and 10 pM Paclitaxel.DETAILED DESCRIPTION OF THE DISCLOSUREMulti-specific binding polypeptide structures

[0158] Disclosed herein are multi-specific binding polypeptides (e.g., multi-specific antibodies) that comprise a cell-specific antigen binding moiety that specifically binds to a cellspecific antigen and a TNF-related apoptosis-inducing ligand (TRAIL) receptors binding moiety. In some instances, the multi-specific binding polypeptide (e.g., the multi-specific antibody) binds to and modulate killing of a cell by apoptosis and immunogenic cell death. In some instances, the multi-specific binding polypeptide comprises a single polypeptide complex comprising at least two distinct non-overlapping binding domains, each of the distinct binding domains being specific for distinct non-overlapping antigens or epitopes. The antigen or epitope can be on different molecules (e.g., different proteins), or non-overlapping portions of the same molecule. A single polypeptide complex encompasses single polypeptides, polypeptides that dimerize or multimerize to form a binding unit (e.g., heavy chain and light chain of an IgG, two heavy chain and light chain pairs forming a mature IgG), polypeptides that are distinct but held together by covalent (e.g., disulfide bonds, linker molecules) or non-covalent interactions (e.g., biotin-streptavidin; affinity interactions; charge interactions, knob-in-hole).

[0159] The multi-specific binding polypeptides described herein, in certain embodiments, comprise antibodies or binding fragments of antibodies. Among the provided multi-specific binding polypeptides are monoclonal antibodies, multi-specific antibodies, for example, bispecific antibodies, and antibody fragments. The antibodies include antibody-conjugates and molecules comprising the antibodies, such as chimeric molecules. Thus, an antibody includes, but is not limited to, full-length and native antibodies, as well as fragments and portion thereof retaining the binding specificities thereof, such as any specific binding portion thereof including those having any number of, immunoglobulin classes and / or isotypes (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and IgM); and biologically relevant (antigen-binding) fragments or specific binding portions thereof, including but not limited to Fab, F(ab')2, Fv, and scFv (single chain or related entity). A monoclonal antibody is generally one within a composition of substantially homogeneous antibodies; thus, any individual antibodies comprised within the monoclonal antibody composition are identical except for possible naturally occurring mutations that may be present in minor amounts. A polyclonal antibody is a preparation that includes different antibodies of varying sequences that generally are directed against two or moredifferent determinants (epitopes). The monoclonal antibody can comprise a human IgGl constant region. The monoclonal antibody can comprise a human IgG4 constant region.

[0160] In some embodiments, a multi-specific binding polypeptide described herein comprises a full-length antibody, an appended antibody, a bispecific fusion protein, or a bispecific antibody conjugate. In some cases, the multi-specific binding polypeptide comprises a nanobody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab’)2, F(ab’)2-scFv2. scFv-KIH, Fab-scFv-Fc, tetravalent HC Ab, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, or intrabody. In some cases, the multi-specific binding polypeptide comprises an appended antibody. In some cases, the multi-specific binding polypeptide comprises a bispecific fusion protein. In some cases, the multi-specific binding polypeptide comprises a bispecific antibody conjugate. In some cases, the multi-specific binding polypeptide comprises an appended antibody further comprising a conjugate (e.g., a payload such as a cytotoxic moiety).

[0161] The multi-specific binding polypeptides (e.g., the multi-specific antibody) of the current disclosure have certain structural attributes. FIG. 1 A shows a specific non-limiting embodiment of one such multi-specific binding polypeptide (e.g., a multi-specific antibody). The polypeptide minimally comprises a first binding moiety that binds a cell-specific antigen. This binding moiety comprises, for example, a VH / VL pair of a monoclonal antibody specific for the cell-specific antigen. The polypeptide also comprises a second binding moiety comprising a single-chain variable region (scFv) that binds to a TNF-related apoptosis-inducing ligand (TRAIL) receptor. While the first binding moiety is described as being specific for a cellspecific antigen, and the second binding moiety is described as being specific for a TRAIL receptor, this order can be reversed with the first binding moiety binding to a TRAIL receptor and the second binding moiety binding to a cell-specific antigen. FIG. 1 A also shows optional features for the polypeptides described herein, including an Fc region and a polypeptide linker (e.g., a flexible polypeptide linker) coupling the first binding moiety and the second binding moiety. In one embodiment, the multi-specific binding polypeptide (e.g., the multi-specific antibody) comprises a first binding moiety that comprises a monoclonal IgG antibody and a second binding moiety that comprises an scFv, wherein the scFv antibody is coupled to the C- terminus of one or both of the Fc regions of the monoclonal antibody. In certain embodiments, the IgG comprises an IgGi isotype. In certain embodiments, the IgG comprises an IgG2 isotype. In certain embodiments, the IgG comprises an IgG4 isotype. In certain embodiments, the polypeptide linker comprises a poly-Ala linker, a poly-Gly linker, or a combination of poly-Ala and poly-Gly residues. In some instances, the polypeptide linker comprises (Gly4Ser)n, whereinn is an integer from 1 to 10. In some instances, the n is an integer from 1 to 6, 1 to 4, or 1 to 2. In some cases, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, n is 1, 2, 3, 4, 5, or 6. In some cases, n is 1, 2, 3, or 4. In some cases, the polypeptide linker comprises GGGSGGGS (SEQ ID NO: 1). In some cases, the polypeptide linker comprises GGGSGGGSGGGS (SEQ ID NO: 2). In some cases, the polypeptide linker comprises GGGSGGGSGGGSGGGS (SEQ ID NO: 3). In certain embodiments, the Fc region has been modified to increase half-life in circulation. In certain embodiments, the half-life is extended to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 28, 30, 60, or more days. In certain embodiments, the half-life is extended to at least 7, 14 or 21 days. In certain embodiments, the Fc region has been modified to increase antibody dependent cell-cytotoxicity (ADCC). In certain embodiments, the Fc region that has been modified to reduce the affinity for human neonatal Fc receptor (FcRn). In certain embodiments, the Fc region that has been modified to reduce neutropenia. In certain embodiments, one or both hinge regions have been modified.

[0162] In addition to the configuration shown in FIG. 1 A other configurations are envisioned. Exemplary multi-specific binding polypeptide configurations include, but are not limited to: (1) a Light chain c-terminal scFv format, wherein an scFv is coupled to the C- terminus of one or more light chains of an antibody, as in US 2013 / 0216543; (2) a dual-variable domain (DVD) antibody, wherein the light chain and heavy chain variable regions comprise a distinct second binding domain, as in US 8,258,268; (3) a knob-in-hole bispecific, wherein one heavy chain constant region comprises an engineered hole, and one heavy chain constant region comprises an engineered knob, as in Merchant et al. “An efficient route to human bispecific IgG.” Nat Biotechnol. 1998 Jul;16(7):677-81, and WO 2016 / 172485; (4) dual scFvs, wherein two scFvs with different binding specificities are joined by a linker; and (5) dual F(ab’)2 wherein two F(ab’)s with different binding specificities are joined by a linker or chemical crosslinking. Any bispecific antibody format can be used as a multi-specific binding polypeptide of the current disclosure. See Spiess, etal., Molecular Immunology, 67(2): 95-106 (2015). In certain embodiments, the multi-specific binding polypeptide is a bispecific of a format selected from the list consisting of: IgG-scFv, nanobody, diabody, Dual-affinity Re-targeting Antibody (DART), TandAb, scDiabody, scDiabody-CH3, triple body, mini-antibody, minibody, TriBi minibody, scFv-CH3 KIH, Cross-mab Knob-in-hole (KIH), Fab-scFv, scFv-CH-CL-scFv, F(ab’)2, F(ab’)2- scFv2. scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, and intrabody.

[0163] In some embodiments, the multi-specific binding polypeptide (e.g., the multi-specific antibody) comprises a bispecific format described in Godar, et al., “Therapeutic bispecific antibody formats: a patent applications review (1994-2017),” Expert Opinion on TherapeuticPatents, 28(3): 251-276 (2018).

[0164] In some embodiments, the multi-specific binding polypeptide (e.g., the multi-specific antibody) comprises a bispecific format described in Labrijn, et al., “Bispecific antibodies: a mechanistic review of the pipeline,” Nature Reviews 18: 585-608 (2019).

[0165] In certain embodiments, the cell-specific antigen binding moiety specifically binds to a cell-specific antigen. In some instances, the cell-specific antigen may be a tumor antigen or an autoimmunity-related antigen. In some instances, the cell-specific antigen may include one or more of CD33, HSP90, HER2, GPC3, FOLR1, CD38, CD20, TROP2, CEA, FLT3, CD30, CSPG4, CDH17, CLND18.2, CD22, SigLec 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 ,15, CD70, CD123, PD-L1, PDL-2, PD-1, CTLA-4, CD19, LGR5, CD3001d, CD300 family of antigens, CD 120a, CD 120b, BCMA, Pre-B cell receptor, TNF, membrane-bound TNF, IL6, membrane bound IL6, CD25, CD69, CD71, CD40L CD44, HLA-DR, CD28, CD62L, CD95, CD137, CD80, CD86, CD23, CD27, CD 19, CD21, CD 137, BLIMp- 1, all TNFR-family proteins, TNFR1, TNFR2, CD40, CD134, BAFF-R, DR3, CD270, GITR, RANK, TACI, all GPI- anchored proteins, and all lipid raft antigens, among others.Multi-specific Antibodies

[0166] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises a cell-specific antigen binding moiety comprising sequences at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of sequences (e.g., CDRs or VH / VL sequences) selected from Tables 1 and / or 2.Table 1 - Antigen Binding Moiety Variable Heavy Sequences*The notation “a” in front of an exemplary tumor-associated antigen denotes an antibody that specifically binds to the tumor-associated antigen. For example, aTR0P2 refers to an antibody that specifically binds to TR0P2, etc.#The underlined sequences denote the respective HCDR1, HCDR2, and HCDR3. For example, the first underlined sequence in aTR0P2 denotes HCDR1; the second underlined sequence in aTR0P2 denotes HCDR2; and the third underlined sequence in aTR0P2 denotes HCDR3, etc.Table 2 - Antigen Binding Moiety Variable Light Sequences*The notation “a” in front of an exemplary tumor-associated antigen denotes an antibody that specifically binds to the tumor-associated antigen.#The underlined sequences denote the respective LCDR1, LCDR2, and LCDR3. For example, the first underlined sequence in aTR0P2 denotes LCDR1; the second underlined sequence in aTR0P2 denotes LCDR2; and the third underlined sequence in aTR0P2 denotes LCDR3.

[0167] In some embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises a cell-specific antigen binding moiety comprising an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NOs: 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 118, or 122; and an immunoglobulin light chain variable region at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NOs: 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 126, or 130. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain variable region and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NOs: 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 118, or 122 . In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain variable region and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NOs: 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 126, or 130. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region,CDRs region, or a combination thereof, of the immunoglobulin heavy chain variable region but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target cell-specific antigen. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain variable region but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target cell-specific antigen.

[0168] In some embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises a cell-specific antigen binding moiety comprising an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 7; and an immunoglobulin light chain variable region at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 47. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain variable region and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 7. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain variable region and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 47. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain variable region but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target cell-specific antigen. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain variable region but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target cell-specific antigen.

[0169] In some embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises a cell-specific binding moiety comprising an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 11; and an immunoglobulin light chain variable region at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the aminoacid sequence set forth in SEQ ID NO: 51. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain variable region and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 11. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain variable region and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 51. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain variable region but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target cell-specific antigen. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain variable region but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target cell-specific antigen.

[0170] In some embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises a cell-specific binding moiety comprising an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 15; and an immunoglobulin light chain variable region at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 55. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain variable region and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 15. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain variable region and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 55. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain variable region but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target cell-specific antigen. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprisesconservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain variable region but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target cell-specific antigen.

[0171] In some embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises a cell-specific binding moiety comprising an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 19; and an immunoglobulin light chain variable region at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 59. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain variable region and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 19. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain variable region and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 59. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain variable region but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target cell-specific antigen. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain variable region but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target cell-specific antigen.

[0172] In some embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises a cell-specific antigen binding moiety comprising an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 23; and an immunoglobulin light chain variable region at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 63. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain variable region and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 23. In some instances, theamino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain variable region and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 63. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain variable region but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target cell-specific antigen. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain variable region but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target cell-specific antigen.

[0173] In some embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises a cell-specific antigen binding moiety comprising an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 118; and an immunoglobulin light chain variable region at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 126. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain variable region and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 118. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain variable region and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 126. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain variable region but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target cell-specific antigen. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain variable region but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target cell-specific antigen.

[0174] In some embodiments, a multi-specific binding polypeptide (e.g., a multi-specificantibody) described herein comprises a cell-specific antigen binding moiety comprising an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 122; and an immunoglobulin light chain variable region at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 130. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain variable region and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 122. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain variable region and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 130. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain variable region but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target cell-specific antigen. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain variable region but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target cell-specific antigen.

[0175] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises a cell-specific antigen binding moiety comprising sequences (e.g., CDRs or VH / VL sequences) derived from a known antibody such as Gemtzumumab (CD33); Vadastuximab (CD33); 6H8 (HSP90), RS7, MAAP-9001a, 7E6, 4F6 (TROP2); Codrituzumab / GC33 / RO5137382, HN3, YP7, HS20, 4G5, MDX-1414 (GPC3); Trastuzumab (HER2); brentuximab, ramucirumab, iratumumab, olinvacimab, vesencumab (CD30); dinutuximab, Hu3F8, MAb-3F8, MORAb-028 / MT228, KM666 (GD2); mirvetuximab / huFR107, farletuzumab, MORAb-003, SP8166, 26B3.F2, HuRA15 (FOLR1); daratumumab, isatuximab, mezagitamab (CD38), Inotuzumab (CD22), Avelumab (PDL1), atezolizumab (PDL1), durvalumab (PDL1) or IMC-EB10 (FLT3).

[0176] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises a TRAIL receptor binding moiety comprising sequences at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of sequences (e.g., CDRs or VH / VL sequences) selected from Tables 3 and / or 4.Table 3 - TRAIL Receptor Moiety Variable Heavy Sequences*The notation “a” in front of an exemplary antigen expressed on an immunosuppressive cell denotes an antibody that specifically binds to the antigen expressed on an immunosuppressive cell.#The underlined sequences denote the respective HCDR1, HCDR2, and HCDR3.Table 4 - TRAIL Receptor Moiety Variable Light Sequences*The notation “a” in front of an exemplary antigen expressed on an immunosuppressive cell denotes an antibody that specifically binds to the antigen expressed on an immunosuppressive cell.#The underlined sequences denote the respective LCDR1, LCDR2, and LCDR3.

[0177] In some embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises TRAIL receptor binding moiety comprising an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 87 and SEQ ID NO: 134; and an immunoglobulin light chain variable region at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 91 and SEQ ID NO: 138. In some instances, the amino aciddifferences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain variable region and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 87 and SEQ ID NO: 134. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain variable region and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NOs: 91 and SEQ ID NO: 138. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain variable region but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target TRAIL receptor. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain variable region but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target TRAIL receptor.

[0178] In certain embodiments, the TRAIL receptor binding moiety of the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises sequences (e.g., CDRs or VH / VL sequences) derived from a known antibody such as: Conatumumab, Tigatuzumab, Drozitumab, Lexatumumab, Benufutamab, Zaptuzumab, KMTR2, LBY135 (TRAIL-R2); Mapatumumab (TRAIL-R1).

[0179] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth under the heavy chain (HC) sequences of Table 5

[0180] ; and an immunoglobulin light chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth under the light chain (LC) sequences of Table 5. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in the respective HC sequence in Table 5. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in the respective LC sequence in Table 5. In some cases, the amino acid differences contributing tothe at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multispecific antibody) retains binding to a target tumor antigen and / or a target antigen expressed on an immunosuppressive cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target tumor antigen and / or a target antigen expressed on an immunosuppressive cell.

[0181] As shown below in Table 5, the underlined portion in the HC sequences indicate the scFv portion of the multi-specific antibody. The respective linker sequences in the heavy chain are shown in lower cases. The LALA mutation is bolded.Table 5 - Multi-Specific Binding Polypeptide Heavy and Light Sequences

[0182] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 92; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 93. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 92. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 93. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0183] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 94; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 95. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 94. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 95. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0184] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consistof the amino acid sequence set forth in SEQ ID NO: 96; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 97. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 96. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 97. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0185] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 98; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 99. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 98. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 99. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%,95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0186] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 100; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 101. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 100. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 101. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0187] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 102; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 103 In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 102. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 103. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0188] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 104; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 105. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 104. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 105. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0189] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consistof the amino acid sequence set forth in SEQ ID NO: 106; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 107. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 106. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 107. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0190] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 108; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 109. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 108. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 109. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%,95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0191] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 110; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 111. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 110. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 111. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0192] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 139; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 140. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 139. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 140. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0193] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 141; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 142. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 141. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 142. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0194] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consistof the amino acid sequence set forth in SEQ ID NO: 143; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 93. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 143. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 93. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0195] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 144; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 103. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 144. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 103. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%,95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0196] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 145; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 101. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 145. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 101. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0197] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 146; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 140. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 146. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 140. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0198] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 147; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 142. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 147. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 142. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0199] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consistof the amino acid sequence set forth in SEQ ID NO: 148; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 111. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 148. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 111. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0200] In certain embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) described herein comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 149; and an immunoglobulin light chain at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 99. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin heavy chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 149. In some instances, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity resides in a framework region of the immunoglobulin light chain and the CDRs remain unchanged relative to the CDRs set forth in SEQ ID NO: 99. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin heavy chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell. In some cases, the amino acid differences contributing to the at least about 80%, 85%, 90%,95%, 97%, 98%, or 99% sequence identity comprises conservative substitutions in the framework region, CDRs region, or a combination thereof, of the immunoglobulin light chain but the multi-specific binding polypeptide (e.g., the multi-specific antibody) retains binding to a target antigen expressed on a cell.

