Bispecific antibody targeting EMR2 (CD312) and the t-cell receptor TRBV19

Abstract

Provided herein are multispecific antibodies, including bispecific antibodies that specifically bind to EMR2 and TRBV19 (also known as Vβ17), and monospecific antibodies that specifically bind to EMR2, and multispecific antigen-binding fragments thereof. Also described are related polynucleotides capable of encoding the provided multispecific antibodies or multispecific antigen-binding fragments, cells expressing the provided multispecific antibodies or multispecific antigen-binding fragments, as well as associated vectors and detectably labeled multispecific antibodies or multispecific antigen-binding fragments. In addition, methods of producing and using the provided multispecific antibodies and multispecific antigen-binding fragments are described.

Description

BISPECIFIC ANTIBODY TARGETING EMR2 (CD312) AND THE T-CELL RECEPTOR TRBV19CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 651,800, filed on May 24, 2024, the disclosure of which is herein incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, is named JBI6897WOPCTl_SL.xml created on May 5, 2025 and is 199,843 bytes in size.TECHNICAL FIELD

[0003] The disclosure provided herein relates to bispecific antibodies that specifically bind epidermal-growth-factor-like module-containing mucin-like hormone receptor-like 2 (EMR2) and the TRBV19 receptor (also known as V017) on T cells and monospecific antibodies that specifically bind EMR2.BACKGROUND

[0004] Acute myeloid leukemia (AML) and myelodysplastic neoplasms (MDS; also known as myelodysplastic syndrome) are highly aggressive hematological malignancies characterized by clonal expansion of stem and myeloid progenitor cells. The current standard of care (SoC) for fit patients with AML is aggressive induction combination chemotherapy followed by high-dose chemotherapy and / or allogeneic transplantation. For AML patients that relapse or are refractory to SoC, targeted therapy is often combined with alternative regimens. While some patients achieve complete remission, 50% to 70% eventually relapse within 3 years and succumb to the disease. The current SoC for fit patients with MDS depends on the risk stratification; higher risk (HR) disease is managed with decitabine or azacytidine followed by allogeneic transplantation while lower-risk disease is managed with growth factors and supportive care until progression. Efforts to develop immunotherapies for AML and HR MDS have been challenging due to limited clinical activity, severe cytokine release syndrome (CRS), and significant toxicitiesowing to the broader expression of AML targets on healthy myeloid cells and nonhematopoietic tissues.SUMMARY

[0005] In one aspect, the disclosure provides an antibody or an antigen-binding fragment thereof, that specifically binds the G-protein-coupled receptor auto-proteolysis inducing (GAIN) domain and / or the GPCR proteolytic site (GPS) motif of epidermal-growth-factor-like modulecontaining mucin- like hormone receptor 2 (EMR2).

[0006] In some embodiments, the GAIN domain comprises amino acid residues D261-Q478 of SEQ ID NO: 205.

[0007] In some embodiments, the GAIN domain comprises the amino acid sequence SEQ ID NO: 215.

[0008] In any of the foregoing embodiments, the GPS motif comprises the amino acid sequence SEQ ID NO: 216.

[0009] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, binds to an epitope comprising SEQ ID NO: 217 or SEQ ID NO: 218.

[0010] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, binds to human EMR2 with a dissociation constant (KD) between about 0.01 nM to about 5 nM.

[0011] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, binds to human EMR2 with an half maximal effective concentration (ECso) between about 0.1 nM to about 15 nM.

[0012] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, is or comprises a fragment antigen-binding (Fab), a F(ab')2 fragment, F(ab)'3 fragments, a single-chain variable fragment (scFv), a bis-scFv, a (scFv)2, a stapled scFv (spFv), a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a singledomain antibody (sdAb), an Immunoglobulin New Antigen Receptor (Ig NAR), a single heavy chain antibody, a camelid antibody, a shark antibody, or a chemically modified derivative thereof.

[0013] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, comprises a Fab.

[0014] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, further comprises a first constant Ig domain of the heavy chain (CHI) domain.

[0015] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, does not comprise a CHI domain.

[0016] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, comprises a scFv or a spFv.

[0017] In some embodiments, the scFv or spFv comprises a signal sequence, a heavy chain variable sequence, a GS-Linker, and a light chain variable sequence.

[0018] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, further comprises a fragment crystallizable (Fc) domain.

[0019] In some embodiments, the Fc domain of the antibody or the antigen-binding fragment thereof, is an IgA, an IgG, an IgE, or an IgM. In some embodiments, the Fc domain of the antibody or the antigen-binding fragment thereof, is an IgG. In some embodiments, the IgG is IgGl or IgG4.

[0020] In some of the foregoing embodiments, the Fc domain comprises one or more different mutations which promote heterodimerization.

[0021] In some of the foregoing embodiments, the Fc domain comprises mutations T366S, L368A and Y407V (EU numbering) or mutation T366W (EU numbering).

[0022] In some of the foregoing embodiments, the Fc domains of the first heavy chain (HC1) and / or the second heavy chain (HC2) further comprise one or more mutations which reduce Fc binding to a Fey receptor. In some embodiments, the Fey receptor is FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, and / or FcyRIIIB. In some of the foregoing embodiments, the Fc domain comprises one or more mutations selected from L234A, L235A, and D265S (EU numbering). In some of the foregoing embodiments, the Fc domain comprises mutations L234A, L235A, and D265S (EU numbering).

[0023] In some of the foregoing embodiments, the Fc domain further comprises one or more mutations which reduce Fc binding to protein A. In some of the foregoing embodiments, the Fc domain comprises mutations H435R and / or Y436F (EU numbering). In some of the foregoing embodiments, the Fc domain comprises mutations H435R and Y436F (EU numbering).

[0024] In some of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, comprises a humanized antibody, or an antigen binding fragment thereof, a humanantibody or an antigen binding fragment thereof, a murine antibody or an antigen binding fragment thereof, a chimeric antibody or an antigen binding fragment thereof, a monospecific antibody or a monospecific antigen binding fragment thereof, a bispecific antibody or a bispecific antigen binding fragment thereof, or a multispecific antibody or a multispecific antigen binding fragment thereof.

[0025] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, comprises: a) a heavy chain complementarity determining region (HCDR) 1, a HCDR2 and a HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 63, and a light chain complementarity determining region (LCDR) 1 , a LCDR2 and a LCDR3 of the light chain variable region (VL) of SEQ ID NO: 64; b) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 95, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 96; c) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 127, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 128; or d) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 191, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 192.

[0026] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of: a) SEQ ID NO: 33, 34, 35, 48, 49, and 50, respectively; b) SEQ ID NO: 36, 37, 38, 51, 52, and 53, respectively; c) SEQ ID NO: 39, 40, 41, 54, 55, and 56, respectively; d) SEQ ID NO: 42, 43, 44, 57, amino acid sequence EVS, and SEQ ID NO: 59, respectively; e) SEQ ID NO: 45, 46, 47, 60, 61, and 62, respectively; f) SEQ ID NO: 65, 66, 67, 80, 81, and 82, respectively; g) SEQ ID NO: 68, 69, 70, 83, 84, and 85, respectively; h) SEQ ID NO: 71, 72, 73, 86, 87, and 88, respectively; i) SEQ ID NO: 74, 75, 76, 89, amino acid sequence AAI, and SEQ ID NO: 91, respectively; j) SEQ ID NO: 77, 78, 79, 92, 93, and 94, respectively; k) SEQ ID NO: 97, 98, 99, 112, 113, and 114, respectively; 1) SEQ ID NO: 100, 101, 102, 115, 116, and 117, respectively; m) SEQ ID NO: 103, 104, 105, 118, 119, and 120, respectively; n) SEQ ID NO: 106, 107, 108, 121, amino acid sequence DNN, and SEQ ID NO: 123, respectively; o) SEQ ID NO: 109, 110, 111, 124, 125, and 126, respectively; p) SEQ ID NO: 161, 162, 163, 176, 177, and 178, respectively; q) SEQ ID NO: 164, 165, 166, 179, 180, and 181, respectively; r) SEQ ID NO: 167, 168, 169, 182, 18, and 184, respectively; s) SEQ ID NO: 170, 171, 172, 185, ammo acid sequence EVS, and SEQ ID NO: 187, respectively; or t) SEQ ID NO: 173, 174, 175, 188, 189, and 190, respectively.

[0027] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, comprises a variable heavy chain region (VH) comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 63; b) the VH of SEQ ID NO: 95; c) the VH of SEQ ID NO: 127; or d) the VH of SEQ ID NO: 191.

[0028] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, comprises or further comprises a variable light chain region (VL) comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 64; b) the VL of SEQ ID NO: 96; c) the VL of SEQ ID NO: 128; or d) the VL of SEQ ID NO: 192.

[0029] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, comprises: a) the VH of SEQ ID NO: 63 and the VL of SEQ ID NO: 64; b) the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 96; c) the VH of SEQ ID NO: 127 and the VL of SEQ ID NO: 128; or d) the VH of SEQ ID NO: 191 and the VL of SEQ ID NO: 192.

[0030] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, comprises a scFv or a spFV which comprises, from the N- to C-terminus, a VH, a linker (L) and a in the format VH-L-VL or the VL, a linker (L) and a VH in the format VL-L-VH.

[0031] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 191, the VL comprises the amino acid sequence of SEQ ID NO: 192, and the L comprises SEQ ID NO: 221.

[0032] In some embodiments, the linker comprises SEQ ID NO: 221.

[0033] In any of the foregoing embodiments, the heavy chain (HC) comprises the amino acid sequence of SEQ ID NO: 194, 196, 198, or 202.

[0034] In any of the foregoing embodiments, the light chain (LC) comprises the amino acid sequence of SEQ ID NO: 195, 197 or 199.

[0035] In some of the foregoing embodiments, a) the HC1 comprises the amino acid sequence of SEQ ID NO: 194 and the first light chain (LC1) comprises the amino acid sequence of SEQ ID NO: 195; b) the HC1 comprises the amino acid sequence of SEQ ID NO: 196 and the LC1 comprises the amino acid sequence of SEQ ID NO: 197; or c) the HC1 comprises the amino acidsequence of SEQ ID NO: 198 and the LC1 comprises the amino acid sequence of SEQ ID NO: 199.

[0036] In another general aspect, the disclosure provides an antibody or an antigen-binding fragment thereof, that binds to the same epitope of EMR2 as the antibody or the antigen-binding fragment thereof, according to any of the embodiments described herein.