[0201] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to CD33 / SIGLEC3 and a binding moiety that specifically binds to TRAIL-R2. In certain embodiments the moiety that binds to CD33 / SIGLEC3 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 7; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 47. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 87; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 91. In certain embodiments, the heavy chain of the multi-specific binding polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 92; and the light chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 93. SEQ ID NO: 92 and 93 form a bispecific molecule depicted as in FIG. 1A and FIG. 2A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multispecific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce the affinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., a modification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia (e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., a modification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0202] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds CD33 comprising a light chain variable region and a heavy chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgG that specifically binds CD33 comprises a CDR1 with the amino acid sequence set forth as DSNIH (SEQ ID NO: 4), a CDR2 with the amino acid sequence set forth as YIYPYNGGTDYNQKFKN (SEQ ID NO: 5), and a CDR3 with the amino acid sequence setforth as GNPWLAY (SEQ ID NO: 6), and the light chain variable region of the IgG that specifically binds CD33 comprises a CDR1 with the amino acid sequence set forth as RASESLDNYGIRFLT (SEQ ID NO: 44), a CDR2 with the amino acid sequence set forth as AASNQGS (SEQ ID NO: 45), and a CDR3 with the amino acid sequence set forth as QQTKEVPWS (SEQ ID NO: 46). In some instances, the IgG that specifically binds TRAIL-R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 with the amino acid sequence set forth as DYFWS (SEQ ID NO: 84), a CDR2 with the amino acid sequence set forth as HIHNSGTTYYNPSLKS (SEQ ID NO: 85), and a CDR3 with the amino acid sequence set forth as DRGGDYYYGMDV (SEQ ID NO: 86), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as RASQGISRSYLA (SEQ ID NO: 88), a CDR2 with the amino acid sequence set forth as GASSRAT (SEQ ID NO: 89), and a CDR3 with the amino acid sequence set forth as QQFGSSPWT (SEQ ID NO: 90).

[0203] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to HSP90 and a binding moiety that specifically binds to TRAIL-R2. In certain embodiments the moiety that binds to HSP90 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 11; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 51. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 87; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 91. In certain embodiments, the heavy chain of the multi-specific binding polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 94; and the light chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 95. SEQ ID NO: 94 and 95 form a bispecific molecule depicted as in FIG. 1A and FIG. 9A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multispecific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce the affinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., a modification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia (e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., amodification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0204] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds HSP90 comprising a heavy chain variable region and a light chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgG that specifically binds HSP90 comprises a CDR1 with the amino acid sequence set forth as SYYIH (SEQ ID NO: 8), a CDR2 with the amino acid sequence set forth as WIYPGNVNTKY (SEQ ID NO: 9), and a CDR3 with the amino acid sequence set forth as YGNYPFAY (SEQ ID NO: 10), and the light chain variable region of the IgG that specifically binds HSP90 comprises a CDR1 with the amino acid sequence set forth as RSSQSLVHSNGNTYLH (SEQ ID NO: 48), a CDR2 with the amino acid sequence set forth as KVSNRF (SEQ ID NO: 49), and a CDR3 with the amino acid sequence set forth as SQSTHVPPT (SEQ ID NO: 50). In some instances, the IgG that specifically binds TRAIL-R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 with the amino acid sequence set forth as DYFWS (SEQ ID NO: 84), a CDR2 with the amino acid sequence set forth as HIHNSGTTYYNPSLKS (SEQ ID NO: 85), and a CDR3 with the amino acid sequence set forth as DRGGDYYYGMDV (SEQ ID NO: 86), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as RASQGISRSYLA (SEQ ID NO: 88), a CDR2 with the amino acid sequence set forth as GASSRAT (SEQ ID NO: 89), and a CDR3 with the amino acid sequence set forth as QQFGSSPWT (SEQ ID NO: 90).

[0205] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to HER2 and a binding moiety that specifically binds to TRAIL-R2. In certain embodiments the moiety that binds to HER2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 15; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 55. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 87; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 91. In certain embodiments, the heavy chain of the multi-specific binding polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 96; and thelight chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 97. SEQ ID NO: 96 and 97 form a bispecific molecule depicted as in FIG. 1A and FIG. 8A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multispecific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce the affinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., a modification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia (e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., a modification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0206] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds HER2 comprising a light chain variable region and a heavy chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgG that specifically binds HER2 comprises a CDR1 with the amino acid sequence set forth as DTYIH (SEQ ID NO: 12), a CDR2 with the amino acid sequence set forth as RIYPTNGYTRYADSVKG (SEQ ID NO: 13), and a CDR3 with the amino acid sequence set forth as WGGDGFYAMDY (SEQ ID NO: 14), and the light chain variable region of the IgG that specifically binds HER2 comprises a CDR1 with the amino acid sequence set forth as RASQDVNTAVA (SEQ ID NO: 52), a CDR2 with the amino acid sequence set forth as SASFLYS (SEQ ID NO: 53), and a CDR3 with the amino acid sequence set forth as QQHYTTPPT (SEQ ID NO: 54). In some instances, the IgG that specifically binds TRAIL-R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 with the amino acid sequence set forth as DYFWS (SEQ ID NO: 84), a CDR2 with the amino acid sequence set forth as HIHNSGTTYYNPSLKS (SEQ ID NO: 85), and a CDR3 with the amino acid sequence set forth as DRGGDYYYGMDV (SEQ ID NO: 86), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as RASQGISRSYLA (SEQ ID NO: 88), a CDR2 with the amino acid sequence set forth as GASSRAT (SEQ ID NO: 89), and a CDR3 with the amino acid sequence set forth as QQFGSSPWT (SEQ ID NO: 90).

[0207] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to GPC3 and a binding moiety that specifically binds to TRAIL-R2. In certain embodiments the moiety that binds to GPC3 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 19;and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 59. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 87; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 91. In certain embodiments, the heavy chain of the multi-specific binding polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 98; and the light chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 99. SEQ ID NO: 98 and 99 form a bispecific molecule depicted as in FIG. 1A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multi-specific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce the affinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., a modification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia (e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., a modification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0208] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds GPC3 comprising a light chain variable region and a heavy chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgG that specifically binds GPC3 comprises a CDR1 with the amino acid sequence set forth as DYEMH (SEQ ID NO: 16), a CDR2 with the amino acid sequence set forth as ALDPKTGDTAYSQKFKG (SEQ ID NO: 17), and a CDR3 with the amino acid sequence set forth as FYSYTY (SEQ ID NO: 18), and the light chain variable region of the IgG that specifically binds GPC3 comprises a CDR1 with the amino acid sequence set forth as RSSQSLVHSNRNTYLH (SEQ ID NO: 56), a CDR2 with the amino acid sequence set forth as KVSNRFS (SEQ ID NO: 57), and a CDR3 with the amino acid sequence set forth as SQNTHVPPT (SEQ ID NO: 58). In some instances, the IgG that specifically binds TRAIL-R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 with the amino acid sequence set forth as DYFWS (SEQ ID NO: 84), a CDR2 with the amino acid sequence set forth as HIHNSGTTYYNPSLKS (SEQ ID NO: 85), and a CDR3 with the amino acid sequence set forth as DRGGDYYYGMDV(SEQ ID NO: 86), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as RASQGISRSYLA (SEQ ID NO: 88), a CDR2 with the amino acid sequence set forth as GASSRAT (SEQ ID NO: 89), and a CDR3 with the amino acid sequence set forth as QQFGSSPWT (SEQ ID NO: 90).

[0209] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to FOLR1 and a binding moiety that specifically binds to TRAIL-R2. In certain embodiments the moiety that binds to FOLR1 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 23; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 63. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 87; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 91. In certain embodiments, the heavy chain of the multi-specific binding polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 100; and the light chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 101. SEQ ID NO: 100 and 101 form a bispecific molecule depicted as in FIG. 1A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multi-specific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce the affinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., a modification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia (e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., a modification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0210] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds FOLR1 comprising a heavy chain variable region and a light chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgG that specifically binds FOLR1 comprises a CDR1 with the amino acid sequence set forth as GYFMN (SEQ ID NO: 20), a CDR2 with the amino acid sequence set forth as RIHPYDGDTFYNQKFQG (SEQ ID NO: 21), and a CDR3 with the amino acid sequence setforth as YDGSRAMDY (SEQ ID NO: 22), and the light chain variable region of the IgG that specifically binds F0LR1 comprises a CDR1 with the amino acid sequence set forth as KASQSVSFAGTSLMH (SEQ ID NO: 60), a CDR2 with the amino acid sequence set forth as RASNLEA (SEQ ID NO: 61), and a CDR3 with the amino acid sequence set forth as QQSREYPYT (SEQ ID NO: 62). In some instances, the IgG that specifically binds TRAIL-R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 with the amino acid sequence set forth as DYFWS (SEQ ID NO: 84), a CDR2 with the amino acid sequence set forth as HIHNSGTTYYNPSLKS (SEQ ID NO: 85), and a CDR3 with the amino acid sequence set forth as DRGGDYYYGMDV (SEQ ID NO: 86), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as RASQGISRSYLA (SEQ ID NO: 88), a CDR2 with the amino acid sequence set forth as GASSRAT (SEQ ID NO: 89), and a CDR3 with the amino acid sequence set forth as QQFGSSPWT (SEQ ID NO: 90).

[0211] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to CD38 and a binding moiety that specifically binds to TRAIL-R2. In certain embodiments the moiety that binds to CD38 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 27; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 67. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 87; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 91. In certain embodiments, the heavy chain of the multi-specific binding polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 102; and the light chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 103. SEQ ID NO: 102 and 103 form a bispecific molecule depicted as in FIG. 1A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multi-specific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce the affinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., a modification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia (e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., amodification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0212] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds CD38 comprising a heavy chain variable region and a light chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgG that specifically binds CD38 comprises a CDR1 with the amino acid sequence set forth as SFAMS (SEQ ID NO: 24), a CDR2 with the amino acid sequence set forth as AISGSGGGTYYADSVKG (SEQ ID NO: 25), and a CDR3 with the amino acid sequence set forth as DKILWFGEPVFDY (SEQ ID NO: 26), and the light chain variable region of the IgG that specifically binds CD38 comprises a CDR1 with the amino acid sequence set forth as RASQSVSSYLA (SEQ ID NO: 64), a CDR2 with the amino acid sequence set forth as DASNRAT (SEQ ID NO: 65), and a CDR3 with the amino acid sequence set forth as QQRSNWPPT (SEQ ID NO: 66). In some instances, the IgG that specifically binds TRAIL-R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 with the amino acid sequence set forth as DYFWS (SEQ ID NO: 84), a CDR2 with the amino acid sequence set forth as HIHNSGTTYYNPSLKS (SEQ ID NO: 85), and a CDR3 with the amino acid sequence set forth as DRGGDYYYGMDV (SEQ ID NO: 86), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as RASQGISRSYLA (SEQ ID NO: 88), a CDR2 with the amino acid sequence set forth as GASSRAT (SEQ ID NO: 89), and a CDR3 with the amino acid sequence set forth as QQFGSSPWT (SEQ ID NO: 90).

[0213] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to CD20 and a binding moiety that specifically binds to TRAIL-R2. In certain embodiments the moiety that binds to CD20 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 31; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 71. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 87; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 91. In certain embodiments, the heavy chain of the multi-specific binding polypeptide comprises an amino acid sequence at least about 80%,85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 104; and the light chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 105. SEQ ID NO: 104 and 105 form a bispecific molecule depicted as in FIG. 1A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multi-specific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce the affinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., a modification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia (e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., a modification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0214] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds CD20 comprising a heavy chain variable region and a light chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgG that specifically binds CD20 comprises a CDR1 with the amino acid sequence set forth as TSYNMH (SEQ ID NO: 28), a CDR2 with the amino acid sequence set forth as AIYPGNGDTSYNQKFKG (SEQ ID NO: 29), and a CDR3 with the amino acid sequence set forth as STYYGGDWYFNV (SEQ ID NO: 30), and the light chain variable region of the IgG that specifically binds CD20 comprises a CDR1 with the amino acid sequence set forth as RASSSVSYIH (SEQ ID NO: 68), a CDR2 with the amino acid sequence set forth as ATSNLAS (SEQ ID NO: 69), and a CDR3 with the amino acid sequence set forth as QQWTSNPPT (SEQ ID NO: 70). In some instances, the IgG that specifically binds TRAIL-R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 with the amino acid sequence set forth as DYFWS (SEQ ID NO: 84), a CDR2 with the amino acid sequence set forth as HIHNSGTTYYNPSLKS (SEQ ID NO: 85), and a CDR3 with the amino acid sequence set forth as DRGGDYYYGMDV (SEQ ID NO: 86), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as RASQGISRSYLA (SEQ ID NO: 88), a CDR2 with the amino acid sequence set forth as GASSRAT (SEQ ID NO: 89), and a CDR3 with the amino acid sequence set forth as QQFGSSPWT (SEQ ID NO: 90).

[0215] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to TROP2 and a binding moiety that specifically binds to TRAIL-R2. In certain embodiments the moiety that binds to TROP2 comprises a heavy chain variable region that comprises an amino acid sequence at least about80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 35; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 75. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 87; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 91. In certain embodiments, the heavy chain of the multi-specific binding polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 106; and the light chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 107. SEQ ID NO: 106 and 107 form a bispecific molecule depicted as in FIG. 1A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multi-specific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce the affinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., a modification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia (e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., a modification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0216] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds TROP2 comprising a heavy chain variable region and a light chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgG that specifically binds TROP2 comprises a CDR1 with the amino acid sequence set forth as NYGMN (SEQ ID NO: 32), a CDR2 with the amino acid sequence set forth as WINTYTGEPTYTDDFKG (SEQ ID NO: 33), and a CDR3 with the amino acid sequence set forth as GGFGSSYWYFDV (SEQ ID NO: 34), and the light chain variable region of the IgG that specifically binds TROP2 comprises a CDR1 with the amino acid sequence set forth as KASQDVSIAVA (SEQ ID NO: 72), a CDR2 with the amino acid sequence set forth as SASYRYT (SEQ ID NO: 73), and a CDR3 with the amino acid sequence set forth as QQHYITPLT (SEQ ID NO: 74). In some instances, the IgG that specifically binds TRAIL-R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 with the amino acid sequence set forth as DYFWS (SEQ ID NO: 84), a CDR2 with the amino acid sequence set forth as HIHNSGTTYYNPSLKS(SEQ ID NO: 85), and a CDR3 with the amino acid sequence set forth as DRGGDYYYGMDV (SEQ ID NO: 86), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as RASQGISRSYLA (SEQ ID NO: 88), a CDR2 with the amino acid sequence set forth as GASSRAT (SEQ ID NO: 89), and a CDR3 with the amino acid sequence set forth as QQFGSSPWT (SEQ ID NO: 90).

[0217] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to CEA and a binding moiety that specifically binds to TRAIL-R2. In certain embodiments the moiety that binds to CEA comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 39; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 79. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 87; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 91. In certain embodiments, the heavy chain of the multi-specific binding polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 108; and the light chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 109. SEQ ID NO: 108 and 109 form a bispecific molecule depicted as in FIG. 1A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multi-specific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce the affinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., a modification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia (e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., a modification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0218] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds CEA comprising a heavy chain variable region and a light chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgG that specifically binds CEA comprises a CDR1 with the amino acid sequence set forth as TYWMS (SEQ ID NO: 36), a CDR2 with the amino acid sequence set forth asEIHPDSSTINYAPSLKD (SEQ ID NO: 37), and a CDR3 with the amino acid sequence set forth as ASLYFGFPWFAY (SEQ ID NO: 38), and the light chain variable region of the IgG that specifically binds CEA comprises a CDR1 with the amino acid sequence set forth as KASQDVGTSVA (SEQ ID NO: 76), a CDR2 with the amino acid sequence set forth as WTSTRHT (SEQ ID NO: 77), and a CDR3 with the amino acid sequence set forth as QQYSLYRS (SEQ ID NO: 78). In some instances, the IgG that specifically binds TRAIL-R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 with the amino acid sequence set forth as DYFWS (SEQ ID NO: 84), a CDR2 with the amino acid sequence set forth as HIHNSGTTYYNPSLKS (SEQ ID NO: 85), and a CDR3 with the amino acid sequence set forth as DRGGDYYYGMDV (SEQ ID NO: 86), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as RASQGISRSYLA (SEQ ID NO: 88), a CDR2 with the amino acid sequence set forth as GASSRAT (SEQ ID NO: 89), and a CDR3 with the amino acid sequence set forth as QQFGSSPWT (SEQ ID NO: 90).

[0219] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to FLT3 and a binding moiety that specifically binds to TRAIL-R2. In certain embodiments the moiety that binds to FLT3 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 43; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 83. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 87; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 91. In certain embodiments, the heavy chain of the multi-specific binding polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 110; and the light chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 111. SEQ ID NO: 110 and 111 form a bispecific molecule depicted as in FIG. 1A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multi-specific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce the affinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., a modification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia(e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., a modification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0220] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds FLT3 comprising a heavy chain variable region and a light chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgG that specifically binds FLT3 comprises a CDR1 with the amino acid sequence set forth as SYYMH (SEQ ID NO: 40), a CDR2 with the amino acid sequence set forth as IINPSGGSTSYAQKFQG (SEQ ID NO: 41), and a CDR3 with the amino acid sequence set forth as GVGAHDAFDI (SEQ ID NO: 42), and the light chain variable region of the IgG that specifically binds FLT3 comprises a CDR1 with the amino acid sequence set forth as RSSQSLLHSNGNNYLD (SEQ ID NO: 80), a CDR2 with the amino acid sequence set forth as LGSNRAS (SEQ ID NO: 81), and a CDR3 with the amino acid sequence set forth as MQGTHPAIS (SEQ ID NO: 82). In some instances, the IgG that specifically binds TRAIL-R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 with the amino acid sequence set forth as DYFWS (SEQ ID NO: 84), a CDR2 with the amino acid sequence set forth as HIHNSGTTYYNPSLKS (SEQ ID NO: 85), and a CDR3 with the amino acid sequence set forth as DRGGDYYYGMDV (SEQ ID NO: 86), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as RASQGISRSYLA (SEQ ID NO: 88), a CDR2 with the amino acid sequence set forth as GASSRAT (SEQ ID NO: 89), and a CDR3 with the amino acid sequence set forth as QQFGSSPWT (SEQ ID NO: 90).

[0221] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to CD22 and a binding moiety that specifically binds to TRAIL-R2. In certain embodiments the moiety that binds to CD22 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 118; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 126. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 87; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 91. In certain embodiments, the heavy chain ofthe multi-specific binding polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 139 ; and the light chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 140. SEQ ID NO: 139 and 140 form a bispecific molecule depicted as in FIG. 1A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multi-specific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce the affinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., a modification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia (e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., a modification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0222] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds CD22 comprising a heavy chain variable region and a light chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgG that specifically binds CD22 comprises a CDR1 with the amino acid sequence set forth as NYWIH (SEQ ID NO: 115), a CDR2 with the amino acid sequence set forth as GINPGNNYATYRRKFQG (SEQ ID NO: 116), and a CDR3 with the amino acid sequence set forth as EGYGNYGAWFAY (SEQ ID NO: 117), and the light chain variable region of the IgG that specifically binds CD22 comprises a CDR1 with the amino acid sequence set forth as RSSQSLANSYGNTFLS (SEQ ID NO: 123), a CDR2 with the amino acid sequence set forth as GISNRFS (SEQ ID NO: 124 ), and a CDR3 with the amino acid sequence set forth as LQGTHQPYT (SEQ ID NO: 125). In some instances, the IgG that specifically binds TR.AIL- R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 with the amino acid sequence set forth as DYFWS (SEQ ID NO: 84), a CDR2 with the amino acid sequence set forth as HIHNSGTTYYNPSLKS (SEQ ID NO: 85), and a CDR3 with the amino acid sequence set forth as DRGGDYYYGMDV (SEQ ID NO: 86), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as RASQGISRSYLA (SEQ ID NO: 88), a CDR2 with the amino acid sequence set forth as GASSRAT (SEQ ID NO: 89), and a CDR3 with the amino acid sequence set forth as QQFGSSPWT (SEQ ID NO: 90).

[0223] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to PDL1 and a binding moiety that specifically binds to TRAIL-R2. In certain embodiments the moiety that binds to PDL1comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 122; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 130. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 87; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 91. In certain embodiments, the heavy chain of the multi-specific binding polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 141; and the light chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 142. SEQ ID NO: 141 and 142 form a bispecific molecule depicted as in FIG. 1A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multi-specific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce the affinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., a modification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia (e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., a modification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0224] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds PDL1 comprising a heavy chain variable region and a light chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgG that specifically binds PDL1 comprises a CDR1 with the amino acid sequence set forth as SYIMM (SEQ ID NO: 119), a CDR2 with the amino acid sequence set forth as SIYPSGGITFYADTVKG (SEQ ID NO: 120), and a CDR3 with the amino acid sequence set forth as IKLGTVTTVDY (SEQ ID NO: 120), and the light chain variable region of the IgG that specifically binds PDL1 comprises a CDR1 with the amino acid sequence set forth as TGTSSDVGGYNYVS (SEQ ID NO: 127), a CDR2 with the amino acid sequence set forth as DVSNRPS (SEQ ID NO: 128), and a CDR3 with the amino acid sequence set forth as SSYTSSSTRV (SEQ ID NO: 129). In some instances, the IgG that specifically binds TRAIL-R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 with the amino acid sequence set forth as DYFWS(SEQ ID NO: 84), a CDR2 with the amino acid sequence set forth as HIHNSGTTYYNPSLKS (SEQ ID NO: 85), and a CDR3 with the amino acid sequence set forth as DRGGDYYYGMDV (SEQ ID NO: 86), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as RASQGISRSYLA (SEQ ID NO: 88), a CDR2 with the amino acid sequence set forth as GASSRAT (SEQ ID NO: 89), and a CDR3 with the amino acid sequence set forth as QQFGSSPWT (SEQ ID NO: 90).

[0225] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to CD33 / SIGLEC3 and a binding moiety that specifically binds to TRAIL-R2. In certain embodiments the moiety that binds to CD33 / SIGLEC3 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 7; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 47. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 134; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 138. In certain embodiments, the heavy chain of the multi-specific binding polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 143; and the light chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 93. SEQ ID NO: 143 and 93 form a bispecific molecule depicted as in FIG. 1A and FIG. 2A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multi-specific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce the affinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., a modification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia (e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., a modification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0226] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds CD33 comprising a light chain variable region and a heavy chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgG that specifically binds CD33 comprises a CDR1 with the amino acid sequence set forth asDSNIH (SEQ ID NO: 4), a CDR2 with the amino acid sequence set forth as YIYPYNGGTDYNQKFKN (SEQ ID NO: 5), and a CDR3 with the amino acid sequence set forth as GNPWLAY (SEQ ID NO: 6), and the light chain variable region of the IgG that specifically binds CD33 comprises a CDR1 with the amino acid sequence set forth as RASESLDNYGIRFLT (SEQ ID NO: 44), a CDR2 with the amino acid sequence set forth as AASNQGS (SEQ ID NO: 45), and a CDR3 with the amino acid sequence set forth as QQTKEVPWS (SEQ ID NO: 46). In some instances, the IgG that specifically binds TRAIL-R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 with the amino acid sequence set forth as DYGMS (SEQ ID NO: 131), a CDR2 with the amino acid sequence set forth as GINWNGGSTGYADSVKG (SEQ ID NO: 132), and a CDR3 with the amino acid sequence set forth as ILGAGRGWYFDL (SEQ ID NO: 133), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as QGDSLRSYYAS (SEQ ID NO: 135), a CDR2 with the amino acid sequence set forth as GKNNRPS (SEQ ID NO: 136), and a CDR3 with the amino acid sequence set forth as NSRDSSGNHVV (SEQ ID NO: 137).

[0227] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to CD38 and a binding moiety that specifically binds to TRAIL-R2. In certain embodiments the moiety that binds to CD38 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 27; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 67. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 134; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 138. In certain embodiments, the heavy chain of the multi-specific binding polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 144; and the light chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 103. SEQ ID NO: 144 and 103 form a bispecific molecule depicted as in FIG. 1A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multi-specific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce the affinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., amodification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia (e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., a modification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0228] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds CD38 comprising a heavy chain variable region and a light chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgG that specifically binds CD38 comprises a CDR1 with the amino acid sequence set forth as SFAMS (SEQ ID NO: 24), a CDR2 with the amino acid sequence set forth as AISGSGGGTYYADSVKG (SEQ ID NO: 25), and a CDR3 with the amino acid sequence set forth as DKILWFGEPVFDY (SEQ ID NO: 26), and the light chain variable region of the IgG that specifically binds CD38 comprises a CDR1 with the amino acid sequence set forth as RASQSVSSYLA (SEQ ID NO: 64), a CDR2 with the amino acid sequence set forth as DASNRAT (SEQ ID NO: 65), and a CDR3 with the amino acid sequence set forth as QQRSNWPPT (SEQ ID NO: 66). In some instances, the IgG that specifically binds TRAIL-R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 with the amino acid sequence set forth as DYGMS (SEQ ID NO: 131), a CDR2 with the amino acid sequence set forth as GINWNGGSTGYADSVKG (SEQ ID NO: 132), and a CDR3 with the amino acid sequence set forth as ILGAGRGWYFDL (SEQ ID NO: 133), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as QGDSLRSYYAS (SEQ ID NO: 135), a CDR2 with the amino acid sequence set forth as GKNNRPS (SEQ ID NO: 136), and a CDR3 with the amino acid sequence set forth as NSRDSSGNHVV (SEQ ID NO: 137).

[0229] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to FOLR1 and a binding moiety that specifically binds to TRAIL-R2. In certain embodiments the moiety that binds to FOLR1 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 23; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 63. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 134; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% identical to that set forth in SEQ ID NO: 138. In certain embodiments, the heavy chain of the multi-specific binding polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 145; and the light chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 101. SEQ ID NO: 145 and 101 form a bispecific molecule depicted as in FIG. 1A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multi-specific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce the affinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., a modification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia (e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., a modification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0230] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds FOLR1 comprising a heavy chain variable region and a light chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgG that specifically binds FOLR1 comprises a CDR1 with the amino acid sequence set forth as GYFMN (SEQ ID NO: 20), a CDR2 with the amino acid sequence set forth as RIHPYDGDTFYNQKFQG (SEQ ID NO: 21), and a CDR3 with the amino acid sequence set forth as YDGSRAMDY (SEQ ID NO: 22), and the light chain variable region of the IgG that specifically binds FOLR1 comprises a CDR1 with the amino acid sequence set forth as KASQSVSFAGTSLMH (SEQ ID NO: 60), a CDR2 with the amino acid sequence set forth as RASNLEA (SEQ ID NO: 61), and a CDR3 with the amino acid sequence set forth as QQSREYPYT (SEQ ID NO: 62). In some instances, the IgG that specifically binds TRAIL-R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 with the amino acid sequence set forth as DYGMS (SEQ ID NO: 131), a CDR2 with the amino acid sequence set forth as GINWNGGSTGYADSVKG (SEQ ID NO: 132), and a CDR3 with the amino acid sequence set forth as ILGAGRGWYFDL (SEQ ID NO: 133), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as QGDSLRSYYAS (SEQ ID NO: 135), a CDR2 with the amino acid sequence set forth as GKNNRPS (SEQ ID NO: 136), and a CDR3 with the amino acid sequence set forth as NSRDSSGNHVV (SEQ ID NO: 137).

[0231] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to CD22 and a binding moiety thatspecifically binds to TRAIL-R2. In certain embodiments the moiety that binds to CD22 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 118; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 126. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 134; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 138. In certain embodiments, the heavy chain of the multi-specific binding polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 146; and the light chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 140. SEQ ID NO: 146 and 140 form a bispecific molecule depicted as in FIG. 1A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multi-specific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce the affinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., a modification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia (e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., a modification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0232] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds CD22 comprising a heavy chain variable region and a light chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgG that specifically binds CD22 comprises a CDR1 with the amino acid sequence set forth as NYWIH (SEQ ID NO: 115), a CDR2 with the amino acid sequence set forth as GINPGNNYATYRRKFQG (SEQ ID NO: 116), and a CDR3 with the amino acid sequence set forth as EGYGNYGAWFAY (SEQ ID NO: 117), and the light chain variable region of the IgG that specifically binds CD22 comprises a CDR1 with the amino acid sequence set forth as RSSQSLANSYGNTFLS (SEQ ID NO: 123), a CDR2 with the amino acid sequence set forth as GISNRFS (SEQ ID NO: 124), and a CDR3 with the amino acid sequence set forth as LQGTHQPYT (SEQ ID NO: 125). In some instances, the IgG that specifically binds TRAIL-R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavychain variable region comprises a CDR1 with the amino acid sequence set forth as DYGMS (SEQ ID NO: 131), a CDR2 with the amino acid sequence set forth as GINWNGGSTGYADSVKG (SEQ ID NO: 132), and a CDR3 with the amino acid sequence set forth as ILGAGRGWYFDL (SEQ ID NO: 133), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as QGDSLRSYYAS (SEQ ID NO: 135), a CDR2 with the amino acid sequence set forth as GKNNRPS (SEQ ID NO: 136), and a CDR3 with the amino acid sequence set forth as NSRDSSGNHVV (SEQ ID NO: 137).

[0233] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to PDL1 and a binding moiety that specifically binds to TRAIL-R2. In certain embodiments the moiety that binds to PDL1 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 122; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 130. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 134; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 138. In certain embodiments, the heavy chain of the multi-specific binding polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 147; and the light chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 142. SEQ ID NO: 147 and 142 form a bispecific molecule depicted as in FIG. 1A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multi-specific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce the affinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., a modification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia (e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., a modification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0234] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds PDL1 comprising a heavy chain variable region and a light chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgGthat specifically binds PDL1 comprises a CDR1 with the amino acid sequence set forth as SYIMM (SEQ ID NO: 119), a CDR2 with the amino acid sequence set forth as SIYPSGGITFYADTVKG (SEQ ID NO: 120), and a CDR3 with the amino acid sequence set forth as IKLGTVTTVDY (SEQ ID NO: 120), and the light chain variable region of the IgG that specifically binds PDL1 comprises a CDR1 with the amino acid sequence set forth as TGTSSDVGGYNYVS (SEQ ID NO: 127), a CDR2 with the amino acid sequence set forth as DVSNRPS (SEQ ID NO: 128), and a CDR3 with the amino acid sequence set forth as SSYTSSSTRV (SEQ ID NO: 129). In some instances, the IgG that specifically binds TRAIL-R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 with the amino acid sequence set forth as DYGMS (SEQ ID NO: 131), a CDR2 with the amino acid sequence set forth as GINWNGGSTGYADSVKG (SEQ ID NO: 132), and a CDR3 with the amino acid sequence set forth as ILGAGRGWYFDL (SEQ ID NO: 133), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as QGDSLRSYYAS (SEQ ID NO: 135), a CDR2 with the amino acid sequence set forth as GKNNRPS (SEQ ID NO: 136), and a CDR3 with the amino acid sequence set forth as NSRDSSGNHVV (SEQ ID NO: 137).

[0235] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to FLT3 and a binding moiety that specifically binds to TRAIL-R2. In certain embodiments the moiety that binds to FLT3 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 43; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 83. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 134; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 138. In certain embodiments, the heavy chain of the multi-specific binding polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 148; and the light chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 111. SEQ ID NO: 148 and 111 form a bispecific molecule depicted as in FIG. 1A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multi-specific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce theaffinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., a modification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia (e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., a modification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0236] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds FLT3 comprising a heavy chain variable region and a light chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgG that specifically binds FLT3 comprises a CDR1 with the amino acid sequence set forth as SYYMH (SEQ ID NO: 40), a CDR2 with the amino acid sequence set forth as IINPSGGSTSYAQKFQG (SEQ ID NO: 41), and a CDR3 with the amino acid sequence set forth as GVGAHDAFDI (SEQ ID NO: 42), and the light chain variable region of the IgG that specifically binds FLT3 comprises a CDR1 with the amino acid sequence set forth as RSSQSLLHSNGNNYLD (SEQ ID NO: 80), a CDR2 with the amino acid sequence set forth as LGSNRAS (SEQ ID NO: 81), and a CDR3 with the amino acid sequence set forth as MQGTHPAIS (SEQ ID NO: 82). In some instances, the IgG that specifically binds TRAIL-R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 with the amino acid sequence set forth as DYGMS (SEQ ID NO: 131), a CDR2 with the amino acid sequence set forth as GINWNGGSTGYADSVKG (SEQ ID NO: 132), and a CDR3 with the amino acid sequence set forth as ILGAGRGWYFDL (SEQ ID NO: 133), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as QGDSLRSYYAS (SEQ ID NO: 135), a CDR2 with the amino acid sequence set forth as GKNNRPS (SEQ ID NO: 136), and a CDR3 with the amino acid sequence set forth as NSRDSSGNHVV (SEQ ID NO: 137).

[0237] In certain embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) comprises a binding moiety that specifically binds to GPC3 and a binding moiety that specifically binds to TRAIL-R2. In certain embodiments the moiety that binds to GPC3 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 19; and a light chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 59. In certain embodiments the moiety that binds to TRAIL-R2 comprises a heavy chain variable region that comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 134; and a light chain variable regionthat comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 138. In certain embodiments, the heavy chain of the multi-specific binding polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 149; and the light chain comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to that set forth in SEQ ID NO: 99. SEQ ID NO: 149 and 99 form a bispecific molecule depicted as in FIG. 1A that can be conjugated to a cytotoxic moiety as in FIG. IB. In some instances, the multi-specific binding polypeptide (e.g., the multi-specific antibody) further comprises one or more Fc modifications (e.g., substitutions) to reduce the affinity for human neonatal Fc receptor (FcRn), to reduce ADCC functionality (e.g., a modification at L234, L235, P238, or P331, or a combination thereof), to reduce neutropenia (e.g., a modification at L234, S239, S442, or a combination thereof), to enhance ADCC (e.g., a modification at S239, A330, 1332, or a combination thereof), and / or to modulate hinge region rigidity (e.g., a modification at S228).