[0037] In another general aspect, the disclosure provides an antibody or an antigen-binding fragment thereof, that competes for binding to the same epitope of EMR2 with the antibody or the antigen-binding fragment thereof, according to any of the embodiments described herein.

[0038] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, is a monospecific antibody.

[0039] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, is a bispecific antibody which specifically binds to EMR2 and to a second antigen.

[0040] In some embodiments, the second antigen is T cell receptor beta variable 19 (TRBV19).

[0041] In an aspect, the disclosure provides an isolated polynucleotide encoding the antibody or the antigen-binding fragment thereof, according to any of the embodiments described herein.

[0042] In some embodiments, the isolated polynucleotide comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 63; b) the VH of SEQ ID NO: 95; c) the VH of SEQ ID NO: 127; or d) the VH of SEQ ID NO: 191.

[0043] In some embodiments, the isolated polynucleotide comprises or further comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 64; b) the VL of SEQ ID NO: 96; c) the VL of SEQ ID NO: 128; or d) the VL of SEQ ID NO: 192.

[0044] In any of the foregoing embodiments, the isolated polynucleotide comprises a sequence comprising a nucleotide sequence encoding: a) the amino acid sequence of SEQ ID NO: 63 and / or SEQ ID NO: 64; b) the amino acid sequence of SEQ ID NO: 95 and / or SEQ ID NO: 96; c) the amino acid sequence of SEQ ID NO: 127 and / or SEQ ID NO: 128; or d) the amino acid sequence of SEQ ID NO: 191 and / or the SEQ ID NO: 192.

[0045] In some embodiments, the isolated polynucleotide comprises a sequence encoding a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 194, 196, 198, or 202.

[0046] In some embodiments, the isolated polynucleotide comprises a sequence encoding a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 195, 197, or 199.

[0047] In any of the foregoing embodiments, the isolated polynucleotide comprises a nucleotide sequence encoding: a) the amino acid sequence of SEQ ID NO: 194 and / or SEQ ID NO: 195; b) the amino acid sequence of SEQ ID NO: 196 and / or SEQ ID NO: 197; c) the amino acid sequence of SEQ ID NO: 198 and / or the amino acid sequence of SEQ ID NO: 199; or d) the amino acid sequence of SEQ ID NO: 202.

[0048] In another general aspect, the disclosure provides a vector comprising the isolated polynucleotide according to any of the foregoing embodiments.

[0049] In some embodiments, the isolated polynucleotide is operably linked to an expression control sequence.

[0050] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpes simplex virus vector, and a poxvirus vector.

[0051] In another general aspect, the disclosure provides a pharmaceutical composition comprising (i) the antibody or the antigen-binding fragment thereof, according to any of the embodiments described herein, or the isolated polynucleotide according to any of the embodiments described herein, or the vector according to any of the embodiments described herein, and (ii) a pharmaceutically acceptable carrier or excipient.

[0052] In an aspect, the disclosure provides a host cell expressing the antibody or the antigenbinding fragment thereof, according to any of the embodiments described herein.

[0053] In some embodiments, the cell is a hybridoma.

[0054] In some embodiments, the antibody, or the antigen-binding fragment thereof, is recombinantly produced.

[0055] In another general aspect, the disclosure provides a host cell comprising the isolated polynucleotide according to any of the embodiments described herein or the vector according to of the embodiments described herein.

[0056] In another general aspect, the disclosure provides a method for treating a cancer in a subject in need thereof, the method comprising administering to the subject a therapeuticallyeffective amount of the antibody or the antigen-binding fragment thereof, according to any of the embodiments described herein, or the isolated polynucleotide according to any of the embodiments described herein, or the vector according to any of the embodiments described herein, or the pharmaceutical composition according to any of the embodiments described herein, or the host cell according to any of the embodiments described herein.

[0057] In another general aspect, the disclosure provides a method for inducing cytotoxicity of a cancer cell or redirecting immune or T cells to a cancer cell, said method comprising administering to the cell an effective amount of the antibody or the antigen-binding fragment thereof, according to any of the embodiments described herein, or the polynucleotide according to any of the embodiments described herein, or the vector according to any of the embodiments described herein, or the pharmaceutical composition of any of the embodiments described herein, or the host cell according to any of the embodiments described herein, wherein said effective amount is sufficient to inhibit the growth or proliferation of the cancer cell.

[0058] In some embodiments, the cancer cell is in a subject and the antibody or the binding fragment, the polynucleotide, the vector, the pharmaceutical composition, or the host cell is administered to the subject.

[0059] In some embodiments, said administration is conducted ex vivo.

[0060] In some embodiments, the cancer is an EMR2-expressing cancer. In some embodiments, the EMR2-expressing cancer is a hematological cancer. In some embodiments, the hematological cancer is a myeloid malignancy. In some embodiments, the hematological cancer is an acute myeloid leukemia (AML), a chronic myelogenous leukemia (CML), or myelodysplastic neoplasms (MDS).

[0061] In any of the foregoing embodiments, the methods further comprise administering a second therapeutic agent.

[0062] In some embodiments, the second therapeutic agent is a surgery, a chemotherapy, an androgen deprivation therapy, a radiation, or any combination thereof.

[0063] In another general aspect, the disclosure provides an antibody according to any of the embodiments described herein for use in the method according to any of the embodiments described herein.

[0064] In another general aspect, the disclosure provides an isolated polynucleotide according to any of the embodiments described herein for use in the method according to any of the embodiments described herein.

[0065] In another general aspect, the disclosure provides a vector according to any of the embodiments described herein for use in the method according to any of the embodiments described herein.

[0066] In another general aspect, the disclosure provides a pharmaceutical composition according to the embodiments described herein for use in the method according to any of the embodiments described herein.

[0067] In another general aspect, the disclosure provides a host cell according to any of the embodiments described herein for use in the method according to any of the embodiments described herein.

[0068] In another general aspect, the disclosure provides a method for generating the antibody or the antigen-binding fragment thereof, according to any of the embodiments described herein, wherein said method comprises culturing the host cell according to any of the embodiments described herein and isolating said antibody or the antigen-binding fragment thereof.

[0069] In another general aspect, the disclosure provides a kit comprising (i) the antibody or the antigen-binding fragment thereof, according to any of the embodiments described herein and / or the polynucleotide according to any of the embodiments described herein, and / or the vector according to any of the embodiments described herein, and / or the pharmaceutical composition of any of the embodiments described herein, and / or the host cell according to any of the embodiments described herein, and (ii) packaging for the same and / or instructions for use.

[0070] In another general aspect, the disclosure provides a bispecific antibody or a bispecific antigen-binding fragment thereof, that specifically binds (i) TRBV19 with a first antigen-binding site and (ii) GAIN domain and / or GPS motif of EMR2 with a second antigen-binding site.

[0071] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment comprises: a) a first heavy chain (HC1); b) optionally a first light chain (LC1); c) a second heavy chain (HC2); and d) optionally a second light chain (LC2), wherein (i) the HC1 and the LC1 form a first antigen-binding site that specifically binds a first antigen, (ii) the HC2 and the LC2 form a second antigen-binding site that specifically binds a second antigen, (iii) the HC1 andHC2 each comprise a Fc domain comprising a CH2-CH3 domain; and wherein the first antigen is TRBV19, and the second antigen is EMR2.

[0072] In some embodiments, the second antigen-binding site specifically binds the GAIN domain and / or GPS motif of EMR2.

[0073] In some embodiments, the GAIN domain comprises amino acid residues D261-Q478 of human EMR2 (SEQ ID NO: 205).

[0074] In some embodiments, the GAIN domain comprises the amino acid sequence of SEQ ID NO: 215.

[0075] In any of the foregoing embodiments, the GPS motif comprises the amino acid sequence SEQ ID NO: 216.

[0076] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, binds to an epitope comprising SEQ ID NO: 217 or SEQ ID NO: 218.

[0077] In any of the foregoing embodiments, the first antigen-binding site comprises a Fab or a scFv or a spFv.

[0078] In any of the foregoing embodiments, the Fc of the first antigen-binding site comprises a CHI domain.

[0079] In any of the foregoing embodiments, the second antigen-binding site comprises a Fab or a scFv or a spFv.

[0080] In any of the foregoing embodiments, the Fc of the second antigen-binding site further comprises a CHI domain.

[0081] In any of the foregoing embodiments, the first antigen-binding site comprises a Fab and the second antigen-binding site comprises a scFv or a spFv.

[0082] In any of the foregoing embodiments, the first antigen-binding site comprises a scFv or a spFv and the second antigen-binding site comprises a Fab.

[0083] In any of the foregoing embodiments, the Fc domain of the antibody or the antigenbinding fragment thereof, is an IgA, an IgG, an IgE, or an IgM.

[0084] In some embodiments, the Fc domain of the antibody or the antigen-binding fragment thereof, is an IgG. In some embodiments, the IgG is IgGl or IgG4.

[0085] In any of the foregoing embodiments, the Fc domains of HC1 and HC2 comprise one or more different mutations which promote heterodimerization.

[0086] In any of the foregoing embodiments, the Fc domain of the HC1 comprises mutations T366S, L368A and Y407V (EU numbering) and the Fc domain of the HC2 comprises mutation T366W (EU numbering). In any of the foregoing embodiments, the Fc domain of the HC2 comprises mutations T366S, L368A and Y407V (EU numbering) and the Fc domain of the HC1 comprises mutation T366W (EU numbering).

[0087] In any of the foregoing embodiments, the Fc domains of HC1 and / or HC2 further comprise one or more mutations which reduce Fc binding to a Fey receptor. In any of the foregoing embodiments, the Fey receptor is FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, and / or FcyRIIIB. In any of the foregoing embodiments, the Fc domains of HC1 and / or HC2 each comprise one or more mutations selected from L234A, L235A, and D265S (EU numbering). In any of the foregoing embodiments, the Fc domains of HC1 and HC2 each comprise mutations L234A, L235A, and D265S (EU numbering).

[0088] In any of the foregoing embodiments, the Fc domains of HC1 or HC2 further comprises one or more mutations which reduce Fc binding to protein A. In any of the foregoing embodiments, the Fc domains of HC1 or HC2 comprise mutations H435R and / or Y436F (EU numbering). In any of the foregoing embodiments, the Fc domain of HC1 comprises mutations H435R and Y436F (EU numbering). In any of the foregoing embodiments, the Fc domain of HC2 comprises mutations H435R and Y436F (EU numbering).

[0089] In any of the foregoing embodiments, the HC1 or HC2 comprise mutation C220S (EU numbering). In any of the foregoing embodiments, the HC1 comprises mutation C220S (EU numbering). In any of the foregoing embodiments, the HC2 comprises mutation C220S (EU numbering).

[0090] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, is a humanized antibody or an antigen binding fragment thereof, a human antibody or an antigen binding fragment thereof, a murine antibody or an antigen binding fragment thereof, a chimeric antibody or an antigen binding fragment thereof, or a chemically modified derivative thereof.

[0091] In any of the foregoing embodiments, the first antigen-binding site that specifically binds TRBV19 comprises: a) a HCDR1, a HCDR2 and a HCDR3 of the VH of SEQ ID NO: 31 and a LCDR1, a LCDR2 and a LCDR3 of the VL of SEQ ID NO: 32; or b) a HCDR1, a HCDR2, and aHCDR3 of the VH of SEQ ID NO: 159 and a LCDR1 , LCDR2, and LCDR3 of the VL of SEQ ID NO: 160.

[0092] In any of the foregoing embodiments, the first antigen-binding site that specifically binds TRBV19 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of: a) SEQ ID NOs: 1, 2, 3, 16, 17, and 18, respectively; b) SEQ ID NOs: 4, 5, 6, 19, 20, and 21, respectively; c) SEQ ID NOs: 7, 8, 9, 22, 23, and 24, respectively; d) SEQ ID NOs: 10, 11, 12, 25, amino acid sequence KVS, and SEQ ID NO: 27, respectively; e) SEQ ID NOs: 13, 14, 15, 28, 29, and 30, respectively,; f) SEQ ID NOs: 129, 130, 131, 144, 145, and 146, respectively,; g) SEQ ID NOs: 132, 133, 134, 147, 148, and 149, respectively,; h) SEQ ID NOs: 135, 136, 137, 150, 151, and 152, respectively,; i) SEQ ID NOs: 138, 139, 140, 153, 154, and 155, respectively, for IMGT numbering; or j) SEQ ID NOs: 141, 142, 143, 156, 157, and 158, respectively.

[0093] In any of the foregoing embodiments, the first antigen-binding site that specifically binds TRBV19 comprises a VH comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: the VH of SEQ ID NO: 31 ; or the VH of SEQ ID NO: 159.

[0094] In any of the foregoing embodiments, the first antigen-binding site that specifically binds TRBV19 comprises or further comprises a VL comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: the VL of SEQ ID NO: 32; or the VL of SEQ ID NO: 160.

[0095] In any of the foregoing embodiments, the first antigen-binding site that specifically binds TRBV19 comprises: the VH of SEQ ID NO: 31 and the VL of SEQ ID NO: 32; or the VH of SEQ ID NO: 159 and the VL of SEQ ID NO: 160.

[0096] In any of the foregoing embodiments, the second antigen-binding site that specifically binds EMR2 comprises: a) a HCDR1, a HCDR2 and a HCDR3 of the VH of SEQ ID NO: 63, and a LCDR1, a LCDR2 and a LCDR3 of the VL of SEQ ID NO: 64; b) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 95, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 96; c) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 127, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 128; or d) a HCDR1, a HCDR2,and a HCDR3 of the VH of SEQ ID NO: 191, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 192.

[0097] In any of the foregoing embodiments, the second antigen-binding site that specifically binds EMR2 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of: a) SEQ ID NO: 33, 34, 35, 48, 49, and 50, respectively; b) SEQ ID NO: 36, 37, 38, 51, 52, and 53, respectively; c) SEQ ID NO: 39, 40, 41, 54, 55, and 56, respectively; d) SEQ ID NO: 42, 43, 44, 57, amino acid sequence EVS, and SEQ ID NO: 59, respectively; e) SEQ ID NO: 45, 46, 47, 60, 61, and 62, respectively; f) SEQ ID NO: 65, 66, 67, 80, 81, and 82, respectively; g) SEQ ID NO: 68, 69, 70, 83, 84, and 85, respectively; h) SEQ ID NO: 71, 72, 73, 86, 87, and 88, respectively; i) SEQ ID NO: 74, 75, 76, 89, amino acid sequence AAI, and SEQ ID NO: 91, respectively; j) SEQ ID NO: 77, 78, 79, 92, 93, and 94, respectively; k) SEQ ID NO: 97, 98, 99, 112, 113, and 114, respectively; 1) SEQ ID NO: 100, 101, 102, 115, 116, and 117, respectively; m) SEQ ID NO: 103, 104, 105, 118, 119, and 120, respectively; n) SEQ ID NO: 106, 107, 108, 121, amino acid sequence DNN, and SEQ ID NO: 123, respectively; o) SEQ ID NO: 109, 110, 111, 124, 125, and 126, respectively; p) SEQ ID NO: 161, 162, 163, 176, 177, and 178, respectively; q) SEQ ID NO: 164, 165, 166, 179, 180, and 181, respectively; r) SEQ ID NO: 167, 168, 169, 182, 18, and 184, respectively; s) SEQ ID NO: 170, 171, 172, 185, ammo acid sequence EVS, and SEQ ID NO:187, respectively; or t) SEQ ID NO: 173, 174, 175, 188, 189, and 190, respectively.

[0098] In any of the foregoing embodiments, the bispecific antibody or the bispecific antigenbinding fragment thereof, comprises a VH comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 63; b) the VH of SEQ ID NO: 95; c) the VH of SEQ ID NO: 127; or d) the VH of SEQ ID NO: 191.

[0099] In any of the foregoing embodiments, the bispecific antibody or the bispecific antigenbinding fragment thereof, comprises or further comprises a VL comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 64; b) the VL of SEQ ID NO: 96; c) the VL of SEQ ID NO: 128; or d) the VL of SEQ ID NO: 192.

[0100] In any of the foregoing embodiments, the first antigen-binding site and / or the second antigen-binding site that specifically binds EMR2 comprises: a) the VH of SEQ ID NO: 63 and the VL of SEQ ID NO: 64; b) the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 96; c) the VH of SEQ ID NO: 127 and the VL of SEQ ID NO: 128; or d) the VH of SEQ ID NO: 191 and the VL of SEQ ID NO: 192.

[0101] In any of the foregoing embodiments, the bispecific antibody or the bispecific antigenbinding fragment thereof, comprises a single-chain variable fragment (scFv) or a spFV which scFv or spFV comprises, from the N- to C-terminus, a VH, a linker (L) and a VL in the format VH-L-VL or a VL, a linker and a VH in the format VL-L-VH.

[0102] In any of the foregoing embodiments, the bispecific antibody or the bispecific antigenbinding fragment thereof, comprises the VH comprising the amino acid sequence of SEQ ID NO: 159, the VL comprises the amino acid sequence of SEQ ID NO: 160, and the L comprises SEQ ID NO: 221.

[0103] In any of the foregoing embodiments, the bispecific antibody or the bispecific antigenbinding fragment thereof, comprises the VH comprising the amino acid sequence of SEQ ID NO: 191, the VL comprises the amino acid sequence of SEQ ID NO: 192, and the L comprises SEQ ID NO: 221.

[0104] In any of the foregoing embodiments, the bispecific antibody or the bispecific antigenbinding fragment thereof, comprises the L comprising SEQ ID NO: 221.

[0105] In any of the foregoing embodiments, the first antigen-binding site specifically binds to TRBV19 with a dissociation constant (KD) that is between about 15 nM to about 200 nM.

[0106] In any of the foregoing embodiments, the first antigen-binding site specifically binds to TRBV19 with an ECso between about 1 nM to about 100 nM.

[0107] In any of the foregoing embodiments, the second antigen-binding site specifically binds to EMR2 with a dissociation constant (KD) that is between about 0.01 nM to about 5 nM.

[0108] In any of the foregoing embodiments, the second antigen-binding site specifically binds to EMR2 with an ECso between about 0.1 nM to about 15 nM.

[0109] In any of the foregoing embodiments, the bispecific antibody or the bispecific antigenbinding fragment thereof, comprises the HC1 comprising the amino acid sequence of SEQ ID NO: 193 or 200.

[0110] In any of the foregoing embodiments, the bispecific antibody or the bispecific antigenbinding fragment thereof, comprises the LC1 comprising the amino acid sequence of SEQ ID NO: 201.

[0111] In any of the foregoing embodiments, the bispecific antibody or the bispecific antigenbinding fragment thereof, comprises a) the HC1 comprising the amino acid sequence of SEQ ID NO: 193; or b) the HC1 comprising the amino acid sequence of SEQ ID NO: 200 and the LC1 comprising the amino acid sequence of SEQ ID NO: 201.

[0112] In any of the foregoing embodiments, the bispecific antibody or the bispecific antigenbinding fragment thereof, comprises the HC2 comprising the amino acid sequence of SEQ ID NO: 194, 196, 198, or 202.

[0113] In any of the foregoing embodiments, the bispecific antibody or the bispecific antigenbinding fragment thereof, comprises the LC2 comprising the amino acid sequence of SEQ ID NO: 195, 197, or 199.

[0114] In any of the foregoing embodiments, the bispecific antibody or the bispecific antigenbinding fragment thereof, comprises a) the HC2 comprising the amino acid sequence of SEQ ID NO: 194 and the LC2 comprising the amino acid sequence of SEQ ID NO: 195; b) the HC2 comprising the amino acid sequence of SEQ ID NO: 196 and the LC1 comprising the amino acid sequence of SEQ ID NO: 197; c) the HC2 comprising the amino acid sequence of SEQ ID NO: 198 and the LC1 comprising the amino acid sequence of SEQ ID NO: 199; or d) the HC2 comprising the amino acid sequence of SEQ ID NO: 202.

[0115] In any of the foregoing embodiments, the bispecific antibody or the bispecific antigenbinding fragment thereof, comprises a) the HC1 comprising the amino acid sequence of SEQ ID NO: 193, the HC2 comprising the amino acid sequence of SEQ ID NO: 194, and the LC2 comprising the amino acid sequence of SEQ ID NO 195; b) the HC1 comprising the amino acid sequence of SEQ ID NO: 193, the HC2 comprising the amino acid sequence of SEQ ID NO: 196, and the LC2 comprising the amino acid sequence of SEQ ID NO: 197; c) the HC1 comprising the amino acid sequence of SEQ ID NO: 193, the HC2 comprising the amino acid sequence of SEQ ID NO: 198, and the LC2 comprising the amino acid sequence of SEQ ID NO: 199; or d) the HC1 comprising the amino acid sequence of SEQ ID NO: 200, the LC1 comprising the amino acid sequence of SEQ ID NO: 201, and the HC2 comprising the amino acid sequence of SEQ ID NO: 202.