[0238] In certain embodiments, the multi-specific binding polypeptide comprises an IgG that specifically binds GPC3 comprising a light chain variable region and a heavy chain variable region, and an IgG that specifically binds TRAIL-R2 comprising a heavy chain variable region and a light chain variable region. In some instances, the heavy chain variable region of the IgG that specifically binds GPC3 comprises a CDR1 with the amino acid sequence set forth as DYEMH (SEQ ID NO: 16), a CDR2 with the amino acid sequence set forth as ALDPKTGDTAYSQKFKG (SEQ ID NO: 17), and a CDR3 with the amino acid sequence set forth as FYSYTY (SEQ ID NO: 18), and the light chain variable region of the IgG that specifically binds GPC3 comprises a CDR1 with the amino acid sequence set forth as RSSQSLVHSNRNTYLH (SEQ ID NO: 56), a CDR2 with the amino acid sequence set forth as KVSNRFS (SEQ ID NO: 57), and a CDR3 with the amino acid sequence set forth as SQNTHVPPT (SEQ ID NO: 58). In some instances, the IgG that specifically binds TRAIL-R2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 with the amino acid sequence set forth as DYGMS (SEQ ID NO: 131), a CDR2 with the amino acid sequence set forth as GINWNGGSTGYADSVKG (SEQ ID NO: 132), and a CDR3 with the amino acid sequence set forth as ILGAGRGWYFDL (SEQ ID NO: 133), and the light chain variable region comprises a CDR1 with the amino acid sequence set forth as QGDSLRSYYAS (SEQ ID NO: 135), a CDR2 with the amino acid sequence set forth as GKNNRPS (SEQ ID NO: 136), and a CDR3 with the amino acid sequence set forth as NSRDSSGNHVV (SEQ ID NO: 137). The affinities of the cellspecific antigen binding moiety and the TRAIL receptors binding moiety can be tuned to reduceoff-target cytotoxic effects. In certain embodiments, the affinity of the cell-specific antigen binding moiety is greater than the affinity of the TRAIL receptors binding moiety. In certain embodiments, the affinity of the cell-specific antigen binding moiety is about 19.3-fold, about 22.38-fold, about 8.6-fold, about >9800-fold, greater than the affinity of the TRAIL receptors binding moiety. In certain embodiments, the affinity of the cell-specific antigen is less than about 0.75 nM, about 0.84 nM, about < 0.001 nM, or about 1.4 nM. In certain embodiments, the affinity of TRAIL receptors binding moiety is greater than about 14.5 nM, about 18.8 nM, about 9.8 nM, or about 12 nM. In certain embodiments, the off-rate (kd (1 / s)) of TNF-related apoptosis-inducing ligand receptors binding moiety is higher than the off-rate (kd (1 / s)) of cellspecific antigen binding moiety. In certain embodiments, the off-rate (kd (1 / s)) of TRAIL receptors binding moiety is at least 100-fold higher than the off-rate (kd (1 / s)) of cell-specific antigen binding moiety. In some cases, the off-rate (kd (1 / s)) of TRAIL receptors binding moiety is about 1.53 x 10'2, about 1.90 x 10'2, about 1.0 x 10'2, or about 1.2 x 10'2. In some cases, the affinity of the TRAIL receptors binding moiety is less than the affinity of the cellspecific antigen binding moiety by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, or higher.

[0239] In certain embodiments, the multi-specific binding polypeptides (e.g., multi-specific antibodies) of the current disclosure have an ECso of killing (e.g., at least about 60%) of a cell of less than about 100 nM, less than about 75 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 0.1 nM, less than about 0.01 nM, less than about 0.001 nM.

[0240] In some embodiments, the multi-specific binding polypeptides (e.g., multi-specific antibodies) are altered to increase or decrease their glycosylation (e.g., by altering the amino acid sequence such that one or more glycosylation sites are created or removed). A carbohydrate attached to an Fc region of an antibody may be altered. Native antibodies from mammalian cells typically comprise a branched, biantennary oligosaccharide attached by an N-linkage to Asn297 of the CH2 domain of the Fc region (See e.g., Wright et al. TIBTECH 15:26-32 (1997)). The oligosaccharide can be various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, sialic acid, fucose attached to a GlcNAc in the stem of the biantennar oligosaccharide structure. Modifications of the oligosaccharide in an antibody can be made, for example, to create antibody variants with certain improved properties. Antibody glycosylation variants can have improved ADCC and / or CDC function. In some embodiments, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined bycalculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn297 (See e.g., WO 08 / 077546). Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues; See e.g., Edelman et al. Proc Natl Acad Set USA. 1969 May; 63(l):78-85). However, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants can have improved ADCC function (See e.g., Okazaki et al. J. Mol. Biol. 336:1239- 1249 (2004); and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)). Cell lines, e.g., knockout cell lines and methods of their use can be used to produce defucosylated antibodies, e.g., Lecl3 CHO cells deficient in protein fucosylation and alpha- 1,6-fucosyltransferase gene (FUT8) knockout CHO cells (See e.g., Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., BiotechnoL Bioeng., 94(4):680-688 (2006)). Other antibody glycosylation variants are also included (See e.g., U.S. Pat. No. 6,602,684).

[0241] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the multi-specific binding polypeptides provided herein, thereby generating an Fc region variant. An Fc region herein is a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. An Fc region includes native sequence Fc regions and variant Fc regions. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0242] In some embodiments, the multi-specific binding polypeptides (e.g., multi-specific antibodies) of this disclosure comprise Fc variants that possess some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity) but retains FcRn binding ability. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No.5,500,362 and 5,821,337. Alternatively, non-radioactive assays methods may be employed (e.g., ACTI™ and CytoTox 96® non-radioactive cytotoxicity assays). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC), monocytes, macrophages, and Natural Killer (NK) cells.

[0243] Antibodies and multi-specific binding polypeptides can have increased half-lives andimproved binding to the neonatal Fc receptor (FcRn) (See e.g., US 2005 / 0014934). Such antibodies can comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn, and include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 according to the EU numbering system (See e.g., U.S. Pat. No. 7,371,826). Other examples of Fc region variants are also contemplated (See e.g., Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos. 5,648,260 and 5,624,821; and WO94 / 29351).

[0244] In some embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) comprises an Fc region that has been modified to reduce the affinity for human neonatal Fc receptor (FcRn).

[0245] In some embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) comprises an Fc region that has been modified to reduce antibody-dependent cellular cytotoxicity (ADCC). In some instances, the Fc region comprises a modification at L234, L235, P238, or P331, or a combination thereof, wherein L234, L235, P238, and P331 correspond to positions 234, 235, 238, and 331 of a wild-type IgGl, according to the EU numbering convention. In some instances, the Fc region comprises a modification at L234, L235, P238, and P331, wherein L234, L235, P238, and P331 correspond to positions 234, 235, 238, and 331 of a wild-type IgGl, according to the EU numbering convention. In some instances, the Fc region comprises L234A, L235A, P238S, P331S, or a combination thereof. In some cases, the Fc region comprises L234A, L235A, P238S, and P331S. In some embodiments described herein, the Fc region multi-specific binding polypeptide comprises a combination of L234A and L235A mutations, also known as the LAL A mutation (See, SEQ ID NOs: 92,96, 98, 100, 102, 104, and 106 in Table 5, for example).

[0246] In some embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) comprises an Fc region that has been modified to reduce neutropenia. In some instances, the Fc region comprises a modification at L234, S239, S442, or a combination thereof, wherein L234, S239, and S442 correspond to positions 234, 239, 442 of a wild-type IgGl, according to the EU numbering convention. In some instances, the Fc region comprises L234F, S239C, S442C, or a combination thereof.

[0247] In some embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) comprises an Fc region that has been modified to enhance antibody-dependent cellular cytotoxicity (ADCC). In some instances, the Fc region comprises a modification at S239, A330, 1332, or a combination thereof, wherein S239, A330, and 1332 correspond to positions 239, 330, and 332 of a wild-type IgGl, according to the EU numbering convention. In some instances, the Fc region comprises S239D, A330L, I332E, or a combination thereof. In some cases, the Fcregion comprises S239D, A330L, and I332E.

[0248] In some embodiments, a multi-specific binding polypeptide (e.g., a multi-specific antibody) comprises a modification to a hinge region. In some instances, the hinge region comprises a modification at S228, wherein S228 correspond to position 228 of a wild-type IgG4, according to the EU numbering convention. In some instances, the hinge region comprises S228P.

[0249] In some embodiments, it may be desirable to create cysteine variant multi-specific binding polypeptides (e.g., multi-specific antibodies), in which one or more residues of an antibody are substituted with cysteine residues. In some embodiments, the substituted residues occur at accessible sites of the antibody. Reactive thiol groups can be positioned at sites for conjugation to other moieties, such as drug moieties or linker drug moieties, to create an immunoconjugate. In some embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; Al 14 (Kabat numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.

[0250] In some embodiments, multi-specific binding polypeptides (e.g., multi-specific antibodies) provided herein may be further modified to contain additional non-proteinaceous moieties that are known and available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly- 1,3 -di oxolane, poly-1, 3, 6-trioxane, ethyl ene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n vinyl pyrrolidone)polyethylene glycol, polypropylene glycol homopolymers, polypropylen oxide / ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if two or more polymers are attached, they can be the same or different molecules.

[0251] In some embodiments, the multi-specific binding polypeptides (e.g., multi-specific antibodies) provided herein further comprise a polyethylene glycol molecule, comprising, e.g., an average molecule weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.

[0252] In some embodiments, the polyalkylene oxide (e.g., PEG) is a discrete PEG, in which the discrete PEG is a polymeric PEG comprising more than one repeating ethylene oxide units. In some embodiments, a discrete PEG (dPEG) comprises from 2 to 60, from 2 to 50, or from 2 to 48 repeating ethylene oxide units. In some embodiments, a dPEG comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42, 48, 50 or more repeating ethylene oxide units. In such embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibodies) provided herein further comprises a discrete PEG comprising, e.g., from 2 to 60, from 2 to 50, or from 2 to 48 repeating ethylene oxide units.

[0253] In some embodiments, also disclosed herein is a nucleic acid encoding a multispecific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in Table 5; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in Table 5.

[0254] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NOs: 7 or 75-77; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 8.

[0255] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 92; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 93.

[0256] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 94; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 95.

[0257] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the aminoacid sequence set forth in SEQ ID NO: 96; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 97.

[0258] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 98; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 99.

[0259] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 100; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 101.

[0260] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 102; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 103.

[0261] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 104; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 105.

[0262] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 106; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 107.

[0263] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acidsequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 108; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 109.

[0264] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 110; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 111.

[0265] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 139; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 140.

[0266] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 141; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 142.

[0267] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 143; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 93.

[0268] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 144; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 103.

[0269] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specificbinding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 145; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 101.

[0270] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 146; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 140.

[0271] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 147; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 142.

[0272] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 148; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 111.

[0273] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 149; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 99.

[0274] In some embodiments, disclosed herein is a nucleic acid encoding a multi-specific binding polypeptide comprising a VH and a VL disclosed in Tables 1 and 2 and optionally in combination with a VH and a VL of Tables 3 and 4.

[0275] In some embodiments, additionally disclosed herein is a vector comprising a nucleic acid of encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100%identical to or consist of the amino acid sequence set forth in Table 5; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in Table 5. In some cases, the vector comprises a viral vector (e.g., a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a herpes simplex virus vector, or a chimeric viral vector). In additional cases, the vector comprises a non-viral vector.

[0276] In some embodiments, additionally disclosed herein is a multi-specific binding polypeptides (e.g., multi-specific antibodies) that binds to a cell-specific antigen and a TNF- related apoptosis-inducing ligand receptors conjugated to a payload and kills the target cell both by activation of TRAIL receptor mediated apoptosis and also by receptor-mediated internalization of the toxin.Methods of Target Selection

[0277] In certain embodiments, described herein is a method of selecting a cell-specific antigen and a TRAIL receptor for generating a multi-specific polypeptide molecule (e.g., a multi-specific antibody) for the treatment of a disease, for example, cancer or an autoimmune disease. In some embodiments, the method comprises the steps: a. selecting cell-specific antigens expressed in the disease cells that are detected with intensity score of +2 to +3 determined by IHC, and / or selecting antigens expressed in the disease cells that are detected in tumors with expression level score of RNASeqV2 (Log) > 5; b. selecting TRAIL receptors expressed in the disease cells that are detected with intensity score of +2 to +3 determined by IHC, and / or selecting receptors expressed in the disease cells that are detected in tumors with expression level score of RNASeqV2 (Log) > 5; and c. selecting antigens / receptors in respective cell types that are detected in the same cell type with expression level score of RNASeqV2 (Log)> 5, and / or selecting antigens / receptors in respective cell types that are detected in the same cell type with intensity score of +2 to +3 determined by IHC; wherein the selected targets in respective cell types are expressed in non-disease tissues with intensity score 0 to +1 as determined by IHC, and / or the selected targets in respective cell types are expressed in non-disease tissues with expression level score of RNASeqV2 (Log) < 4. In certain embodiments, the receptors are selected by analyzing disease genome databases, for example, cancer genome databases or an autoimmune disease genome database, that reports the expression level of receptors by estimating RNA level, protein level and staining intensities byIHC. In certain embodiments, the receptors are selected by analyzing genome databases using data analytics software that executes steps a-c.Production of multi-specific binding polypeptides

[0278] Included herein are methods to manufacture a multi-specific binding polypeptide (e.g., a multi-specific antibody). The multi-specific binding polypeptides (e.g., multi-specific antibodies) can be produced by several methods known in the art. For example, the multispecific binding polypeptides can be encoded by nucleic acid(s). This nucleic acid can be, for example, a plasmid or viral vector that is then transferred to a suitable cell line such as, for example, a eukaryotic host cell or a prokaryotic host cell.

[0279] Exemplary mammalian host cells include 293T cell line, 293A cell line, 293FT cell line, 293F cells, 293 H cells, A549 cells, MDCK cells, CHO DG44 cells, CHO-S cells, CH0-K1 cells, Expi293F™ cells, Flp-In™ T-REx™ 293 cell line, Flp-In™-293 cell line, Flp-In™-3T3 cell line, Flp-In™-BHK cell line, Flp-In™-CHO cell line, Flp-In™-CV-l cell line, Flp-In™- Jurkat cell line, FreeStyle™ 293-F cells, FreeStyle™ CHO-S cells, GripTite™ 293 MSR cell line, GS-CHO cell line, HCC-38, HCC-1806, HepaRG™ cells, MCF-7, MDA-MB-468, MDA- MB-231, SK-BR-3, T-REx™ Jurkat cell line, Per.C6 cells, T-REx™-293 cell line, T-REx™- CHO cell line, and T-REx™-HeLa cell line.

[0280] In some embodiments, a eukaryotic host cell is an insect host cell. Exemplary insect host cell include Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expresSF+® cells.

[0281] In some embodiments, a eukaryotic host cell is a yeast host cell. Exemplary yeast host cells include Pichia pastoris yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33, and Saccharomyces cerevisiae yeast strain such as INVScl.

[0282] In some embodiments, a eukaryotic host cell is a plant host cell. In some embodiments, the plant cells comprise a cell from algae. Exemplary plant cell lines include strains from Chlamydomonas reinhardtii 137c, or Synechococcus elongatus PPC 7942.

[0283] In some embodiments, a host cell is a prokaryotic host cell. Exemplary prokaryotic host cells include BL21, Maehl™, DH10B™, TOP10, DH5a, DHIOBac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F’, INVaF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™.

[0284] In some embodiments, the plasmid vector includes a vector from bacteria (e.g., E. coli), insects, yeast (e.g., Pichia pastoris), algae, or mammalian source. Bacterial vectors include, for example, pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQEvector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-l, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC -MAT-2.

[0285] Insect vectors include, for example, pFastBacl, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 MIO, pVL1393 Mi l, pVL1393 M12, FLAG vectors such as pPolh-FLAGl or pPolh-MAT 2, or MAT vectors such as pPolh-MATl, or pPolh-MAT2.

[0286] Yeast vectors include, for example, Gateway® pDEST™ 14 vector, Gateway® pDEST™ 15 vector, Gateway® pDEST™ 17 vector, Gateway® pDEST™ 24 vector, Gateway® pYES-DEST52 vector, pBAD-DEST49 Gateway® destination vector, pAO815 Pichia vector, pFLDl Pichi pastoris vector, pGAPZA, B, & C Pichia pastoris vector, pPIC3.5K Pichia vector, pPIC6 A, B, & C Pichia vector, pPIC9K Pichia vector, pTEFl / Zeo, pYES2 yeast vector, pYES2 / CT yeast vector, pYES2 / NT A, B, & C yeast vector, or pYES3 / CT yeast vector. Algae vectors include, for example, pChlamy-4 vector or MCS vector.

[0287] Mammalian vectors include, for example, transient expression vectors or stable expression vectors. Exemplary mammalian transient expression vectors include p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a,b,c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG- Myc-CMV 24, pCMV-FLAG-MATl, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP- CMV 4. Exemplary mammalian stable expression vectors include pFLAG-CMV 3, p3xFLAG- CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.

[0288] A cell line comprising the nucleic acid encoding the multi-specific binding polypeptide can then be cultured under suitable conditions such that the multi-specific binding polypeptide is expressed and secreted into the cell supernatant. The cell supernatant can be subjected to one or more steps that include filtration, centrifugation, precipitation, purification by ion exchange, protein A / G affinity, dialysis, desalting, or buffer exchange.

[0289] In some embodiments, an expression vector comprising the nucleotide sequence of a multi-specific binding polypeptide is transferred to a host cell by conventional techniques (e.g., viral transduction, electroporation, liposomal transfection, calcium phosphate precipitation), and the transfected cells are then cultured to produce the multi-specific binding polypeptide. In specific embodiments, the expression of the multi-specific binding polypeptide is regulated by a constitutive, an inducible, or a tissue specific promoter.

[0290] In some embodiments, a variety of host-expression vector systems are utilized toexpress a multi-specific binding polypeptide described herein. Such host-expression systems represent vehicles, by which the coding sequences of the multi-specific binding polypeptide (e.g., multi-specific antibody) is produced and subsequently purified, but also represent cells that are, when transformed or transfected with the appropriate nucleotide coding sequences, express an multi-specific binding polypeptide in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coll and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing multi-specific binding polypeptide coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing multi-specific binding polypeptide coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing multi-specific binding polypeptide coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing multi-specific binding polypeptide coding sequences; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5K promoter).

[0291] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some embodiments, cell lines that stably express a multi-specific binding polypeptide (e.g., a multi-specific antibody) are optionally engineered. Rather than using expression vectors that contain viral origins of replication, host cells are transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells are then allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn are cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express the multi-specific binding polypeptide.

[0292] In some embodiments, a number of selection systems are used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11 :223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes are employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistanceare used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Set. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Set. USA 78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926- 932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5): 155-215) and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30: 147). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1).

[0293] In some embodiments, the expression levels of a multi-specific binding polypeptide (e.g., a multi-specific antibody) are increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing a multi-specific binding polypeptide is amplifiable, an increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of the multi-specific binding polypeptide, production of the multi-specific binding polypeptide will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0294] In some embodiments, the multi-specific binding polypeptide (e.g., multi-specific antibody) is produced under a cell-free system. In some embodiments, a cell-free system comprises a mixture of cytoplasmic and / or nuclear components from a cell and is suitable for in vitro nucleic acid synthesis. In some embodiments, a cell-free system utilizes prokaryotic cell components. In other embodiments, a cell-free system utilizes eukaryotic cell components. Nucleic acid synthesis is obtained in a cell-free system based on, for example, Drosophila cell, Xenopus egg, Archaea, or HeLa cells. Exemplary cell-free systems include E. coli S30 Extract system, E. coli T7 S30 system, or PURExpress®, XpressCF, and XpressCF+.Cytotoxic payloads

[0295] In certain embodiments, the multi-specific binding polypeptides (e.g., multi-specific antibodies) of the current disclosure can be conjugated to payloads (e.g., cytotoxic moieties) asshown in FIG. IB. These cytotoxic moieties are payloads specifically targeted to kill both tumors and immunosuppressive cells. In certain embodiments, the multi-specific polypeptide molecule comprises at least one cytotoxic moiety. In other embodiments, the multi-specific polypeptide molecule comprises two or more cytotoxic moieties.

[0296] In some embodiments, the cytotoxic payload comprises a microtubule disrupting agent. Exemplary microtubule disrupting agents include, but are not limited to, 2- methoxyestradiol, chaicones, colchicine, combretastatin, dictyostatin, discodermolide, eleutherobin, epothilone, laulimalide, peloruside, podophyllotoxin, taxane, cryptophycin, halichondrin, maytansine, phomopsin, rhizoxin, spongistatin, tubulysin, vinca alkaloid, noscapinoid, auristatin, dolastain, or derivatives or analogs thereof. In some embodiments, the cytotoxic payload is combretastatin or a derivative or analog thereof. In some embodiments, an analog of combretastatin is ombrabulin. In some embodiments, the epothilone is epothilone B, patupilone, ixabepilone, sagopilone, BMS-310705, or BMS-247550. In some embodiments, the tubulysin is a tubulysin analog or derivative such as described in U.S. Patent Nos. 8580820 and 8980833 and in U.S. Publication Nos. 20130217638, 20130224228, and 201400363454. In some embodiments, the maytansine is a maytansinoid. In some embodiments, the maytansinoid is DM1, DM4, or ansamitocin. In some embodiments, the maytansinoid is DM1. In some embodiments, the maytansinoid is DM4. In some embodiments, the maytansinoid is ansamitocin. In some embodiments, the maytansinoid is a maytansionid derivative or analog such as described in U.S. Patent Nos. 5208020, 5416064, 7276497, and 6716821 or U.S. Publication Nos. 2013029900 and US20130323268. In some embodiments, the taxane is paclitaxel or docetaxel. In some embodiments, the vica alkaloid is vinblastine, vincristine, vindesine, vinorelbine, desoxy vincaminol, vincaminol, vincamajine, vineridine, vinburnine, vinpocetine, or vincamine.

[0297] In some embodiments, the cytotoxic payload is a dolastatin, or a derivative or analog thereof. In some embodiments, the dolastatin is dolastatin 10 or dolastatin 15, or derivatives or analogs thereof. In some embodiments, the dolastatin 10 analog is auristatin, soblidotin, symplostatin 1, or symplostatin 3. In some embodiments, the dolastatin 10 analog is auristatin or an auristatin derivative. In some embodiments, the auristatin or auristatin derivative is auristatin E (AE), auristatin F (AF), auristatin E5 -benzoyl valeric acid ester (AEVB), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), or monomethyl auristatin D (MMAD), auristatin PE, or auristatin PYE. In some embodiments, the auristatin derivative is monomethyl auristatin E (MMAE). In some embodiments, the auristatin derivative is monomethyl auristatin F (MMAF). In some embodiments, the auristatin is an auristatin derivative or analog such as described in U.S. Patent No. 6884869, 7659241, 7498298, 7964566,7750116, 8288352, 8703714 and 8871720. In some embodiments, the dolastatin 15 analog is cemadotin or tasidotin.

[0298] In some embodiments, the cytotoxic payload comprises a DNA modifying agent. In some embodiments, the DNA modifying agent comprises amsacrine, anthracycline, camptothecin, doxorubicin, duocarmycin, enediyne, etoposide, indolinobenzodiazepine, netropsin, teniposide, pyrrolobenzodiazepine, or derivatives or analogs thereof. In some embodiments, the anthracycline is doxorubicin, daunorubicin, epirubicin, idarubicin, mitomycin-C, dactinomycin, mithramycin, nemorubicin, pixantrone, sabarubicin, or valrubicin. In some embodiments, the analog of camptothecin is topotecan, irinotecan, silatecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan, rubitecan, or SN-38. In some embodiments, the duocarmycin is duocarmycin A, duocarmycin Bl, duocarmycin B2, duocarmycin Cl, duocarmycin C2, duocarmycin D, duocarmycin SA, or CC-1065. In some embodiments, the enediyne is a calicheamicin, esperamicin, or dynemicin A.

[0299] In some embodiments, the cytotoxic payload is pyrrolobenzodiazepine. In some embodiments, the pyrrolobenzodiazepine is anthramycin, abbeymycin, chicamycin, DC-81, mazethramycin, neothramycins A, neothramycin B, porothramycin, prothracarcin, sibanomicin (DC- 102), sibiromycin, or tomaymycin. In some embodiments, the pyrrolobenzodiazepine is a tomaymycin derivative, such as described in U.S. Patent Nos. 8404678 and 8163736. In some embodiments, the pyrrolobenzodiazepine is such as described in U.S. Patent Nos. 8426402, 8802667, 8809320, 6562806, 6608192, 7704924, 7067511, US7612062, 7244724, 7528126, 7049311, 8633185, 8501934, and 8697688 and U.S. Publication No. US20140294868.

[0300] In some embodiments, the pyrrolobenzodiazepine is a pyrrolobenzodiazepine dimer. In some embodiments, the PBD dimer is a symmetric dimer. Examples of symmetric PBD dimers include, but are not limited to, SJG-136 (SG-2000), ZC-423 (SG2285), SJG-720, SJG- 738, ZC-207 (SG2202), and DSB-120. In some embodiments, the PBD dimer is an unsymmetrical dimer. Examples of unsymmetrical PBD dimers include, but are not limited to, SJG-136 derivatives such as described in U.S. Patent Nos. 8697688 and 9242013 and U.S. Publication No. 20140286970.

[0301] In certain embodiments, the at least one cytotoxic moiety comprises an auristatin derivative, maytansine, a maytansinoid, a taxane, a calicheamicin, cemadotin, a duocarmycin, a pyrrolobenzodiazepine (PBD), or a tubulysin. the auristatin derivative is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). In certain embodiments, the maytansinoid is DM1 (emtansine). In certain embodiments, the maytansinoid is DM2 (mertansine) or DM4 (ravtansine / soravtansine). In certain embodiments, the pyrrolobenzodiazepine is a pyrrolobenzodiazepine dimer.

[0302] The cytotoxic moiety can be conjugated to the multi-specific binding polypeptide at a suitable stoichiometry. In certain embodiments, the cytotoxic moiety is conjugated at a ratio of about 1:1; about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 18:1, or about 20:1 cytotoxic moiety:multi-specific binding polypeptide.Linkers

[0303] In certain embodiments, the cytotoxic moiety is conjugated to lysine residues, cysteine residues, or lysine and cysteine residues by a suitable linker molecule. In certain embodiments, the linker is a cleavable linker. In certain embodiments, the linker is a pH- cleavable linker. In certain embodiments, the linker is an enzyme-cleavable linker. In certain embodiments, the linker is a protease-sensitive linker. In certain embodiments, the enzyme cleavage linker is a valine-citruline linker. In certain embodiments, the linker is a self- immolative linker. In certain embodiments, the linker is a non-cleavable linker. In certain embodiments, the linker comprises a zero-length linker, a homobifunctional linker, a heterobifunctional linker, a di-peptide linker, a spacer, a maleimide-based conjugating moiety, or a combination thereof. In certain embodiments, the linker comprises a polymer. In certain embodiments, the polymer comprises a linear or branched polyethylene glycol. In certain embodiments, the linker is a peptide. In certain embodiments, the linker is a peptidomimetic linker. In certain embodiments, the linker is a thioether linker.

[0304] Exemplary homobifuctional linkers include, but are not limited to, Lomant' s reagent dithiobis (succinimidylpropionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl proprionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene glycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl- 3,3'-dithiobispropionimidate (DTBP), l,4-di-3'-(2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), such as e.g. l,5-difluoro-2,4-dinitrobenzene or l,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'- dinitrophenylsulfone (DFDNPS), bis-[P-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3 '-dimethylbenzidine, benzidine, a,a'-p-diaminodiphenyl, diiodo- p-xylene sulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene- bis(iodoacetamide).

[0305] In some embodiments, the linker comprises a heterobifunctional linker. Exemplaryheterobifunctional linker include, but are not limited to, amine-reactive and sulfhydryl crosslinkers such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N- succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble-long-chain N- succinimidyl 3-(2-pyridyldithio) propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-a- methyl-a-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[a-methyl-a-(2- pyridyldithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate (sMCC), sulfosuccinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N- hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs), N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB), sulfosuccinimidyl(4- iodoacteyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(y- maleimidobutyryloxy)succinimide ester (GMBs), N-(y-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6- (((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4- (((iodoacetyl)amino)methyl)cyclohexane-l -carboxylate (sIAC), succinimidyl 6-((((4- iodoacetyl)amino)methyl)cyclohexane-l-carbonyl)amino) hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl -reactive and sulfhydryl-reactive cross-linkers such as 4-(4-N- maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-l- carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive and photoreactive cross-linkers such as N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs- AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA), sulfosuccinimidyl- (4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p- azidosalicylamido)ethyl- 1,3 '-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'- azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'- nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB- NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl- 1,3 '-dithiopropionate (sAND), N- succinimidyl-4(4-azidophenyl)l, 3 '-dithiopropionate (sADP), N-sulfosuccinimidyl(4- azidophenyl)- 1,3 '-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-l,3'- dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate (sulfo- sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3- trifluoropropi onate (PNP-DTP), sulfhydryl-reactive and photoreactive cross-linkers such asl-(p- Azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p-azidosalicylamido)butyl]-3 '-(2 '-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4- maleimide carbonyl -reactive and photoreactive cross-linkers such as p-azidobenzoyl hydrazide (ABH), carboxylate-reactive and photoreactive cross-linkers such as 4-(p- azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive cross-linkers such as p-azidophenyl glyoxal (APG).

[0306] In some embodiments, the linker comprises a reactive functional group. In some embodiments, the reactive functional group comprises a nucleophilic group that is reactive to an electrophilic group present on a binding moiety. Exemplary electrophilic groups include carbonyl groups — such as aldehyde, ketone, carboxylic acid, ester, amide, enone, acyl halide or acid anhydride. In some embodiments, the reactive functional group is aldehyde. Exemplary nucleophilic groups include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0307] In some embodiments, the linker comprises a maleimide group. In some embodiments, the maleimide group is also referred to as a maleimide spacer. In some embodiments, the maleimide group further encompasses a caproic acid, forming maleimidocaproyl (me). In some embodiments, the linker comprises maleimidocaproyl (me). In some embodiments, the linker is maleimidocaproyl (me). In other embodiments, the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate (sMCC) or sulfosuccinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate (sulfo-sMCC) described above.

[0308] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some embodiments, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalysis of tiosuccinimide ring hydrolysis, thereby eliminating maleimide from undergoing an elimination reaction through a retro-Michael reaction. In some embodiments, the self-stabilizing maleimide is a maleimide group described in Lyon, et al., “Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates,” Nat. Biotechnol.32(10): 1059-1062 (2014). In some embodiments, the linker comprises a self-stabilizing maleimide. In some embodiments, the linker is a self-stabilizing maleimide.

[0309] In some embodiments, the linker comprises a peptide moiety. In some embodiments, the peptide moiety comprises at least 2, 3, 4, 5, 6, 7, 8, or more amino acid residues. In some embodiments, the peptide moiety is a cleavable peptide moiety (e.g., either enzymatically or chemically). In some embodiments, the peptide moiety is a non-cleavable peptide moiety. In some embodiments, the peptide moiety comprises Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 112), Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit,Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu- Ala-Leu (SEQ ID NO: 113), or Gly-Phe- Leu-Gly (SEQ ID NO: 144). In some embodiments, the linker comprises a peptide moiety such as: Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 112), Phe-Lys, Val-Lys, Gly-Phe- Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala- Leu-Ala-Leu (SEQ ID NO: 113), or Gly-Phe-Leu-Gly (SEQ ID NO: 114). In some embodiments, the linker comprises Val-Cit. In some embodiments, the linker is Val-Cit.

[0310] In some embodiments, the linker comprises a benzoic acid group, or its derivatives thereof. In some embodiments, the benzoic acid group or its derivatives thereof comprise paraaminobenzoic acid (PAB A). In some embodiments, the benzoic acid group or its derivatives thereof comprise gamma-aminobutyric acid (GABA).

[0311] In some embodiments, the linker comprises one or more of a maleimide group, a peptide moiety, and / or a benzoic acid group, in any combination. In some embodiments, the linker comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In some embodiments, the maleimide group is maleimidocaproyl (me). In some embodiments, the peptide group is val-cit. In some embodiments, the benzoic acid group is PAB A. In some embodiments, the linker comprises a mc-val-cit group. In some embodiments, the linker comprises a val-cit-PABA group. In additional embodiments, the linker comprises a mc-val-cit-PABA group.

[0312] In some embodiments, the linker is a self-immolative linker or a self-elimination linker. In some embodiments, the linker is a self-immolative linker. In other embodiments, the linker is a self-elimination linker (e.g., a cyclization self-elimination linker). In some embodiments, the linker comprises a linker described in U.S. Patent No. 9,089,614 or PCT Publication No. WO2015038426.

[0313] In some embodiments, the linker is a traceless linker or a linker in which after cleavage does not leave behind a linker moiety (e.g., an atom or a linker group) to a payload or to a multi-specific binding polypeptide described herein. Exemplary traceless linkers include, but are not limited to, germanium linkers, silicium linkers, sulfur linkers, selenium linkers, nitrogen linkers, phosphorus linkers, boron linkers, chromium linkers, or phenylhydrazide linker. In some embodiments, the linker is a traceless aryl-triazene linker as described in Hejesen, et al., “A traceless aryl-triazene linker for DNA-directed chemistry,” Org Biomol Chem 11(15): 2493-2497 (2013). In some embodiments, the linker is a traceless linker described in Blaney, et al., “Traceless solid-phase organic synthesis,” Chem. Rev. 102: 2607-2024 (2002). In some embodiments, a linker is a traceless linker as described in U.S. Patent No. 6,821,783.

[0314] In some embodiments, the linker is a dendritic type linker. In some embodiments, the dendritic type linker comprises a branching, multifunctional linker moiety. In someembodiments, the dendritic type linker comprises PAMAM dendrimers.

[0315] In some embodiments, the linker is an acid cleavable linker. In some embodiments, the acid cleavable linker comprises a hydrazone linkage, which is susceptible to hydrolytic cleavage. In some embodiments, the acid cleavable linker comprises a thiomaleamic acid linker. In some embodiments, the acid cleavable linker is a thiomaleamic acid linker as described in Castaneda, et al, “Acid-cleavable thiomaleamic acid linker for homogeneous antibody-drug conjugation,” Chem. Commun. 49: 8187-8189 (2013).

[0316] In some embodiments, the linker is a linker described in U.S. Patent Nos. 6,884,869;7,498,298; 8,288,352; 8,609,105; or 8,697,688; U.S. Patent Publication Nos. 2014 / 0127239; 2013 / 028919; 2014 / 286970; 2013 / 0309256; 2015 / 037360; or 2014 / 0294851; or PCT Publication Nos. WO2015057699; W02014080251; WO2014197854; W02014145090; or WO2014177042.Conjugation Chemistry

[0317] Various conjugation reactions are described herein and contemplated for reacting a multi-specific binding polypeptide described herein (e.g., a multi-specific antibody, optionally a bispecific antibody) with a payload (e.g., a cytotoxic payload). In some embodiments, the reaction occurs at a natural (“canonical”) amino acid in the multi-specific binding polypeptide described herein (e.g., the multi-specific antibody, optionally the bispecific antibody). In some embodiments, the amino acid for conjugation is a natural amino acid found in a wild type sequence, or alternatively the amino acid has been mutated. In some embodiments, a conjugation reaction comprises formation of a disulfide bond at a cysteine residue. In some embodiments, a conjugation reaction comprises a 1,4 Michael addition reaction of a cysteine or lysine. In some embodiments, a conjugation reaction comprises a cyanobenzothiazole ligation of a cysteine. In some embodiments, a conjugation reaction comprises crosslinking with an acetone moiety, such as l,3-dichloro-2-propionone. In some embodiments, a conjugation reaction comprises a 1,4 Michael addition to a dehydroalanine, formed by reaction of cysteine with O- mesitylenesulfonylhydroxylamine. In some embodiments a conjugation reaction comprises reaction of a tyrosine with a triazolinedione (TAD), or TAD derivative. In some embodiments a conjugation reaction comprises reaction of a tryptophan with a rhodium carbenoid.