[0116] In another general aspect, the disclosure provides a bispecific antibody or a bispecific antigen-binding fragment thereof, that specifically binds (i) TRBV19 with a first antigen-binding site and (ii) the GAIN domain and / or the GPS motif of EMR2 with a second antigen-binding site, wherein the first antigen-binding site that specifically binds TRBV19 comprises a HCDR1, a HCDR2 and a HCDR3 of the VH of SEQ ID NO: SEQ ID NO: 159 and a LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 160 and wherein the second antigen-binding site that specifically binds EMR2 comprises a HCDR1 , a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 95, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 96.

[0117] In any of the foregoing embodiments, wherein the first antigen-binding site that specifically binds TRBV19 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of: SEQ ID NOs: 129, 130, 131, 144, 145, and 146, respectively; SEQ ID NOs: 132, 133, 134, 147, 148, and 149, respectively; SEQ ID NOs: 135, 136, 137, 150, 151, and 152, respectively; SEQ ID NOs: 138, 139, 140, 153, 154, and 155, respectively; or SEQ ID NOs: 141, 142, 143, 156, 157, and 158, respectively; and wherein the second antigen-binding site that specifically binds EMR2 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NO: 65, 66, 67, 80, 81, and 82, respectively; SEQ ID NO: 68, 69, 70, 83, 84, and 85, respectively; SEQ ID NO: 71, 72, 73, 86, 87, and 88, respectively; SEQ ID NO: 74, 75, 76, 89, amino acid sequence AAI, and SEQ ID NO: 91, respectively; or SEQ ID NO: 77, 78, 79, 92, 93, and 94, respectively.

[0118] In any of the foregoing embodiments, the first antigen-binding site that specifically binds TRBV19 comprises the VH of SEQ ID NO: 159 and the VL of SEQ ID NO: 160 and the second antigen-binding site that specifically binds EMR2 comprises the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 96.

[0119] In any of the foregoing embodiments, the bispecific antibody or the bispecific antigenbinding fragment thereof, comprises an HC1 comprising the amino acid sequence of SEQ ID NO: 193, an HC2 comprising the amino acid sequence of SEQ ID NO: 198, and an LC2 comprising the amino acid sequence of SEQ ID NO: 199.

[0120] In another general aspect, the disclosure provides a bispecific antibody or a bispecific antigen-binding fragment thereof, that binds to the same epitope as the bispecific antibody or the bispecific antigen-binding fragment thereof, according to any of the embodiments described herein.

[0121] In another general aspect, the disclosure provides a bispecific antibody or a bispecific antigen-binding fragment thereof, that competes for binding to the same epitope with the bispecific antibody or the bispecific antigen-binding fragment thereof, according to any of the embodiments described herein.

[0122] In another general aspect, the disclosure provides a bispecific antibody, or a bispecific antigen-binding fragment thereof, wherein the bispecific antibody, or the bispecific antigenbinding fragment thereof, comprises an HC1 comprising the amino acid sequence of SEQ ID NO: 193, an HC2 comprising the amino acid sequence of SEQ ID NO: 198, and an LC2 comprising the amino acid sequence of SEQ ID NO: 199.

[0123] In another general aspect, the disclosure provides an isolated polynucleotide encoding the bispecific antibody or the bispecific binding fragment of any of the embodiments described herein.

[0124] In some embodiments, the isolated polynucleotide comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 63; b) the VH of SEQ ID NO: 95; c) the VH of SEQ ID NO: 127; or d) the VH of SEQ ID NO: 191.

[0125] In any of the foregoing embodiments, the isolated polynucleotide comprises or further comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 64; b) the VL of SEQ ID NO: 96; c) the VL of SEQ ID NO: 128; or d) the VL of SEQ ID NO: 192.

[0126] In any of the foregoing embodiments, the isolated polynucleotide comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 31 ; or b) the VH of SEQ ID NO: 159.

[0127] In any of the foregoing embodiments, the isolated polynucleotide comprises or further comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 32; or b) the VL of SEQ ID NO: 160.

[0128] In any of the foregoing embodiments, the isolated polynucleotide comprises a sequence encoding a first antigen-binding site that specifically binds TRBV19, the sequence comprising a nucleotide sequence encoding: a) the amino acid sequence of SEQ ID NO: 31 and / or the amino acid sequence of SEQ ID NO: 32; or b) the amino acid sequence of SEQ ID NO: 159 and / or the amino acid sequence of SEQ ID NO: 160.

[0129] In any of the foregoing embodiments, the isolated polynucleotide comprises a sequence encoding a second antigen-binding site that specifically binds EMR2, the sequence comprising a nucleotide sequence encoding: a) the amino acid sequence of SEQ ID NO: 63 and / or the amino acid sequence of SEQ ID NO: 64; b) the amino acid sequence of SEQ ID NO: 95 and / or the amino acid sequence of SEQ ID NO: 96; c) the amino acid sequence of SEQ ID NO: 127 and / or the amino acid sequence of SEQ ID NO: 128; or d) the amino acid sequence of SEQ ID NO: 191 and / or the amino acid sequence of SEQ ID NO: 192.

[0130] In any of the foregoing embodiments, the isolated polynucleotide comprises a sequence encoding an HC1 comprising the amino acid sequence of SEQ ID NO: 193 or 200.

[0131] In any of the foregoing embodiments, the isolated polynucleotide comprises a sequence encoding a LC1 comprising the amino acid sequence of SEQ ID NO: 201.

[0132] In any of the foregoing embodiments, the isolated polynucleotide comprises a sequence encoding: a) the amino acid sequence of SEQ ID NO: 193; or b) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 201.

[0133] In any of the foregoing embodiments, the isolated polynucleotide comprises a sequence encoding an HC2 comprising the nucleotide sequence of SEQ ID NO: 194, 196, 198, or 202.

[0134] In any of the foregoing embodiments, the isolated polynucleotide comprises a sequence encoding a LC2 comprising the nucleotide sequence of SEQ ID NO: 195, 197, or 199.

[0135] In any of the foregoing embodiments, the isolated polynucleotide comprises a sequence encoding: a) the amino acid sequence of SEQ ID NO: 194 and / or the amino acid sequence of SEQ ID NO: 195; b) the amino acid sequence of SEQ ID NO: 196 and / or the amino acid sequence of SEQ ID NO: 197; c) the amino acid sequence of SEQ ID NO: 198 and / or the amino acid sequence of SEQ ID NO: 199; or d) the amino acid sequence of SEQ ID NO: 202.

[0136] In any of the foregoing embodiments, the isolated polynucleotide comprises a sequence encoding: a) the amino acid sequence of SEQ ID NO: 193, the amino acid sequence of SEQ ID NO: 194 and the amino acid of SEQ ID NO: 195; b) the amino acid sequence of SEQ ID NO: 193, the amino acid sequence of SEQ ID NO: 196 and the amino acid sequence of SEQ ID NO: 197; c) the amino acid sequence of SEQ ID NO: 193, the amino acid sequence of SEQ ID NO: 198 and the amino acid sequence of SEQ ID NO: 199; or d) the amino acid sequence of SEQ ID NO: 200, the amino acid sequence of SEQ ID NO: 201, and the amino acid sequence of SEQ ID NO: 202.

[0137] In another general aspect, the disclosure provides a vector comprising the polynucleotide according to any of the embodiments described herein.

[0138] In some embodiments, the isolated polynucleotide is operably linked to an expression control sequence.

[0139] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpes simplex virus vector, and a poxvirus vector.

[0140] In another general aspect, the disclosure provides a pharmaceutical composition comprising (i) the bispecific antibody or the bispecific antigen-binding fragment thereof, according to any of the foregoing embodiments, or the polynucleotide according to any of the foregoing embodiments, or the vector according to any of the foregoing embodiments, and (ii) a pharmaceutically acceptable carrier or excipient.

[0141] In another general aspect, the disclosure provides a host cell expressing the bispecific antibody or the bispecific antigen-binding fragment thereof, according to any of the embodiments described herein.

[0142] In some embodiments, the cell is a hybridoma.

[0143] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is recombinantly produced.

[0144] In another general aspect, the disclosure provides a host cell comprising the isolated polynucleotide according to any of the embodiments described herein or the vector according to any of the embodiments described herein.

[0145] In another general aspect, the disclosure provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeuticallyeffective amount of the bispecific antibody or the bispecific antigen-binding fragment thereof, according to any of the embodiments described herein, or the polynucleotide according to any of the embodiments described herein, or the vector according to any of the foregoing embodiments, or the pharmaceutical composition according to any of the embodiments described herein.

[0146] In another general aspect, the disclosure provides a method for inducing cytotoxicity of a cancer cell or redirecting immune or T cells to a cancer cell, said method comprising administering to the cell an effective amount of the bispecific antibody or the bispecific antigenbinding fragment thereof, according to any of the embodiments described herein, or the isolated polynucleotide according to any of the embodiments described herein, or the vector according to any of the embodiments described herein, or the pharmaceutical composition according to any of the embodiments described herein, or the host cell according to any of the embodiments described herein, wherein the effective amount is sufficient to inhibit the growth or proliferation of the cancer cell.

[0147] In some embodiments, the cancer cell is in a subject and the bispecific antibody or the bispecific antigen-binding fragment thereof, the polynucleotide, the vector, the pharmaceutical composition, or the host cell is administered to the subject.

[0148] In some embodiments, the administration is conducted ex vivo.

[0149] In another general aspect, the disclosure provides a method of redirecting a T cell to EMR2-expressing cancer cells in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the bispecific antibody or the bispecific antigen-binding fragment thereof, according to any of the embodiments described herein, or the polynucleotide according to any of the embodiments described herein, or the vector according to any of the embodiments described herein, or the pharmaceutical composition according to any of the embodiments described herein, or the host cell according to any of the embodiments described herein.

[0150] In some embodiments, the therapeutically effective amount is sufficient to direct said T cell response to the cancer cells.