[0318] In some embodiments, a multi-specific binding polypeptide described herein (e.g., a multi-specific antibody, optionally a bispecific antibody) is conjugated to a payload described supra by a chemical ligation process. In some instances, the multi-specific binding polypeptide described herein (e.g., the multi-specific antibody, optionally the bispecific antibody) is conjugated to the payload by a native ligation. In some instances, the conjugation is as describedin: Dawson, et al. “Synthesis of proteins by native chemical ligation,” Science 1994, 266, 776- 779; Dawson, et al. “Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives,” J. Am. Chem. Soc. 1997, 119, 4325-4329; Hackeng, et al. “Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology.,” Proc. Natl. Acad. Sci. USA 1999, 96, 10068-10073; or Wu, et al. “Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol,” Angew. Chem. Int. Ed. 2006, 45, 4116-4125. In some instances, the conjugation is as described in U.S. Pat. No. 8,936,910. In some embodiments, the multi-specific binding polypeptide described herein (e.g., the multispecific antibody, optionally the bispecific antibody) is conjugated to the payload either site- specifically or non-specifically via native ligation chemistry.

[0319] In some instances, a multi-specific binding polypeptide described herein (e.g., a multi-specific antibody, optionally a bispecific antibody) is conjugated to a payload described supra by a “Click” chemistry. In some instances, the conjugation reaction comprises a 1,3- dipolar cycloaddition reaction. In some embodiments, the 1,3-dipolar cycloaddition reaction comprises reaction of an azide and a phosphine ("Click" reaction). In some embodiments, the conjugation reaction is catalyzed by copper. In some embodiments, the conjugation reaction comprises reaction of an azide with a strained olefin. In some embodiments, a conjugation reaction comprises reaction of an azide with a strained alkyne. In some embodiments, a conjugation reaction comprises reaction of an azide with a cycloalkyne, for example, OCT, DIFO, DIFBO, DIBO, BARAC, TMTH, or other strained cycloalkyne, the structures of which are shown in Gong, Y., Pan, L. Tett. Lett. 2015, 56, 2123. In some embodiments, a 1,3-dipolar cycloaddition reaction is catalyzed by light ("photoclick"). In some embodiments, a conjugation reaction comprises reaction of a terminal allyl group with a tetrazole and light. In some embodiments, a conjugation reaction comprises reaction of a terminal alkynyl group with a tetrazole and light. In some embodiments, a conjugation reaction comprises reaction of an O- allyl amino acid with a tetrazine and light. In some embodiments, a conjugation reaction comprises reaction of O-allyl tyrosine with a tetrazine and light.

[0320] In some instances, a multi-specific binding polypeptide described herein (e.g., a multi-specific antibody, optionally a bispecific antibody) is conjugated to a payload described supra by a GlyCLICK® site-specific conjugation technology (Life Technologies Corporation). In some instances, the GlyCLICK® site-specific conjugation chemistry comprises a Fc-glycan remodeling which comprises a deglycosylation of the antibody to allow site-specific conjugation using click-chemistry.

[0321] In some instances, a multi-specific binding polypeptide described herein (e.g., a multi-specific antibody, optionally a bispecific antibody) is conjugated to a payload describedsupra by a GlycoConnect™ conjugation technology (Synaffix BV). In some instances, the GlycoConnect™ conjugation technology utilizes enzymatic modification of two naturally occurring glycan anchor points to engage in site-specific conjugation.

[0322] In some instances, a multi-specific binding polypeptide described herein (e.g., a multi-specific antibody, optionally a bispecific antibody) is conjugated to a payload described supra by an immunoglobulin binding peptide. In some instances, the immunoglobulin binding peptide utilizes IgG Fc-affinity reagents to directly conjugate cytotoxic payloads to the multispecific binding polypeptide (e.g., the multi-specific antibody).

[0323] In some instances, a multi-specific binding polypeptide described herein (e.g., a multi-specific antibody, optionally a bispecific antibody) is conjugated to a payload described supra by an AJICAP™ conjugation technology (Ajinomoto Co. Inc.). In some instances, the AJICAP™ conjugation technology utilizes a class of IgG Fc-affinity reagents to directly conjugate one or more cytotoxic payloads to the multi-specific binding polypeptide (e.g., the multi-specific antibody).

[0324] In some instances, a multi-specific binding polypeptide described herein (e.g., a multi-specific antibody, optionally a bispecific antibody) is conjugated to a payload described supra by a conjugation reaction comprising an inverse-electron demand cycloaddition reaction that comprises a diene and a dienophile. In some embodiments, the diene comprises a tetrazine. In some embodiments, the dienophile comprises an alkene. In some embodiments, the dienophile comprises an alkyne. In some embodiments, the alkyne is a strained alkyne. In some embodiments, the alkene is a strained diene. In some embodiments, the alkyne is a transcyclooctyne. In some embodiments, the alkyne is a cyclooctene. In some embodiments, the alkene is a cyclopropene. In some embodiments, the alkene is a fluorocyclopropene. In some embodiments, a conjugation reaction results in the formation of a multi-specific binding polypeptide (e.g., a multi-specific antibody) attached to a linker or payload via a 6-membered ring heterocycle comprising two nitrogen atoms in the ring.

[0325] In some instances, a multi-specific binding polypeptide described herein (e.g., a multi-specific antibody, optionally a bispecific antibody) is conjugated to a payload described supra by a conjugation reaction comprising an olefin metathesis reaction. In some embodiments, a conjugation reaction comprises reaction of an alkene and an alkyne with a ruthenium catalyst. In some embodiments, a conjugation reaction comprises reaction of two alkenes with a ruthenium catalyst. In some embodiments, a conjugation reaction comprises reaction of two alkynes with a ruthenium catalyst. In some embodiments, a conjugation reaction comprises reaction of an alkene or alkyne with a ruthenium catalyst and an amino acid comprising an allyl group. In some embodiments, a conjugation reaction comprises reaction of an alkene or alkynewith a ruthenium catalyst and an amino acid comprising an allyl sulfide or selenide. In some embodiments, a ruthenium catalyst is Hoveda- Grubbs 2nd generation catalyst. In some embodiments, an olefin metathesis reaction comprises reaction of one or more strained alkenes or alkynes.

[0326] In some instances, a multi-specific binding polypeptide described herein (e.g., a multi-specific antibody, optionally a bispecific antibody) is conjugated to a payload described supra by a conjugation reaction comprising a cross-coupling reaction. In some embodiments, cross-coupling reactions comprise transition metal catalysts, such as iridium, gold, ruthenium, rhodium, palladium, nickel, platinum, or other transition metal catalyst and one or more ligands. In some embodiments, transition metal catalysts are water-soluble. In some embodiments, a conjugation reaction comprises a Suzuki-Miyaura cross-coupling reaction. In some embodiments, a conjugation reaction comprises reaction of an aryl halide (or triflate, or tosylate), an aryl or alkenyl boronic acid, and a palladium catalyst. In some embodiments, a conjugation reaction comprises a Sonogashira cross-coupling reaction. In some embodiments, a conjugation reaction comprises reaction of an aryl halide (or triflate, or tosylate), an alkyne, and a palladium catalyst. In some embodiments, cross-coupling reactions result in attachment of a linker or payload to a multi-specific binding polypeptide (e.g., a multi-specific antibody) via a carbon-carbon bond.

[0327] In some instances, a multi-specific binding polypeptide described herein (e.g., a multi-specific antibody, optionally a bispecific antibody) is conjugated to a payload described supra by a site-directed method utilizing a “traceless” coupling technology (Philochem). In some instances, the “traceless” coupling technology utilizes an N-terminal 1,2-aminothiol group on the multi-specific binding polypeptide which is then conjugate with a payload containing an aldehyde group, (see Casi et al., “Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery,” JACS 134(13): 5887-5892 (2012)).

[0328] In some instances, a multi-specific binding polypeptide described herein (e.g., a multi-specific antibody, optionally a bispecific antibody) is conjugated to a payload described supra by a site-directed method utilizing an unnatural amino acid incorporated into the multispecific binding polypeptide (e.g., the multi-specific antibody, optionally the bispecific antibody). In some instances, the unnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In some instances, the keto group of pAcPhe is selectively coupled to an alkoxyamine derivatived conjugating moiety to form an oxime bond, (see Axup etal., “Synthesis of site-specific antibody-drug conjugates using unnatural amino acids,” PNAS 109(40): 16101- 16106 (2012)).

[0329] In some instances, a multi-specific binding polypeptide described herein (e.g., amulti-specific antibody, optionally a bispecific antibody) is conjugated to a payload described supra by a site-directed method utilizing an enzyme-catalyzed process. In some instances, the site-directed method utilizes SMARTag™ technology (Catalent Biologies). In some instances, the SMARTag™ technology comprises generation of a formylglycine (FGly) residue from cysteine by formylgly cine-generating enzyme (FGE) through an oxidation process under the presence of an aldehyde tag and the subsequent conjugation of FGly to an alkylhydraine- functionalized multi-specific binding polypeptide (e.g., functionalized multi-specific antibody, optionally functionalized bispecific antibody) via hydrazino-Pictet-Spengler (HIPS) ligation. In some instances, a 6-amino acid consensus sequence is incorporated into the heavy chain, light chain, or both chains of the multi-specific binding polypeptide (optionally the bispecific antibody) for recognition by the FGE to generate a functional aldehyde group for site-specific conjugation to the payload. In some instances, the 6-amino acid consensus sequence is LCXPXR, wherein X is any amino acid. In some cases, the 6-amino acid consensus sequence is incorporated into the N-terminus, C-terminus, or within a Fc region of the multi-specific binding polypeptide (optionally the bispecific antibody), (see Wu et al., “Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag,” AHS 106(9): 3000-3005 (2009); Agarwal, et al., “A Pictet-Spengler ligation for protein chemical modification,” PNAS 110(1): 46-51 (2013)).

[0330] In some instances, the enzyme-catalyzed process comprises microbial transglutaminase (mTG). In some cases, a multi-specific binding polypeptide described herein (e.g., a multi-specific antibody, optionally a bispecific antibody) is conjugated to a payload described supra utilizing a microbial transglutaminze catalyzed process. In some instances, mTG catalyzes the formation of a covalent bond between the amide side chain of a glutamine within the recognition sequence and a primary amine of a functionalized multi-specific binding polypeptide (e.g., a functionalized multi-specific antibody, optionally a functionalized bispecific antibody). In some instances, mTG is produced from Streptomyces mobarensis. (see Strop et al., “Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates,” Chemistry and Biology 20(2) 161-167 (2013)).

[0331] In some instances, a multi-specific binding polypeptide described herein (e.g., a multi-specific antibody, optionally a bispecific antibody) is conjugated to a payload described supra by a method as described in PCT Publication No. W02014 / 140317, which utilizes a sequence-specific transpeptidase.

[0332] In some instances, a multi-specific binding polypeptide described herein (e.g., a multi-specific antibody, optionally a bispecific antibody) is conjugated to a payload describedsupra by a method as described in U.S. Patent Publication Nos. 2015 / 0105539 and 2015 / 0105540.Methods of Use

[0333] The multi-specific binding polypeptides (e.g., multi-specific antibodies) of the current disclosure are useful in the treatment of a disease or condition. In some instances, the disease or condition is a cancer (e.g., a carcinoma, sarcoma, leukemia, or lymphoma). In certain embodiments, the multi-specific binding polypeptides (e.g., multi-specific antibodies) are for use in a method to treat a cancer. In certain embodiments, the multi-specific binding polypeptides (e.g., multi-specific antibodies) are for use in the manufacture of a medicament for treating a cancer. Many cancers can be treated with the multi-specific binding polypeptides (e.g., multi-specific antibodies) described herein including solid tumors / cancers and hematological malignancies. In certain embodiments, the solid cancer is bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, testicular cancer, or thyroid cancer. In certain embodiments, the hematological malignancy comprises a Hodgkin’s lymphoma or a non-Hodgkin’s lymphoma. Exemplary hematological malignancies include, but are not limited to, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom’s macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt’s lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, or acute myeloid leukemia. In some embodiments, the hematological malignancy comprises chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia, or acute myeloid leukemia.

[0334] In some embodiments, the cancer is a metastatic cancer. In some embodiments, the metastatic cancer is a metastatic solid tumor / cancer. In other embodiments, the metastatic cancer is a metastatic hematologic malignancy. In some embodiments, the metastatic solid tumor comprises metastatic bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, eye cancer, headand neck cancer, kidney cancer, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, testicular cancer, or thyroid cancer. In some embodiments, the metastatic hematologic malignancy comprises metastatic chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom’s macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt’s lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, or acute myeloid leukemia.

[0335] In some embodiments, the cancer is a relapsed or refractory cancer. In some embodiments, the relapsed or refractory cancer is a solid cancer, e.g., a relapsed or refractory bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, testicular cancer, or thyroid cancer. In some embodiments, the relapsed or refractory cancer is a hematologic malignancy, e.g., a relapsed or refractory chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom’s macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt’ s lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, or acute myeloid leukemia.

[0336] In certain embodiments, the cancer is a breast cancer. In some embodiments, the breast cancer is luminal A breast cancer, luminal B breast cancer, triple-negative breast cancer, HER2-enriched breast cancer, or normal-like breast cancer. In some embodiments, the breast cancer is ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), inflammatory breast cancer, lubular carcinoma in situ (LCIS), male breast cancer, Paget’s disease of the Nipple, or phyllodes tumors of the breast. In some embodiments, the IDC comprises tubular carcinoma of the breast, medullary carcinoma of the breast, papillarycarcinoma of the breast, or cribriform carcinoma of the breast. In some embodiments, the breast cancer is triple negative breast cancer. In some embodiments, the breast cancer is a metastatic breast cancer. In additional embodiments, the breast cancer is a relapsed or refractory breast cancer.

[0337] In certain embodiments, the cancer is an ovarian cancer. In some embodiments, the ovarian cancer is epithelial carcinoma, serous carcinoma, small-cell carcinoma, primary peritoneal carcinoma, clear-cell carcinoma, clear-cell adenocarcinoma, endometrioid, malignant mixed mullerian tumor, mucinous, mucinous adenocarcinoma, pseudomyxoma peritonei, undifferentiated epithelial, malignant Brener tumor, transitional cell carcinoma, sex cord-stromal tumor, granulosa cell tumor, adult granulosa cell tumor, juvenile granulosa cell tumor, Sertoli- Leydig cell tumor, sclerosing stromal tumors, germ cell tumor, dysgerminoma, choriocarcinoma, immature (solid) teratoma, mature teratoma (dermoid cyst), yolk sac tumor (endodermal sinus tumor), embryonal carcinoma, polyembryoma, squamous cell carcinoma, mixed tumors, or low malignant potential tumors. In some embodiments, the ovarian cancer is a metastatic ovarian cancer. In additional embodiments, the ovarian cancer is a relapsed or refractory ovarian cancer.

[0338] In certain embodiments, the cancer is a lung cancer. In some embodiments, the lung cancer is non-small cell lung carcinoma (NSCLC) or small cell lung cancer (SCLC). In some embodiments, the lung cancer is a metastatic lung cancer. In additional embodiments, the lung cancer is a relapsed or refractory lung cancer.

[0339] In certain embodiments, the cancer is a liver cancer. In some embodiments, the liver cancer is hepatocellular carcinoma (HCC), cholangiocarcinoma, liver angiosarcoma, or hepatoblastoma. In some instances, the liver cancer is a metastatic liver cancer. In some cases, the liver cancer is a relapsed or refractory liver cancer.

[0340] In certain embodiments, the cancer is a prostate cancer. In some embodiments, the prostate cancer is acinar adenocarcinoma, ductal adenocarcinoma, transitional cell (or urothelial) cancer, squamous cell cancer, small cell prostate cancer, carcinoid in the prostate, or sarcoma in the prostate. In some embodiments, the prostate cancer is a metastatic prostate cancer. In additional embodiments, the prostate cancer is a relapsed or refractory prostate cancer.

[0341] In certain embodiments, the cancer is a brain cancer. In some instances, the brain cancer is glioblastoma (e.g., glioblastoma multiforme or GBM). In some instances, the brain cancer is neuroblastoma. In some cases, the brain cancer is a metastatic brain cancer.

[0342] In certain embodiments, the cancer is a carcinoma.

[0343] In certain embodiments, the cancer is a sarcoma.

[0344] In certain embodiments, the cancer is acute myeloid leukemia, diffuse large B cell lymphoma (DLBCL), bladder urothelial carcinoma, breast carcinoma, triple negative breastcarcinoma, liver hepatocellular carcinoma, cervical squamous cell carcinoma, cholangiocarcinoma, colon adenocarcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, kidney renal papillary cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, NSCLC, SCLC, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, prostate adenocarcinoma, kidney renal clear cell carcinoma, uterine corpus endometrial carcinoma, thyroid carcinoma, stomach adenocarcinoma, rectal adenocarcinoma, sarcoma, testis and germ cell tumors, or uterine carcinosarcoma.

[0345] In some embodiments, the cancer is a pediatric cancer. Exemplary pediatric cancers include, but are not limited to, bone cancer, brain cancer, leukemia, hepatoblastoma, lymphoma (e.g., Hodgkin’s and non-Hodgkin’s lymphoma), neuroblastoma, rhabdomyosarcoma, retinoblastoma, rhabdoid tumor, sarcoma, spinal cord tumor, and Wilms tumor. In some instances, a pediatric cancer occurs in a child less than 18 years age.

[0346] The multi-specific binding polypeptides (e.g., multi-specific antibodies) described herein can be used to treat cancers and / or tumors that are refractory to treatment with immune checkpoint inhibitor therapies, fail treatment with immune checkpoint inhibitor therapies, or respond sub-optimally to treatment with immune checkpoint inhibitor therapies. In certain embodiments, checkpoint inhibitor therapies include therapies that target PD-1, PDL-1, PDL-2, CTLA4, LAG-3, TIM-3, KIR, VISTA or other immune checkpoint receptors. Current checkpoint inhibitor therapies include, for example, nivolumab (Opdivo®), pembrolizumab (Keytruda®), pidilizumab (CT-011), BMS-936559, atezolizumab (MPDL3280A), avelumab, ipilimumab (Yervoy®), or tremelimumab. In certain embodiments, a subject is selected for treatment with the multi-specific binding polypeptides (e.g., multi-specific antibodies) described herein after receiving a checkpoint inhibitor therapy. In certain embodiments, a subject is selected for treatment with the multi-specific binding polypeptides (e.g., multi-specific antibodies) described herein after failing a checkpoint inhibitor therapy. In certain embodiments, failing a checkpoint inhibitor therapy is a lack of a complete response (e.g., complete reduction of tumor or signs of cancer). In certain embodiments, failing a checkpoint inhibitor therapy is a lack of an objective response (e.g., lack of measurable tumor shrinkage or reduction in signs of cancer). In certain embodiments, the multi-specific binding polypeptides (e.g., multi-specific antibodies) are for use in a method to treat a subject that has been administered one or more previous immune checkpoint inhibitor treatments. In certain embodiments, subjects are selected for treatment with the multi-specific binding polypeptides (e.g., multi-specific antibodies) provided herein based upon low expression or an absence of expression of an immune checkpoint inhibitor. In certain embodiments, subjects are selected for treatment with the multispecific binding polypeptides (e.g., multi-specific antibodies) provided herein based upon lowexpression or an absence of expression of PD-1, PDL-1, PDL-2, CTLA4, LAG-3, TIM-3, KIR, VISTA or other immune checkpoint receptors. Low expression or absent expression can be suitably determined using methods such as immunohistochemistry, flow-cytometry, RT-PCR, RNA-Seq, ELISA or western blot. Exemplary assays for PDL-1 diagnostic purposes are known, and, for example, can be found in Udall et al. Diagn Pathol. 2018; 13: 12. In some embodiments, the subject is insensitive to treatment with an immune checkpoint inhibitor, has failed to respond to treatment with an immune checkpoint inhibitor, or who expresses low level of or does not express an immune checkpoint protein. In some instances, the multi-specific binding polypeptides (e.g., multi-specific antibodies) described herein are administered to the subject in conjugation with an immune checkpoint modulator, e.g., simultaneously with an immune checkpoint modulator or sequentially with an immune checkpoint modulator.

[0347] In certain embodiments, the disease or condition is a liver disease or condition. In some cases, the liver disease or condition is non-alcoholic fatty liver disease (NASH) or alcoholic steatohepatitis.

[0348] In some embodiments, the disease or condition is an immune or autoimmune disease. In certain embodiments, the multi-specific binding polypeptides (e.g., multi-specific antibodies) are for use in a method to treat an immune or autoimmune disease. In certain embodiments, the multi-specific binding polypeptides (e.g., multi-specific antibodies) are for use in the manufacture of a medicament for treating an immune or autoimmune disease. Many immune and autoimmune diseases can be treated with the multi-specific binding polypeptides (e.g., multi-specific antibodies) described herein including, but not limited to, Behcet disease, systemic lupus erythematosus (SLE), chronic discoid lupus erythematosus, multiple sclerosis (systemic scleroderma and progressive systemic scleroderma), scleroderma, polymyositis, dermatomyositis, periarteritis nodosa (polyarteritis nodosa and microscopic polyangiitis), aortitis syndrome (Takayasu arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondylarthritis, mixed connective tissue disease, Sjogren syndrome, adultonset Still's disease, vasculitis, allergic granulomatous angiitis, hypersensitivity angiitis, rheumatoid vasculitis, largo-vessel vasculitis, ANCA-associated vasculitis (e.g., granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis), Cogan's syndrome, RS3PE syndrome, temporal arteritis, giant-cell arteritis, polymyalgia rheumatics, fibromyalgia syndrome, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related diseases (e.g., primary sclerosing cholangitis and autoimmune pancreatitis), Guillain-Barre syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, non-alcoholic steatohepatitis, primary biliary cirrhosis, aortitis syndrome, Goodpasture's syndrome, rapidly progressive glomerulonephritis, anti-glomerular basement membrane nephritis, megaloblasticanemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, hyperthyroidism (Graves' disease (Basedow's disease)), Hashimoto's thyroiditis, autoimmune adrenal insufficiency, primary hypothyroidism, Addison's disease, idiopathic Addison's disease (chronic adrenal inefficiency), type I diabetes mellitus, slowly progressive type I diabetes mellitus, localized scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous skin disease, epidermolysis bullosa acquisita, alopecia areata, vitiligo, vitiligo vulgaris, Harada disease, autoimmune optic neuropathy, idiopathic azoospermia, recurrent fetal loss, inflammatory bowel diseases (e.g., ulcerative colitis and Crohn's disease), celiac disease, dermatitis, atopic dermatitis, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, pulmonary alveolar proteinosis, autoimmune hemorrhagic disease XIII, relapsing polychondritis, sarcoidosis, ankylosing spondylitis, severe asthma, chronic urticaria, transplantation immunity, familial Mediterranean fever, eosinophilic sinusitis, dilated cardiomyopathy, food allergy, systemic mastocytosis, amyotrophic lateral sclerosis or inclusion body myositis, systemic sclerosis and sclerosis, respiratory distress syndrome, meningitis, encephalitis, staphylocitis, eczema, asthma, atherosclerosis, leukocyte adhesion deficiency, Raynaud's syndrome, juvenile-onset diabetes, Reiter's disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, acute idiopathic thrombocytopenia purpura and chronic idiopathic thrombocytopenia purpura, Hashimoto thyroiditis, Wegener's granulomatosis, Omen's syndrome, Chronic renal failure, acute infectious mononuclear disease, HIV, and Epstein-Barr virus (EBV).

[0349] The multi-specific binding polypeptides (e.g., multi-specific antibodies) described herein can be used to selectively eliminate disease cells. In some cases, the disease cells are cancer cells. In some cases the disease cells are infected cells.

[0350] The multi-specific binding polypeptides (e.g., multi-specific antibodies) described herein can be used to selectively eliminate senescent cells. In some cases, the senescent cells are chemotherapy induced senescent cells. In some embodiments, the senescent cells are age-related senescent cells.

[0351] The choice of cell-specific antigen and TRAIL receptor binding moiety of the multispecific binding polypeptide can be dependent upon the type of disease or cancer treated.Cell Killing by TRAIL-R2-mediated Apoptosis

[0352] TRAIL receptor 2 (also referred to as TRAIL-R2, death receptor 5, DR5, tumor necrosis factor receptor superfamily member 10B, or TNFRSF10B), is a cell surface receptor of the Tumor Necrosis Factor (TNF)-receptor superfamily that binds to TRAIL and mediatesapoptosis via an intracellular Death Domain (DD). In some instances, oligomerization of TRAIL-R2 occurs prior to activation of the apoptotic signaling pathway. As utilized herein, oligomerization of TRAIL-R2 encompasses two or more TRAIL-R2 monomers, optionally two, three, four, five, six, or more TRAIL-R2 monomers. In some cases, TRAIL-R2 dimerization occurs prior to activation of the apoptotic signaling pathway. In such cases, oligomerization (e.g., dimerization) is facilitated by a disulfide bond between, e.g., Cys209 of each TRAIL-R2 monomer.

[0353] In some embodiments, oligomerization of TRAIL-R2 occurs in a lipid raft of a target cell. In some instances, two or more TRAIL-R2 monomers, optionally two, three, four, five, six, or more TRAIL-R2 monomers oligomerizes in the lipid raft. In such instances and upon oligomerization, the apoptotic signaling pathway is activated within the target cell. In some cases, dimerization of the TRAIL-R2 occurs in a lipid raft of the target cell, optionally facilitated by a disulfide bond between, e.g., Cys209 of each TRAIL-R2 monomer. I...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A multi-specific binding polypeptide comprising a TNF-related apoptosis-inducing ligand (TRAIL) receptor binding moiety that specifically binds to a TRAIL receptor and a cell-specific antigen binding moiety that specifically binds to a cell-specific antigen, wherein the off-rate (kd (1 / s)) of the TRAIL receptor binding moiety is higher than the off-rate (kd (1 / s)) of the cellspecific antigen binding moiety.

2. The multi-specific binding polypeptide of claim 1, wherein the off-rate (kd (1 / s)) of the TRAIL receptor binding moiety is at least 20-fold higher than the off-rate (kd (1 / s)) of cellspecific antigen binding moiety.

3. The multi-specific binding polypeptide of claim 1 or claim 2, wherein a first binding affinity between the TRAIL receptor binding moiety and the TRAIL receptor is less than a second binding affinity between the cell-specific antigen binding moiety and the cell-specific antigen.

4. The multi-specific binding polypeptide of claim 3, wherein the first binding affinity is less than the second binding affinity by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, or higher.

5. The multi-specific binding polypeptide of any of claims 1-4, wherein the TRAIL receptor binding moiety has a KD for TRAIL receptor that is lower than the KD of the cell-specific antigen binding moiety for the antigen expressed on the cell.

6. The multi-specific binding polypeptide of any of claims 1-4, wherein cell killing potency of the polypeptide decreases with decreasing binding affinity to the tumor antigen and increasing rate of dissociation from the tumor antigen.

7. The multi-specific binding polypeptide of any of claims 1-6, wherein the cell-specific antigen comprises CD33, HSP90, F0LR1, HER2, GPC3, GD2, FLT3, CD20, CEA, CD38, CD22, PDL1, or TR0P2.

8. The multi-specific binding polypeptide of any of claims 1-7, wherein the TRAIL receptor comprises TRAIL-R2 (DR5) or TRAIL-R1 (DR4).

9. The multi-specific binding polypeptide of any of claims 1-8, wherein the multi-specific binding polypeptide comprises an Fc portion.

10. The multi-specific binding polypeptide of any of claims 1-9, wherein the multi-specific binding polypeptide comprises an Fc region that has been modified to reduce the affinity for human neonatal Fc receptor (FcRn).

11. The multi-specific binding polypeptide of any of claims 1-10, wherein the multi-specific binding polypeptide comprises an Fc region including a modification to reduce antibodydependent cellular cytotoxicity (ADCC), wherein the modification optionally comprises L234, L235, P238, or P331, or a combination thereof, wherein L234, L235, P238, and P331 correspond to positions 234, 235, 238, and 331 of a wild-type IgGl, according to the EU numbering convention.

12. The multi-specific binding polypeptide of claim 11, wherein the Fc region comprises an L234A, L235A, P238S, or P331 S modification, or a combination thereof.

13. The multi-specific binding polypeptide of any of claims 1-12, wherein the multi-specific polypeptide comprises an Fc region that has been modified to reduce neutropenia.

14. The multi-specific binding polypeptide of any of claims 1-13, wherein the multi-specific binding polypeptide comprises an Fc region including a modification at L234, S239, S442, or a combination thereof, wherein L234, S239, and S442 correspond to positions 234, 239, 442 of a wild-type IgGl, according to the EU numbering convention.

15. The multi-specific binding polypeptide of claim 14, wherein the Fc region comprises an L234F, S239C, or S442C modification, or a combination thereof.

16. The multi-specific binding polypeptide of any of claims 1-15, wherein the multi-specific binding polypeptide comprises an Fc region including an L234A modification and an L235A modification.

17. The multi-specific binding polypeptide of any of claims 1-15, wherein the multi-specific polypeptide comprises a modification to a hinge region.

18. The multi-specific binding polypeptide of any of claims 1-16, wherein the multi-specific polypeptide comprises a hinge region having a modification at S228, wherein S228 correspond to position 228 of a wild-type IgG4, according to the EU numbering convention.

19. The multi-specific binding polypeptide of any of claims 1-17, wherein the multi-specific polypeptide comprises a hinge region having an S228P modification.

20. The multi-specific binding polypeptide of any of claims 1-18, wherein the multi-specific polypeptide is a full-length antibody, an appended antibody, chimeric antibody, a bispecific fusion protein, a bispecific antibody conjugate, or an antigen binding fragment thereof.

21. The multi-specific binding polypeptide of any of claims 1-18, wherein the multi-specific binding polypeptide comprises an Fc region that has been modified to enhance antibodydependent cellular cytotoxicity (ADCC).

22. The multi-specific binding polypeptide of claim 21, wherein the Fc region comprises a modification at S239, A330, 1332, or a combination thereof, wherein S239, A330, and 1332 correspond to positions 239, 330, and 332 of a wild-type IgGl, according to the EU numbering convention.

23. The multi-specific binding polypeptide of any of claims 1-22, wherein the multi-specific polypeptide comprises a nanobody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody- CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv- CH-CL-scFv, F(ab’)2, F(ab’)2-scFv2. scFv-KIH, Fab-scFv-Fc, tetravalent HC Ab, scDiabody- Fc, diabody-Fc, tandem scFv-Fc, or intrabody.

24. The multi-specific binding polypeptide of claim 23, wherein the multi-specific binding polypeptide comprises an IgG framework, optionally an IgGl, IgG2, or IgG4 framework, further optionally an IgGl or IgG4 framework.

25. The multi-specific binding polypeptide of any of claims 1-24, wherein the multi-specific binding polypeptide is a humanized antibody.

26. The multi-specific binding polypeptide of any of claims 1-25, wherein the multi-specific binding polypeptide can induce apoptosis in a target cell that expresses the cell-specific antigen.

27. The multi-specific binding polypeptide of any of claims 1-26, wherein the cell-specific antigen is expressed in a cancer cell, immune cell, hyperimmune cell, T-cell, immunosuppressive cell, and / or senescent cell.

28. The multi-specific binding polypeptide of any of claims 1-27, wherein the cell-specific antigen is located in a lipid raft of a cell membrane, in lipid rich microdomains of the cell membrane, and / or on the cell membrane.

29. The multi-specific binding polypeptide of any of claims 1-28, wherein the cell-specific antigen is Glycosylphosphatidylinositol (GPI)-anchored protein.

30. The multi-specific binding polypeptide of any of claims 1-29, wherein the cell-specific antigen is a tumor-specific antigen and / or tumor associated antigen.

31. The multi-specific binding polypeptide of any of claims 1-29, wherein the cell-specific antigen is an immune cell-specific antigen and / or immune cell associated antigen, a hyperimmune cell-specific antigen and / or hyperimmune cell associated antigen, T-cell-specific antigen and / or T-cell associated antigen, or an immunosuppressive cell-specific antigen or and / or immunosuppressive cell associated antigen.

32. The multi-specific binding polypeptide of any of claims 1-31, wherein the TRAIL receptor binding moiety comprises an IgG antibody framework, optionally an IgGl or IgG4 framework.

33. The multi-specific binding polypeptide of any of claims 1-32, wherein the TRAIL receptor binding moiety comprises a full-length antibody or an antigen binding fragment thereof.

34. The multi-specific binding polypeptide of any of claims 1-33, wherein the TRAIL receptor binding moiety comprises a Fab, F(ab)2, single-domain antibody, a single chain variable fragment (scFv), or a nanobody.

35. The multi-specific binding polypeptide of any of claims 1-34, wherein the TRAIL receptor binding moiety is a humanized antibody.

36. The multi-specific binding polypeptide of any of claims 1-35, wherein the TRAIL receptor binding moiety is a chimeric antibody.

37. The multi-specific binding polypeptide of any of claims 1-36, wherein the TRAIL receptor binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 84, HCDR2 comprising SEQ ID NO: 86, and a HCDR3 comprising SEQ ID NO: 87, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 88, a LCDR2 comprising SEQ ID NO: 89, and a LCDR3 comprising SEQ ID NO: 90; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 87; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 91.

38. The multi-specific binding polypeptide of any of claims 1-36, wherein the TRAIL receptor binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 131, HCDR2 comprising SEQ ID NO: 132, and a HCDR3 comprising SEQ ID NO: 133, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 135, a LCDR2 comprising SEQ ID NO: 136, and a LCDR3 comprising SEQ ID NO: 137; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 134; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 138.

39. The multi-specific binding polypeptide of any of claims 1-38, wherein the cell-specific antigen binding moiety comprises an IgG antibody framework, optionally an IgGl or IgG4 framework.

40. The multi-specific binding polypeptide of any of claims 1-39, wherein the cell-specific antigen binding moiety comprises a full-length antibody or an antigen binding fragment thereof.

41. The multi-specific binding polypeptide of any of claims 1-40, wherein the cell-specific antigen binding moiety comprises a Fab, F(ab)2, single-domain antibody, a single chain variable fragment (scFv), or a nanobody.

42. The multi-specific binding polypeptide of any of claims 1-41, wherein the cell-specific antigen binding moiety is a humanized antibody.

43. The multi-specific binding polypeptide of any of claims 1-42, wherein the cell-specific antigen binding moiety is a chimeric antibody.

44. The multi-specific binding polypeptide of any of claims 1-43, wherein the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 118, or 122; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 126, or 130.

45. The multi-specific binding polypeptide of any of claims 1-43, wherein the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 4, a HCDR2 comprising SEQ ID NO: 5, and a HCDR3 comprising SEQ ID NO: 6, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 44, a LCDR2 comprising SEQ ID NO: 45, and a LCDR3 comprising SEQ ID NO: 46; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 47.

46. The multi-specific binding polypeptide of any of claims 1-43, wherein the cell-specific antigen binding moiety comprises:an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 8, a HCDR2 comprising SEQ ID NO: 9, and a HCDR3 comprising SEQ ID NO: 10, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 48, a LCDR2 comprising SEQ ID NO: 49, and a LCDR3 comprising SEQ ID NO: 50; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 11; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 51.

47. The multi-specific binding polypeptide of any of claims 1-43, wherein the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 12, a HCDR2 comprising SEQ ID NO: 13, and a HCDR3 comprising SEQ ID NO: 14, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 52, a LCDR2 comprising SEQ ID NO: 53, and a LCDR3 comprising SEQ ID NO: 54; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 15; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 55.