[0151] In any of the foregoing embodiments, the cancer is an EMR2-expressing cancer. In some embodiments, the EMR2-expressing cancer is a hematological cancer. In some embodiments, the hematological cancer is a myeloid malignancy. In some embodiments, the cancer is AML, chronic myelogenous leukemia (CML), or MDS.

[0152] In any of the foregoing embodiments, the method further comprises administering a second therapeutic agent.

[0153] In some embodiments, the second therapeutic agent is a surgery, a chemotherapy, an androgen deprivation therapy, or a radiation, or any combination thereof.

[0154] In another general aspect, the disclosure provides a bispecific antibody or a bispecific antigen-binding fragment thereof, according to any of the embodiments described herein for use in the method according to any of the embodiments described herein.

[0155] In another general aspect, the disclosure provides an isolated polynucleotide according to any of the embodiments described herein for use in the method according to any of the embodiments described herein.

[0156] In another general aspect, the disclosure provides a vector according to any of the embodiments described herein for use in the method according to any of the embodiments described herein.

[0157] In another general aspect, the disclosure provides a pharmaceutical composition according to any of the embodiments described herein for use in the method according to any of the embodiments described herein.

[0158] In another general aspect, the disclosure provides a host cell according to any of the embodiments described herein for use in the method according to any of the embodiments described herein.

[0159] In another general aspect, the disclosure provides a method for generating the bispecific antibody or the bispecific antigen-binding fragment thereof, according to any of the embodiments described herein, wherein said method comprises culturing the host cell according to any of the embodiments described herein, and isolating the bispecific antibody or the bispecific binding fragment.

[0160] In another general aspect, the disclosure provides a kit comprising (i) the bispecific antibody or the bispecific antigen-binding fragment thereof, according to any of the embodiments described herein, or the isolated polynucleotide according to any of the embodiments described herein, or the vector according to any of the embodiments described herein, or the pharmaceutical composition according to any of the embodiments described herein, or the host cell according to any of the embodiments described herein, and (ii) packaging for the same and / or instructions for use.BRIEF DESCRIPTION OF THE DRAWINGS

[0161] FIG. 1. Frequency of TRBV19+T cells. AML, acute myeloid leukemia; CD, cluster of differentiation; HD, healthy donor; PBMC, peripheral blood mononuclear cells. Healthy-donor PBMCs (n=20) and AML PBMCs (n=16) were commercially purchased and phenotyped for the frequency of TRBV19+ T cells. Data shown as population percentage from total CD3 -expressing cells.

[0162] FIG. 2. Depiction of EMR2 Structure. EMR2 receptor features a 7-pass transmembrane domain (shown in a box), a GAIN domain, and 5 EGF-like domains (labeled as El to E5). EGF, epidermal growth factor; EMR2, GAIN.

[0163] FIG. 3. EMR2 expression in hematological malignancies (mRNA levels). The InforMe database was used to quantitate the relative mRNA levels of EMR2 across hematological malignancies using the GeneLogic Heme Plus 2.0 data set. AML, acute myeloid leukemia; CML, chronic myelogenous leukemia; DLBCL, diffused large B-cell lymphoma; EMR2, epidermal-growth-factor-like module containing mucin-like hormone receptor 2; FL, follicular lymphoma; MCL, mantle cell lymphoma; MDS, myelodysplastic syndrome (also known as myelodysplastic neoplasms); MGUS, monoclonal gammopathy of undetermined significance; MM, multiple myeloma.

[0164] FIG. 4. EMR2 expression on healthy hematopoietic cells and primary AML blasts. Heat maps showing EMR2 expression percentage as assessed by flow cytometry on healthy- donor CD34+ HSCs and progenitor cells (n=8), healthy-donor PBMCs (n=8), healthy-donor peripheral blood (n=2), and AML patient- derived blast detected in the BM (n=14) or PBMCs (n=9). Expression is indicated as EMR2 percentage within each population that was identified based on its respective specific surface markers. For each population receptor density was calculated and depicted. AML, acute myeloid leukemia; BM, bone marrow; CD, cluster of differentiation; CMP, common myeloid progenitor; EMR2, GMP, granulocyte-monocyte progenitor; HD, healthy donor; HSC, hematopoietic stem cell; MEP, megakaryocyte erythrocyte progenitor; MPP, multi-potent progenitor; PB, peripheral blood; PBMC

[0165] FIG. 5. Concentration of soluble EMR2 in HD and AML patient sera. Soluble EMR2 levels in HD and AML patient serum via ELISA showed no significant difference between populations. There was a median soluble EMR2 concentration of 5.00 pM in HD serum and 4.03pM in AML patient serum. AML, ELISA: enzyme-linked immunosorbent assay; EMR2, HD, healthy donors; NS, no significant difference.

[0166] FIG. 6. Bispecific antibody 1-hour, 37°C binding on AML cell lines. Binding of a bispecific or EMR2xNull control or TRBV 19xNull control were incubated with tumor cells for 1 hour at 37°C at a starting concentration of 2,000 nM, diluted serially 1 :3 for 11 points. After secondary detection with AF647-labeled material, the MFI signal was analyzed to generate an EC50 value. Curves are representative of 3 independent experiments. AML, acute myeloid leukemia; ECso, 50% effective concentration; EMR2, MFI, mean fluorescence intensity; V 17, variable domain of T-cell receptor beta chain 17, also referred to as TRBV19.

[0167] FIG. 7. 1-, 3-, 5-, and 24-hour, 37°C kinetic binding of the bispecific antibody and TRBV19xNull on 0CI-AML3 cells. A bispecific antibody or TRBV19xNull control was incubated with OCI- AML-3 cells for 1, 3, 5, and 24 hours at 37°C at concentrations of 3, 30, and 300 nM. After secondary detection with AF647-labeled material, the MFI signal was analyzed to show stable binding of the molecule at various concentrations over time.

[0168] FIG. 8. Bispecific antibody 1-hour, 37°C binding on TRBV19+ T cells. Bispecific antibody or EMR2xNull control were incubated with pan-T cells from 6 healthy donors for 1 hour at 37°C at a starting concentration of 2,000 nM. After secondary detection, the percent TRBV19+ (AF647+) of live cells was analyzed to generate an EC50 value. ECso, 50% effective concentration.

[0169] FIGs. 9A-9B. A representative data set for the EMR2xTRBV19 cytotoxicity assays using 0CI-AML3 AML cell line: (FIG. 9A) T-cell cytotoxicity assay was performed with increasing concentrations of antibody for 72 hours at E:T ratio of 10: 1. (FIG. 9B) PBMC cytotoxicity assay was performed with increasing concentrations of antibody for 96 hours at E:T ratio of 5 : 1.

[0170] FIGs. 10A-10C. Exemplary bispecific amino acid sequences. CDRs using AbM definition shown in bold. Linker connecting TRBV 19 (TRBV 19) VL and VL are underlined. Mature amino acid sequence of an exemplary Heavy Chain 1 (FIG. 10A). Mature amino acid sequence of an exemplary Light Chain 2 (FIG. 10B). Mature amino acid sequence of an exemplary Heavy Chain 2 (FIG. 10C).

[0171] FIG. 11. T-cell-mediated cytotoxicity. Cancer cell cytotoxicity was assessed in a flowcytometry-based assay at 3 and 6 days with pan-T cells as effector cells (n=4-5 different healthydonors) cocultured with EMR2+ (i.e., 0CI-AML3, 0CI-AML5, 0CI-AML2) or EMR2- (i.e., OCI-LYIO, n=3) AML cell lines. Absolute E:T ratio shown is 10:1 (relative E:T ratio 0.5: 1). EMR2, E:T ratio, effector-to-target ratio

[0172] FIGS. 12A-12B. The bispecific antibody induced T-cell activation and expansion in the presence of different cancer cell lines. (FIG. 12A) T-cell activation and (FIG. 12 B) T-cell expansion were assessed by flow cytometry at Day 3 and Day 6 upon coculture with a panel of antigen-positive or -negative cancer cell lines at an absolute E:T ratio of 10: 1 (relative E:T ratio of 0.5: 1). T-cell activation was determined through the expression (%) of the late T-cell activation marker CD25. T-cell expansion was assessed by T-cell absolute counts, normalized to the untreated wells. Data from 4 to 5 different healthy T-cell donors were pooled and represented as mean ± SEM. CD, cluster of differentiation; EMR2, E:T ratio, effector-to-target ratio; SEM, standard error of the mean;.

[0173] FIGs. 13A-13C. The bispecific antibody induced selective TRB VI 9+T-cell activation in the presence of different cancer cell lines. T-cell activation was assessed by flow cytometry at Day 3 and Day 6 upon coculture with a panel of antigen-positive or -negative cancer cell lines at an absolute E:T ratio of l0: l (relative E:T ratio of 0.5:l). (FIG. 13 A) TRBV19+T-cell expansion was assessed by measuring the percentage of TRBV19+T cells. T-cell activation was determined for both (FIG. 13B) TRBV19+and (FIG. 13C) TRBV19- T-cell subpopulations. The expression (%) of the late T-cell activation marker CD25 was used to identify the activated T cells. Data from 4 to 5 different healthy T-cell donors for EMR2+cell lines and 3 healthy donors for EMR2- cell lines were pooled and represented as mean ± SEM. CD, cluster of differentiation; E:T ratio, effector-to-target ratio; SEM, standard error of the mean.

[0174] FIGs. 14A-14D. Intracellular cytokine production upon stimulation with the bispecific antibody. Characterization of TRBV19+T-cell profile upon the bispecific antibody treatment was assessed in a flow-cytometry-based assay. 0CI-AML3 cells and pan-T cells (n=6 different healthy donors) were cocultured at an absolute E:T ratio of 10: 1 (relative E:T ratio for TRBV19+T cells of 0.5:1) for 48 hours. (FIG. 14A) Efficacy was evaluated by determining cytotoxicity to cancer cells and granzyme B expression on pan-T cells and TRBV19+T cells. Profiling of cytokine expression was determined for (FIG. 14B) IFN-y, (FIG. 14C) IL-2, and (FIG. 14D) TNF-a by assessing the expression of the respective cytokine on pan-T cells and TRBV19+T cells. The intensity of cytokine expression is indicated as signal MFI and normalized to untreatedconditions. CD, cluster of differentiation; E:T ratio, effector-to-target ratio; IFN, interferon; IL, interleukin; MFI, mean fluorescence intensity; TNF, tumor necrosis factor.

[0175] FIGs. 15A-15D. Intracellular cytokine production upon stimulation with the bispecific antibody compared to a control comparator antibody A. Characterization of TRBV19+T-cell profile upon bispecific antibody or control comparator antibody A treatments was assessed in a flow- cytometry-based assay. 0CI-AML3 cells and pan-T cells (n=6 different healthy donors) were cocultured at a relative E:T ratio for TRBV19+T cells of 0.5: 1 for 48 hours. (FIG. 15 A) T - cell-mediated cytotoxicity was evaluated by determining granzyme B expression on pan-T cells and TRBV19+T cells. Profiling of cytokine expression was determined for (FIG. 15B) IFN-y, (FIG. 15C) IL-2, and (FIG. 15D) TNF-a by assessing the expression of the respective cytokine on pan-T cells and TRBV19+T cells. The intensity of cytokine expression is indicated as signal MFI in the positive population and normalized to untreated conditions. CD, cluster of differentiation; EMR2, E:T ratio, effector-to-target ratio; Gzmb, granzyme B; IFN, interferon; IL, interleukin; MFI, mean fluorescence intensity; TNF, tumor necrosis factor;

[0176] FIGs. 16A-16F. Therapeutic efficacy (in vitro cytotoxicity assay) of the bispecific antibody in a time-course PBMC-based cytotoxicity assay. (FIG. 16A) Cancer cell cytotoxicity of the bispecific antibody was assessed by flow cytometry at 24, 48, 72, 96, and 120 hours. PBMCs were cocultured with 0CI-AML3 cells at an E:T ratio of 5:1 with total PBMCs as effector cells (relative E: T ratio of ~0.19: 1 for the bispecific antibody and -3.3: 1 for the control comparator antibody A). Cancer cell cytotoxicity was calculated by normalizing the absolute target cell count in the treated conditions to the counts of the untreated wells. T-cell activation was assessed by evaluating the expression of CD25 on either (FIG. 16B) total CD3 T cells or (FIG. 16C) TRBV19+T cells. (FIGS. 16D-16F) NullxTRBV19 control was assessed in parallel. Graphing of data was done in GraphPad Prism 10. Data from 5 healthy donors were pooled and represented as mean ± SEM. CD, cluster of differentiation; E:T ratio, effector-to-target ratio; PBMC, peripheral blood mononuclear cell; SEM, standard error of the mean.

[0177] FIGs. 17A-17C. Cytotoxicity of control comparator antibody A in PBMC assays. As a comparison for assessing the cancer cell cytotoxicity of the bispecific antibody, a control comparator antibody A was used in parallel. Readout of the flow-cytometry-based assay was performed at 24, 48, 72, 96, and 120 hours with 0CI-AML3 as target cancer cells and healthy PBMCs as effector cells (n=5 donors) at an E:T ratio of 5: 1. (FIG. 17A) Cytotoxicity wascalculated by normalizing the absolute target cell count in the treated conditions to the counts of the untreated wells. T-cell activation was assessed by evaluating the expression of CD25 on either (FIG. 17B) total CD3 T cells or (FIG. 17C) TRBV19+T cells. CD, cluster of differentiation; E:T ratio, effector-to-target ratio; LH, light-heavy; PBMC, peripheral blood mononuclear cell; spFv, single-chain fragment variable featuring a ‘stapled’ linker.

[0178] FIG. 18. Time-course evaluation of cytokine production upon bispecific antibody treatment in a PBMC-based cytotoxicity assay. Supernatant were collected at the different time points and analyzed for inflammatory cytokines using MSD Proinflammatory kit. Graphing of data was done in GraphPad Prism 10. Data from 5 different donors were pooled and represented as mean ± SEM. IFN, interferon; IL, interleukin; MSD, Meso Scale Discovery; PBMC, peripheral blood mononuclear cell; SEM, standard error of the mean; TNF, tumor necrosis factor.

[0179] FIG. 19. Time-course evaluation of cytokine production upon control comparator antibody A treatment in a PBMC-based cytotoxicity assay. Supernatant were collected at the different time points and analyzed for inflammatory cytokines using MSD Proinflammatory kit. Graphing of data was done in GraphPad Prism 10. Data from 5 different donors were pooled and represented as mean ± SEM. CD, cluster of differentiation; EMR2, IFN, interferon; IL, interleukin; MSD, Meso Scale Discovery; PBMC, peripheral blood mononuclear cell; SEM, standard error of the mean; TNF, tumor necrosis factor.

[0180] FIGs. 20A-20D. Bispecific antibody cytotoxicity assays on primary AML BM cells. (FIG. 20 A) Cytotoxicity of primary AML BM cells was evaluated in a flow-cytometry-based assay for 24 hours at a relative E:T ratio of 2: 1. Two different T-cell donors were used with different AML BM donors (n=3). (FIG. 20B) Pan-T-cell activation, (FIG. 20C) specific TRBV19+T-cell activation, and (FIG. 20D) TRBV19- T-cell activation were evaluated by measuring the expression of the activation markers CD69 and / or CD25. AML, acute myeloid leukemia; BM, bone marrow; CD, cluster of differentiation; DN, donor; E:T ratio, effector-to- target ratio. FIGs. 20A-20D. Bispecific antibody cytotoxicity assays on primary AML BM cells. (FIG. 20 A) Cytotoxicity of primary AML BM cells was evaluated in a flow-cytometry-based assay for 24 hours at a relative E:T ratio of 2: 1. Two different T-cell donors were used with different AML BM donors (n=3). (FIG. 20B) Pan-T-cell activation, (FIG. 20C) specific TRBV19+T-cell activation, and (FIG. 20D) TRBV19- T-cell activation were evaluated bymeasuring the expression of the activation markers CD69 and / or CD25. AML, acute myeloid leukemia; BM, bone marrow; CD, cluster of differentiation; DN, donor; E:T ratio, effector-to- target ratio.

[0181] FIGs. 21A-21B. Bispecific antibody on-target / off-tumor assessment on primary HSPC and myeloid cells. (FIG. 21 A) CFU assays were performed to evaluate on-target / off-tumor toxicity of the bispecific antibody on healthy-donor CD34+HSPC. Healthy-donor pan-T cells were used as effector cells at a relative E:T ratio of 1 : 1 with CD34+cells (n=5) or 0CI-AML3 cells (n=5) as target cells. After 48 hours of preincubation with a dose range of the bispecific antibody, cells were seeded in semisolid methylcellulose medium for 14 days to support colony formation. The viability of either CD34+cells or 0CI-AML3 cells was assessed by enumerating the number of colonies formed compared to the untreated controls. As shown in the bar graph, the bars alternate CD34+ HSPC cells followed by 0CI-AML3 cells from left to right. (FIG. 21B) Viability of monocytes upon the bispecific antibody treatment was assessed by flow cytometry at 72 hours. PBMCs were cocultured with 0CI-AML3 cells at an E:T ratio of 5: 1 with total PBMCs as effector cells. Monocyte cytotoxicity was calculated by normalizing the absolute target cell count in the treated conditions to the counts of the untreated wells. CD, cluster of differentiation; CFU, colony forming unit; E:T, effector to target; HSCP, hematopoietic stem and progenitor cells; PBMC, peripheral blood mononuclear cell.

[0182] FIG. 22. On-target / off-tumor assessment of control comparator antibody A. Viability of monocytes upon control comparator antibody treatment A was assessed by flow cytometry at 72 hours. PBMCs were cocultured with 0CI-AML3 cells at an E:T ratio of 5: 1 with total PBMCs as effector cells (n=5). Monocyte cytotoxicity was calculated by normalizing the absolute target cell count in the treated conditions to the counts of the untreated wells. CD, cluster of differentiation; EMR2, E:T, effector to target; PBMC, peripheral blood mononuclear cell.

[0183] FIGs. 23A-23D. Effect of the bispecific antibody on M0LM-13-luc disseminated human AML progression in T-cell- humanized mice. Tumor cells were implanted on Day 0. T cells were implanted on Day 3, as indicated by an asterisk. Mice (n=10 / group) bearing established M0LM-13-luc disseminated AML were IP dosed the bispecific antibody or control DPBS as indicated by triangles and an arrow pointing out the triangles on Days 4, 7, 10, 13, 17, 20, 24, 27, 31, and 34. (FIG. 23 A) Logarithmic scale and (FIG. 23 B) linear scale group BLI isgraphed as average radiance mean expressed in p / s / cm2 / sr ± SEM when at least 7 of 10 animals remained in each group. * Denotes statistically significant difference of the bispecific antibody - treated groups over time versus DPBS-treated control group. (FIG. 23C) Percent survival. * Denotes statistically significant difference in survival curves of the bispecific antibody-treated groups versus DPBS-treated control group. (FIG. 23D) Individual BLI plots with number of CRs in each group. Ab, antibody; AML, acute myeloid leukemia; BLI, bioluminescent imaging; CR, complete response; DPBS, Dulbecco’s phosphate-buffered saline; IP, intraperitoneal; luc, luciferase; M, million; p, photons; SEM, standard error of the mean; sr, steradian.

[0184] FIG. 24. Effect of the bispecific antibody on M0LM-13-luc disseminated human AML progression in T-cell- humanized mice; representative ventral BLI images. Representative ventral BLI images from 5 mice per group on the indicated study days. Tumor cells were implanted on Day 0. Mice bearing established M0LM-13-luc disseminated AML were IP dosed with the bispecific antibody at the indicated doses. AML, acute myeloid leukemia; BLI, bioluminescent imaging; DPBS, Dulbecco’s phosphate-buffered saline; luc, luciferase; M, million; p, photons; sr, steradian.

[0185] FIGs. 25A-25B. In vivo efficacy of control comparator antibody A on OCI-AML3-luc disseminated human AML progression in T-cell-humanized mice. Tumor cells were implanted on Day 0 and T cells on Day 6, as indicated by an asterisk. Mice (n=10 / group) bearing established OCI-AML3-luc disseminated AML were IP dosed with control comparator antibody A (1 mg / kg) or control DPBS as indicated by triangles on Days 7, 11, 14, 18, 21, 25, 28, 32, 36, 40, 43, 47, 50, and 54. (FIG. 25 A) Group BLI is graphed as average radiance mean expressed in p / s / cm2 / sr ± SEM when at least 7 of 10 animals remained in each group. * Denotes statistically significant difference of control comparator antibody A-treated groups on Day 33 versus DPBS- treated control group (100% ATGI). (FIG. 25B) Percent survival. * Denotes statistically significant difference in survival curves of control comparator antibody A-treated groups versus DPBS-treated control group (>65% ILS).