48. The multi-specific binding polypeptide of any of claims 1-43, wherein the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 16, a HCDR2 comprising SEQ ID NO: 17, and a HCDR3 comprising SEQ ID NO: 18, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 56, a LCDR2 comprising SEQ ID NO: 57, and a LCDR3 comprising SEQ ID NO: 58; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 19; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 59.

49. The multi-specific binding polypeptide of any of claims 1-43, wherein the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 20, a HCDR2 comprising SEQ ID NO: 21, and a HCDR3 comprising SEQ ID NO: 22,and a VL domain comprising a LCDR1 comprising SEQ ID NO: 60, a LCDR2 comprising SEQ ID NO: 61, and a LCDR3 comprising SEQ ID NO: 62; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 23; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 63.

50. The multi-specific binding polypeptide of any of claims 1-43, wherein the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 24, a HCDR2 comprising SEQ ID NO: 25, and a HCDR3 comprising SEQ ID NO: 26, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 64, a LCDR2 comprising SEQ ID NO: 65, and a LCDR3 comprising SEQ ID NO: 66; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 27; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 67.

51. The multi-specific binding polypeptide of any of claims 1-43, wherein the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 28, a HCDR2 comprising SEQ ID NO: 29, and a HCDR3 comprising SEQ ID NO: 30, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 68, a LCDR2 comprising SEQ ID NO: 69, and a LCDR3 comprising SEQ ID NO: 70; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 31; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 71.

52. The multi-specific binding polypeptide of any of claims 1-43, wherein the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 32, a HCDR2 comprising SEQ ID NO: 33, and a HCDR3 comprising SEQ ID NO: 34, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 72, a LCDR2 comprising SEQ ID NO: 73, and a LCDR3 comprising SEQ ID NO: 74; oran immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 35; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 75.

53. The multi-specific binding polypeptide of any of claims 1-43, wherein the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 36, a HCDR2 comprising SEQ ID NO: 37, and a HCDR3 comprising SEQ ID NO: 38, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 76, a LCDR2 comprising SEQ ID NO: 77, and a LCDR3 comprising SEQ ID NO: 78; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 39; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 79.

54. The multi-specific binding polypeptide of any of claims 1-43, wherein the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 40, a HCDR2 comprising SEQ ID NO: 41, and a HCDR3 comprising SEQ ID NO: 42, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 80, a LCDR2 comprising SEQ ID NO: 81, and a LCDR3 comprising SEQ ID NO: 82; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 43; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 83.

55. The multi-specific binding polypeptide of any of claims 1-43, wherein the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 115, a HCDR2 comprising SEQ ID NO: 116, and a HCDR3 comprising SEQ ID NO: 117, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 123, a LCDR2 comprising SEQ ID NO: 124, and a LCDR3 comprising SEQ ID NO: 125; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forthin SEQ ID NO: 118; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 126.

56. The multi-specific binding polypeptide of any of claims 1-43, wherein the cell-specific antigen binding moiety comprises: an immunoglobulin heavy chain variable region comprising a HCDR1 comprising SEQ ID NO: 119, a HCDR2 comprising SEQ ID NO: 120, and a HCDR3 comprising SEQ ID NO: 121, and a VL domain comprising a LCDR1 comprising SEQ ID NO: 127, a LCDR2 comprising SEQ ID NO: 128, and a LCDR3 comprising SEQ ID NO: 129; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 122; and an immunoglobulin light chain variable region at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 130.

57. The multi-specific binding polypeptide of any of claims 1-56, wherein the TRAIL receptor binding moiety comprises an IgG antibody framework, optionally a full-length IgG antibody framework, the cell-specific antigen binding moiety is an scFv, and the cell-specific antigen binding moiety is coupled to the C-terminus of the TRAIL receptor binding moiety, optionally recombinantly fused to the C-terminus of the TRAIL receptor binding moiety.

58. The multi-specific binding polypeptide of any of claims 1-56, wherein the TRAIL receptor binding moiety comprises an IgG antibody framework, optionally a full-length IgG antibody framework, the cell-specific antigen binding moiety is an scFv, and the cell-specific antigen binding moiety is coupled to the N-terminus of the TRAIL receptor binding moiety, optionally recombinantly fused to the N-terminus of the TRAIL receptor binding moiety.

59. The multi-specific binding polypeptide of any of claims 1-56, wherein the cell-specific antigen binding moiety comprises an IgG antibody framework, optionally a full-length IgG antibody framework, the TRAIL receptor binding moiety is an scFv, and the TRAIL receptor binding moiety is coupled to the C-terminus of the cell-specific antigen binding moiety, optionally recombinantly fused to the C-terminus of the cell-specific antigen binding moiety.

60. The multi-specific binding polypeptide of any of claims 1-56, wherein the cell-specific antigen binding moiety comprises an IgG antibody framework, optionally a full-length IgG antibody framework, the TRAIL receptor binding moiety is an scFv, and the TRAIL receptor binding moiety is coupled to the N-terminus of the cell-specific antigen binding moiety, optionally recombinantly fused to the N-terminus of the cell-specific antigen binding moiety.

61. The multi-specific binding polypeptide of any of claims 1-60, wherein the cell-specific antigen binding moiety is coupled to the TRAIL receptor binding moiety by a polypeptide linker.

62. The multi-specific binding polypeptide of claim 61, wherein the polypeptide linker comprises (Gly4Ser)n, wherein n is an integer from 1 to 10, optionally from 1 to 6, 1 to 4, or 1 to 2, further optionally 1, 2, 3, or 4.

63. The multi-specific binding polypeptide of any of claims 1-62, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in Table 5; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in Table 5.

64. The multi-specific binding polypeptide of any of claims 1-37, 39- 45 and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 92; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 93.

65. The multi-specific binding polypeptide of any of claims 1-37, 39-42, 44, and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 94; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 95.

66. The multi-specific binding polypeptide of any of claims 1-37, 39-42, 47, and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 96; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 97.

67. The multi-specific binding polypeptide of any of claims 1-37, 39-42, 48, and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 98; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 99.

68. The multi-specific binding polypeptide of any of claims 1-37, 39-42, 49, and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 100; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 101.

69. The multi-specific binding polypeptide of any of claims 1-37, 39-42, 50, and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 102; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 103.

70. The multi-specific binding polypeptide of any of claims 1-37, 39-42, 51, and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 104; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 105.

71. The multi-specific binding polypeptide of any of claims 1-37, 39-42, 52, and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 106; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 107.

72. The multi-specific binding polypeptide of any of claims 1-37, 39-42, 53, and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 108; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 109.

73. The multi-specific binding polypeptide of any of claims 1-37, 39-42, 54, and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 110; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 111.

74. The multi-specific binding polypeptide of any of claims 1-37, 39-42, 55, and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 139; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 140.

75. The multi-specific binding polypeptide of any of claims 1-37, 39-42, 56, and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 141; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 142.

76. The multi-specific binding polypeptide of any of claims 1-37, 39-42, and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 143; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 93.

77. The multi-specific binding polypeptide of any of claims 1-37, 39-42, 50, and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 144; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 103.

78. The multi-specific binding polypeptide of any of claims 1-37, 39-42, 49, and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 145; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 101.

79. The multi-specific binding polypeptide of any of claims 1-37, 39-42, 55, and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 146; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 140.

80. The multi-specific binding polypeptide of any of claims 1-37, 39-42, 56, and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 147; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 142.

81. The multi-specific binding polypeptide of any of claims 1-37, 39-42, 54, and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 148; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 111.

82. The multi-specific binding polypeptide of any of claims 1-37, 39-42, 48, and 57-63, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 149; and an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 99.

83. The multi-specific binding polypeptide of any of claims 1-82, wherein the multi-specific binding polypeptide further comprises at least one payload and / or cytotoxic moiety.

84. The multi-specific binding polypeptide of claim 83, wherein the payload and / or cytotoxic moiety comprises an auristatin, an auristatin derivative, maytansine, a maytansinoid, a taxane, a calicheamicin, cemadotin, a duocarmycin, a pyrrolobenzodiazepine (PBD), tubulysin, dexamethasone, or dasatinib.

85. The multi-specific binding polypeptide of claim 83, wherein the auristatin derivative is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

86. The multi-specific binding polypeptide of claim 83, wherein the maytansinoid is DM1, DM2, or DM4.

87. The multi-specific binding polypeptide of claim 83, wherein the pyrrolobenzodiazepine is a pyrrolobenzodiazepine dimer.

88. The multi-specific binding polypeptide of claim 83, wherein the at least one payload and / or cytotoxic moiety is attached to the multi-specific binding polypeptide via a linker, optionally a cleavable linker or a non-cleavable linker.

89. The multi-specific binding polypeptide of claim 88, wherein the linker comprises a pH- sensitive linker, a protease-sensitive linker, a self-immolative linker, or a combination thereof.

90. The multi-specific binding polypeptide of claim 88, wherein the linker comprises a zerolength linker, a homobifunctional linker, a heterobifunctional linker, a di-peptide linker, a spacer, a maleimide-based conjugating moiety, or a combination thereof.

91. The multi-specific binding polypeptide of claim 88, wherein the linker comprises a polymer, a linear or branched polyethylene glycol, a peptide, or a combination thereof.

92. The multi-specific binding polypeptide of claim 88, wherein the linker is a peptidomimetic linker.

93. The multi-specific binding polypeptide of any of claims 83-92, wherein a ratio of the payload and / or cytotoxic moiety to the multi-specific binding polypeptide is about 1 :1, about 2: 1, about 3:1, about 4: 1, about 5:1, about 6: 1, about 7:1, about 8: 1, about 10: 1, or about 12: 1.

94. A pharmaceutical composition comprising the multi-specific binding polypeptide of any of claims 1 to 93, and a pharmaceutically acceptable excipient, carrier, or diluent.

95. The pharmaceutical composition of claim 94, further comprising an additional therapeutic agent selected from the group consisting of Mylotarg, Venetoclax, Daratumumab, Cisplatin, Doxorubicin, Paclitaxel, or a combination thereof.

96. The pharmaceutical composition of claim 94 or 95, wherein the pharmaceutical composition is formulated for parenteral administration, optionally for subcutaneous, intramuscular, or intravenous administration.

97. A nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 92; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 93.

98. A nucleic acid encoding a multi-specific binding polypeptide comprising animmunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 94; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 95.

99. A nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 96; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 97.

100. A nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 98; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 99.

101. A nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 100; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 101.

102. A nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 102; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 103.

103. A nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 104; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 105.

104. A nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 106; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 107.

105. A nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 108; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 109.

106. A nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 110; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 111.

107. A nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 139; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 140.

108. A nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 141; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 142.

109. A nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 143; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 93.

110. A nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 144; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 103.

111. A nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 145; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 101.

112. A nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 146; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 140.

113. A nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 147; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 142.

114. A nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 148; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 111.

115. A nucleic acid encoding a multi-specific binding polypeptide comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO:149; and optionally an immunoglobulin light chain at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to or consist of the amino acid sequence set forth in SEQ ID NO: 99.

116. A nucleic acid encoding a multi-specific binding polypeptide of claims 1-93.

117. A vector comprising a nucleic acid of claims 97-116.

118. A cell comprising the nucleic acid of claims 97- 116 or the vector of embodiment 117.

119. A method of inducing a cell killing effect in a target cell population, comprising contacting the target cell population with a multi-specific binding polypeptide of claims 1-93, a pharmaceutical composition of claims 94-97, or a nucleic acid of claims 98-116 for a time sufficient to induce cell kill effect, thereby killing the at least one cell in the target cell population.

120. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the multi-specific binding polypeptide of claims 1-93, a pharmaceutical composition of claims 94-96, or a nucleic acid of claims 97-116.

121. The method of claim 120, wherein the disease or condition is cancer.

122. The method of claim 120 or 121, wherein the disease or condition is bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, hematologic malignancy, kidney cancer, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, testicular cancer, or thyroid cancer.

123. The method of claim 122, wherein the breast cancer is luminal A breast cancer, luminal B breast cancer, triple-negative breast cancer, HER2-enriched breast cancer, normal-like breast cancer, ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC) such as tubular carcinoma of the breast, medullary carcinoma of the breast, papillary carcinoma of the breast, or cribriform carcinoma of the breast, invasive lobular carcinoma (ILC), inflammatory breast cancer, lubular carcinoma in situ (LCIS), male breast cancer, Paget’s disease of the Nipple, or phyllodes tumors of the breast.

124. The method of claim 122, wherein the ovarian cancer is epithelial carcinoma, serous carcinoma, small-cell carcinoma, primary peritoneal carcinoma, clear-cell carcinoma, clear-cell adenocarcinoma, endometrioid, malignant mixed mullerian tumor, mucinous, mucinous adenocarcinoma, pseudomyxoma peritonei, undifferentiated epithelial, malignant Brener tumor, transitional cell carcinoma, sex cord-stromal tumor, granulosa cell tumor, adult granulosa cell tumor, juvenile granulosa cell tumor, Sertoli-Leydig cell tumor, sclerosing stromal tumors, germ cell tumor, dysgerminoma, choriocarcinoma, immature (solid) teratoma, mature teratoma (dermoid cyst), yolk sac tumor (endodermal sinus tumor), embryonal carcinoma, polyembryoma, squamous cell carcinoma, mixed tumors, or low malignant potential tumors.

125. The method of claim 122, wherein the lung cancer is small cell lung cancer (SCLC) or nonsmall cell lung cancer (NSCLC), such as adenocarcinoma, squamous cell cancer, large cell carcinoma, or undifferentiated non-small cell lung cancer.

126. The method of claim 122, wherein the brain cancer is glioblastoma.

127. The method of claim 122, wherein the hematologic malignancy is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom’s macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt’s lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, or acute myeloid leukemia.

128. The method of claim 122, wherein the liver cancer is hepatocellular carcinoma (HCC), cholangiocarcinoma, liver angiosarcoma, or hepatoblastoma.

129. The method of claim 122, wherein the prostate cancer is acinar adenocarcinoma, ductal adenocarcinoma, transitional cell (or urothelial) cancer, squamous cell cancer, small cell prostate cancer, carcinoid in the prostate, or sarcoma in the prostate.

130. The method of any of claims 121-123, wherein the cancer is a metastatic cancer.

131. The method of any of claims 121-123, wherein the cancer relapsed or refractory cancer.

132. The method of claim 120, wherein the disease or condition is an autoimmune disease.

133. The method of claim 132, wherein the autoimmune disease is rheumatoid arthritis, multiple sclerosis (MS), type 1 diabetes, systemic lupus erythematosus (SLE), Graves Disease, or Hashimoto’s thyroiditis.

134. The method of claim 120, wherein the disease or condition is a liver disease or condition.

135. The method of claim 134, wherein the liver disease or condition is non-alcoholic fatty liver disease (NASH) or alcoholic steatohepatitis.

136. The method of any of claims 119-135, further comprising administering an additional therapeutic agent.

137. The method of claim 136, wherein the additional therapeutic agent is a chemotherapeutic agent, radiation, or a combination thereof.

138. The method of claim 136, wherein the additional therapeutic agent is Mylotarg, Venetoclax, Daratumumab, Cisplatin, Doxorubicin, Paclitaxel, or a combination thereof.

139. The method of any of claims 136-138, wherein the multi-specific binding polypeptide and the additional therapeutic agent are administered simultaneously or sequentially.

140. The method of any of claims 136-138, wherein the multi-specific binding polypeptide is administered to the subject prior to administering the additional therapeutic agent.

141. The method of any of claims 136-138, wherein the additional therapeutic agent is administered to the subject prior to administering the multi-specific binding polypeptide.

142. The method of any of claims 136-138, wherein the multi-specific binding polypeptide and the additional therapeutic agent are administered as a combination.

143. The method of any of claims 136-138, wherein the multi-specific binding polypeptide and the additional therapeutic agent are administered as separate dosage forms.

144. The method of any of claims 121-143, wherein the subject has previously been treated with an immune checkpoint inhibitor treatment.

145. The method of any of claims 121-143, wherein the subject is insensitive to treatment with an immune checkpoint inhibitor, has failed to respond to treatment with an immune checkpoint inhibitor, or who expresses low level of or does not express an immune checkpoint protein.

146. The method of any one of claims 121-145, wherein the subject has undergone surgery.

147. The method of any of claims 121-146, wherein the multi-specific binding polypeptide of claims 1-93, the pharmaceutical composition of claims 94-96, or the nucleic acid of claims 97- 116 decreases tumor cells, optionally tumor cell proliferation, in the target cell population by about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30- fold, 40-fold, 50-fold, or more.

148. The method of any of the claims 120-146, wherein the multi-specific binding polypeptide of claims 1-91, the pharmaceutical composition of claims 92-96, or the nucleic acid of claims 97-116 decreases immunosuppressive cells, optionally immunosuppressive cell proliferation in the target cell population by about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more.

149. The method of any of the claims 121-148, wherein the multi-specific binding polypeptide of claims 1-93, the pharmaceutical composition of claim 94-96, or the nucleic acid of claims 97- 116 enhances T-cell proliferation, optionally tumor-infiltrating lymphocyte (TIL) proliferation.

150. The method of any of the claims 120-149, wherein the target cell population is an in vivo target cell population.

151. The method of any of the claims 120-150, wherein the target cell population is within a tumor microenvironment.

152. A kit comprising the multi-specific binding polypeptide of any one of claims 1-93, the nucleic acid of any one of claims 97-116, the vector of claims 117, or the cell of embodiment 118.

153. The kit of claim 152, further comprising an additional therapeutic agent.

154. The kit of claim 153, wherein the additional therapeutic agent is Mylotarg, Venetoclax, Daratumumab, Cisplatin, Doxorubicin, Paclitaxel, or a combination thereof.

155. A kit comprising the pharmaceutical composition of any one of claims 94-96.

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