[0186] FIGs. 26A-26C. Effect of the bispecific antibody on OCI-AML3-luc disseminated human AML progression in T-cell- humanized mice. Tumor cells were implanted on Day 0 and T cells on Day 6, as indicated by an asterisk. Mice (n=10 / group) bearing established OCI- AML3-luc disseminated AML were IP dosed the bispecific antibody or control DPBS asindicated by solid triangles on Days 7, 11, 14, 18, 21, 25, 28, 32, 36, 40, 43, 47, 50 and 54. (FIG. 26A) Logarithmic scale and ( FIG. 26B) linear scale group BLI is graphed as average radiance mean expressed in p / s / cm2 / sr ± SEM when at least 7 of 10 animals remained in each group. * Denotes statistically significant difference of the bispecific antibody -treated groups on Day 33 versus DPBS-treated control group. (FIG. 26C) Percent survival. * Denotes statistically significant difference in survival curves of the bispecific antibody -treated groups versus DPBS- treated control group. Ab, antibody; AML, acute myeloid leukemia; BLI, bioluminescent imaging; DPBS, Dulbecco’s phosphate-buffered saline; IP, intraperitoneal; luc, luciferase; M, million; p, photons; SEM, standard error of the mean; sr, steradian.

[0187] FIG. 27. Effect of the bispecific antibody on OCI-AML3-luc disseminated human AML progression in T-cell- humanized mice; representative dorsal BLI images. Representative dorsal BLI images from 5 mice per group on the indicated study days. Tumor cells were implanted on Day 0. Mice bearing established M0LM-13-luc disseminated AML were IP dosed with the bispecific antibody at the indicated doses. AML, acute myeloid leukemia; BLI, bioluminescent imaging; DPBS, Dulbecco’s phosphate-buffered saline; luc, luciferase; M, million; p, photons; sr, steradian

[0188] FIG. 28. Table of the Biophysical Properties of the bispecific antibodies.

[0189] FIGs. 29A-29C. Binding activity of the bispecific antibodies. Percent Relative Activity of TRBV19 binding (FIG. 29A). Binding to TRBV19+ T cells (FIG. 29B). EMR2 arm binding affinity (FIG 29C).

[0190] FIG. 30. Amino acid sequences of an exemplary bispecific antibody.

[0191] FIG. 31. Amino acid sequences of an exemplary bispecific antibody.

[0192] FIG. 32. Amino acid sequences of an exemplary bispecific antibody.

[0193] FIG. 33. Amino acid sequences of an exemplary bispecific antibody.

[0194] FIG. 34. Amino acid sequences of exemplary Kappa (SEQ ID NO: 222), Lambda(SEQ ID NO: 223), CH (SEQ ID NO: 225) and CHI (SEQ ID NO: 224) regions. Figure discloses the full “huIgGl_Glm(17)” sequence as SEQ ID NO: 227.DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0195] The bispecific molecules of the present disclosure, bind the variable domain of T-cell receptor beta chain 17 (V017; also referred to as TRBV19) expressed on a subtype of T cells andepidermal-growth-factor-like module-containing mucin-like hormone receptor-like 2 (EMR2; also known as adhesion G protein-coupled receptor E2 or ADGRE2) on AML blast cells. The mechanism of action (MoA) of the bispecific molecules of the present disclosure is to selectively engage TRBV19+ T cells and act as a bridge between cancer cells and T cells. TRBV19+ T cells are a subtype of mature and primed viral-specific T lymphocytes with memory phenotype found in approximately 5% of T cells in the peripheral blood of healthy and AML patients. Binding to the TRBV19 receptor on a subset of T cells and the specific antigen on cancer cells (i.e., EMR2) results in TRBV19+ T-cell activation and lysis of cancer cells. This approach differentiates from CD3 T-cell engagers (TCEs) through the recruitment of a selected T-cell subpopulation (i.e., TRBV19+) with the potential to increase the therapeutic index by lowering the risk of severe cytokine release syndrome (CRS).

[0196] Examples of bispecific (EMR2xTRBV19) molecules of the present disclosure include fully human immunoglobulin (Ig)Gl - L234A, L235A, D265S (AAS) bispecific antibodies specifically targeting TRBV19+T cells with one binding arm and EMR2 with the other binding arm. The antibodies feature the AAS mutations in the Fc region (fragment crystallizable [Fc]) to abolish interaction with Fc receptors. The TRB VI 9-engaging arm bound with an affinity of 94 ± 13 nM to recombinant TRBV19 protein by surface plasmon resonance (SPR) and detected TRBV19+T cells with a 50% effective concentration (EC50) of 7.8 nM. The EMR2-binding arm bound EMR2-expressing cells with an affinity range of 27 to 55 nM and showed a stable binding profile over 24 hours. The TRBV19-binding arm is a stabilized single-chain fragment variable featuring a ‘stapled’ linker (spFv), while the EMR2-binding arm is a fragment antigenbinding (Fab).

[0197] The bispecific molecules of the present disclosure exhibited good biophysical properties. In vitro, the bispecific molecules of the present disclosure induced T-cell mediated cytotoxicity to EMR2+AML cancer cell lines. No impact on the viability of tumor-associated antigen (TAA)-negative cells was observed. The bispecific molecules of the present disclosure induced selective activation and expansion of TRBV19+T cells with no / minimal impact on the TRBV19- population.

[0198] The bispecific molecules of the present disclosure showed potent cancer cell cytotoxicity in peripheral blood mononuclear cell (PBMC)-based assays. The bispecific molecules of the present disclosure led to low overall T-cell activation associated with slowerand lower cytokine secretion (i.e., interleukin [IL]- 1 [3, IL-10, and tumor necrosis factor [TNF]-a) than the comparator antibody A.

[0199] The bispecific molecules of the present disclosure induced potent T-cell-mediated cytotoxicity to primary AML bone marrow (BM) cells with minimal overall T-cell activation.

[0200] The bispecific molecules of the present disclosure induced higher cytotoxic activity on AML cell lines than on healthy hematopoietic stem and progenitor cells (HSPCs) or monocytes.

[0201] In vivo, the bispecific molecules of the present disclosure showed robust antitumor efficacy leading to increased survival in M0LM-13-luc and OCI-AML3-luc established models. EMR2 expression was high on macrophage lineages (i.e., monocyte / macrophages / dendritic cells) across various tissues and low on granulocytes (i.e., basophils, eosinophils, and neutrophils).

[0202] In vitro data showed a moderate level of activation of monocytes and neutrophils, and to a lesser extent natural killer (NK) cells. No activation of basophils was observed. No off- target liabilities were observed based on the human cell microarray platform screen (Retrogenix) and an in vitro functional assay on TAA-negative cell lines.Definitions

[0203] Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

[0204] Techniques and procedures described or referenced herein include those that are generally well understood and / or commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3d ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Dtibel eds., 2d ed. 2010).

[0205] In an attempt to help the reader of the present application, the description has been separated in various paragraphs or sections. These separations should not be considered as disconnecting the substance of a paragraph or section from the substance of another paragraph or section. To the contrary, the present description encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated.

[0206] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0207] The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of up to ±10% from the specified value, as such variations are appropriate to perform the disclosed methods. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0208] “Isolated” means a biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins that have been “isolated” thus include nucleic acids and proteins purified by standard purification methods. “Isolated” nucleic acids, peptides and proteins can be part of a composition and still be isolated if such composition is not part of the native environment of the nucleic acid, peptide, orprotein. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. An "isolated” antibody or an antigen-binding fragment, as used herein, is intended to refer to an antibody or an antigenbinding fragment which is substantially free of other antibodies or antigen-binding fragments having different antigenic specificities.

[0209] “Polynucleotide,” synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and doublestranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be singlestranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.

[0210] The meaning of “substantially the same” can differ depending on the context in which the term is used. Because of the natural sequence variation likely to exist among heavy and light chains and the genes encoding them, one would expect to find some level of variation within the amino acid sequences or the genes encoding the antibodies or antigen-binding fragments described herein, with little or no impact on their unique binding properties (e.g., specificity and affinity). Such an expectation is due in part to the degeneracy of the genetic code, as well as to the evolutionary success of conservative amino acid sequence variations, which do not appreciably alter the nature of the encoded protein. Accordingly, in the context of nucleic acid sequences, “substantially the same” means at least 65% identity between two or more sequences. Preferably, the term refers to at least 70% identity between two or more sequences, morepreferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, and more preferably at least 99% or greater identity. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences may e.g. be determined using the algorithm ofE. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988) which has been incorporated into the ALIGN program (version 2.0), using a P AMI 20 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970) algorithm.

[0211] The degree of variation that may occur within the amino acid sequence of a protein without having a substantial effect on protein function is much lower than that of a nucleic acid sequence, since the same degeneracy principles do not apply to amino acid sequences. Accordingly, in the context of an antibody or an antigen-binding fragment, “substantially the same” means antibodies or antigen-binding fragments having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the antibodies or antigen-binding fragments described. Other embodiments include antibodies, or antigen-binding fragments, that have framework, scaffold, or other non-binding regions that do not share significant identity with the antibodies and antigen-binding fragments described herein, but do incorporate one or more CDRs or other sequences needed to confer binding that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences described herein.

[0212] A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations. In some examples provided herein, cells are transformed by transfecting the cells with DNA.

[0213] The terms “express” and “produce” are used synonymously herein, and refer to the biosynthesis of a gene product. These terms encompass the transcription of a gene into RNA. These terms also encompass translation of RNA into one or more polypeptides, and further encompass all naturally occurring post-transcriptional and post-translational modifications. The expression or production of an antibody or an antigen-binding fragment thereof, may be within the cytoplasm of the cell, or into the extracellular milieu such as the growth medium of a cell culture.

[0214] The terms “treating” or “treatment” refer to any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject’s physical or mental well-being, or prolonging the length of survival. The treatment may be assessed by objective or subjective parameters; including the results of a physical examination, neurological examination, or psychiatric evaluations.

[0215] An “effective amount” or “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of an antibody described herein may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or the antibody portion are outweighed by the therapeutically beneficial effects.

[0216] “Antibody” is understood in accordance with its ordinary meaning in the field and encompasses all isotypes of immunoglobulins (IgG, IgA, IgE, IgM, IgD, and IgY) including various monomeric, polymeric and chimeric forms, unless otherwise specified. Specifically encompassed by the term “antibody” are polyclonal antibodies, monoclonal antibodies (mAbs), and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies or human antibodies, as well as multispecific antibodies, bispecific antibodies, single-domain antibodies (sdAb), Immunoglobulin New Antigen Receptor (Ig NARs), single heavy chain antibodies, camelid antibodies, shark antibodies, or chemically modified derivatives thereof. The term “antibody” encompasses the meaning of non-naturally occurring antibody or of an engineeredantibody. “Antigen-binding fragments” are any proteinaceous structure that may exhibit binding affinity for a particular antigen. Antigen-binding fragments include those provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. Some antigen-binding fragments are composed of portions of intact antibodies that retain antigen-binding specificity of the parent antibody molecule. For example, antigen-binding fragments may comprise at least one variable region (either a heavy chain or light chain variable region) or one or more CDRs of an antibody known to bind a particular antigen. Examples of suitable antigen-binding fragments include, without limitation diabodies and single-chain molecules as well as Fab, F(ab’)2, F(ab)'3, Fc, Fabc, and Fv molecules, single chain (Sc) antibodies, single-chain variable fragments (scFv), bis-scFvs, (scFv)2, stapled scFvs (spFv) (see, e.g., Boucher, LE et al., “Stapling” scFv for multispecific biotherapeutics of superior properties, MAbs. 2023; 15(1): 2195517; PCT Int. Publ. No. WO 2023 / 089587; PCT Int. Publ. No. WO 2021 / 030657), individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains or CDRs and other proteins, protein scaffolds, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, a monovalent fragment consisting of the VL, VH, CL and CHI domains, or a monovalent antibody as described in W02007059782, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, a Fd fragment consisting essentially of the VH and CHI domains; a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, a dAb fragment (see, e.g., Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a VH domain and also called domain antibodies (see, e.g., Holt et al; Trends Biotechnol. 2003 Nov.; 21(l l):484-90); camelid or nanobodies (see, e.g., Revets et al; Expert Opin Biol Ther. 2005 Jan.; 5(1): 111-24); an isolated complementarity determining region (CDR), a diabody; a minibody; a triabody; a tetrabody; a disulfide stabilized Fv protein (dsFv); and the like. All antibody isotypes may be used to produce antigen-binding fragments.Additionally, antigen-binding fragments may include non-antibody proteinaceous frameworks that may successfully incorporate polypeptide segments in an orientation that confers affinity for a given antigen of interest, such as protein scaffolds. Antigen-binding fragments may be recombinantly produced or produced by enzymatic or chemical cleavage of intact antibodies. The phrase “an antibody or an antigen-binding fragment thereof’ may be used to denote that agiven antigen-binding fragment incorporates one or more amino acid segments of the antibody referred to in the phrase.

[0217] The terms “CDR”, and its plural “CDRs”, refer to a complementarity determining region (CDR) of which three make up the binding character of a light chain variable region (CDRL1, CDRL2 and CDRL3) and three make up the binding character of a heavy chain variable region (CDRH1, CDRH2 and CDRH3). CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences that comprise scaffolding or framework regions. The exact definitional CDR boundaries and lengths are subject to different classification and numbering systems. CDRs may therefore be referred to by Kabat, Chothia, Contact, IMTG, AbM, or any other boundary definitions. Despite differing boundaries, each of these systems has some degree of overlap in what constitutes the so called “hypervariable regions” within the variable sequences. CDR definitions according to these systems may therefore differ in length and boundary areas with respect to the adjacent framework region. See for example Kabat et al., Sequences of Proteins of Immunological Interest, 5thed. NIH Publication No. 91-3242 (1991); Chothia et al., “Canonical Structures For the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196:901 (1987); and MacCallum et al., “Antibody- Antigen Interactions: Contact Analysis and Binding Site Topography,” J. Mol. Biol. 262:732 (1996)), each of which is hereby incorporated by reference in its entirety.

[0218] Typically, CDRs form a loop structure that can be classified as a canonical structure. The term “canonical structure” refers to the main chain conformation that is adopted by the antigen binding (CDR) loops. From comparative structural studies, it has been found that five of the six antigen binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized by the torsion angles of the polypeptide backbone. Correspondent loops between antibodies may, therefore, have very similar three dimensional structures, despite high amino acid sequence variability in most parts of the loops (Chothia et al., “Canonical Structures For the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196:901 (1987); Chothia et al., “Conformations of Immunoglobulin Hypervariable Regions,” I 342:877 (1989); Martin and Thornton, “Structural Families in Loops of Homologous Proteins: Automatic Classification, Modelling and Application to Antibodies,” J. Mol. Biol. 263:800 (1996), each of which is incorporated by reference in its entirety). Furthermore, there is a relationship between the adopted loop structure and the amino acid sequences surrounding it.The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues residing at key positions within the loop, as well as within the conserved framework (i.e., outside of the loop). Assignment to a particular canonical class can therefore be made based on the presence of these key amino acid residues.

[0219] The term “polypeptide” is used interchangeably with the term “protein” and in its broadest sense refers to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc. As used herein the term “amino acid” refers to either natural and / or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics. A peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.

[0220] “Specifically binds” or “binds specifically” or derivatives thereof when used in the context of antibodies, or antibody fragments, represents binding via domains encoded by immunoglobulin genes or fragments of immunoglobulin genes to one or more epitopes of a protein of interest, without preferentially binding other molecules in a sample containing a mixed population of molecules. Typically, an antibody binds to a cognate antigen with a Ka of less than about 1x1 O'8M, as measured by a surface plasmon resonance assay or a cell-binding assay. Phrases such as “[antigen] -specific” antibody (e.g., EMR2-specific antibody or TRBV19-specific antibody) are meant to convey that the recited antibody specifically binds the recited antigen. As used herein, the term “chimeric” refers to an antibody, or antigen-binding fragment thereof, having at least some portion of at least one variable domain derived from the antibody amino acid sequence of a non-human mammal, a rodent, or a reptile, while the remaining portions of the antibody, or antigen-binding fragment thereof, are derived from a human.

[0221] “Polynucleotide,” synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and doublestranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be singlestranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.

[0222] A “vector” is a replicon, such as plasmid, phage, cosmid, or virus in which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment.

[0223] As used herein, the term “host cell” can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In specific embodiments, the term "host cell" refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, e.g., due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome. The terms “expression” and “production” are used synonymously herein, and refer to the biosynthesis of a gene product. These terms encompass the transcription of a gene into RNA. These terms also encompass translation of RNA into one or more polypeptides, and further encompass all naturally occurring post-transcriptional and post-translational modifications.

[0224] The term “subject” refers to human and non-human animals, including all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. In many embodiments of the described methods, the subject is a human.

[0225] The term “redirect” or “redirecting” as used herein refers to the ability of the described multispecific antibody (e.g., a EMR2xTRBV19 antibody) to traffic the activity of T cells effectively, from its inherent cognate specificity toward reactivity against EMR2 -expressing cells.

[0226] The term “sample” as used herein refers to a collection of similar fluids, cells, or tissues (e.g., surgically resected tumor tissue, biopsies, including fine needle aspiration), isolated from a subject, as well as fluids, cells, or tissues present within a subject. In some embodiments the sample is a biological fluid. Biological fluids are typically liquids at physiological temperatures and may include naturally occurring fluids present in, withdrawn from, expressed or otherwise extracted from a subject or biological source. Certain biological fluids derive from particular tissues, organs or localized regions and certain other biological fluids may be more globally or systemically situated in a subject or biological source. Examples of biological fluids include blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tear drops, feces, sputum, mucosal secretions of the secretory tissues and organs, vaginal secretions, ascites fluids such as those associated with non-solid tumors, fluids of the pleural, pericardial, peritoneal, abdominal and other body cavities, fluids collected by bronchial lavage and the like. Biological fluids may also include liquid solutions contacted with a subject or biological source, for example, cell and organ culture medium including cell or organ conditioned medium, lavage fluids and the like. The term “sample,” as used herein, encompasses materials removed from a subject or materials present in a subject.

[0227] A “EMR2xTRBV19 antibody” is a multispecific antibody, optionally a bispecific antibody, which comprises two different antigen-binding regions, one of which binds specifically to the antigen EMR2 and one of which binds specifically to TRBV19 (also known as V017). The term “multispecific antibody” is used herein in the broadest sense and specifically covers an antibody that has polyepitopic specificity. Multispecific antibodies include, but are not limited to, an antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), where the VHVL unit has polyepitopic specificity, antibodies having two or more VL and VH domains where each VHVL unit binds to a different epitope, antibodies having two or more single variable domains with each single variable domain binding to a different epitope, full length antibodies, and antibodies comprising one or more antibody fragments as well as antibodies comprising antibody fragments that have been linked covalently or non-covalently.

[0228] A multispecific antibody can be a bispecific antibody, diabody, or similar molecule (see for instance PNAS USA 90(14), 6444-8 (1993) for a description of diabodies). The bispecific antibodies, diabodies, and the like, provided herein may bind any suitable target in addition to a portion of epidermal-growth-factor-like module-containing mucin-like hormone receptor 2(EMR2) or T cell receptor (TCR) TRBV19. The term “bispecific antibody” is to be understood as an antibody having two different antigen-binding regions defined by different antibody sequences. This can be understood as different target binding but includes as well as binding to different epitopes in one target.

[0229] A “reference sample” is a sample that may be compared against another sample, such as a test sample, to allow for characterization of the compared sample. The reference sample will have some characterized property that serves as the basis for comparison with the test sample. For instance, a reference sample may be used as a benchmark for EMR2 levels that are indicative of a subject having cancer. The reference sample does not necessarily have to be analyzed in parallel with the test sample, thus in some instances the reference sample may be a numerical value or range previously determined to characterize a given condition, such as EMR2 levels that are indicative of cancer in a subject.

[0230] The term “progression,” as used in the context of progression of EMR2 -expressing cancer, includes the change of a cancer from a less severe to a more severe state. This may include an increase in the number or severity of tumors, the degree of metastasis, the speed with which the cancer is growing or spreading, and the like. For example, “the progression of colon cancer” includes the progression of such a cancer from a less severe to a more severe state, such as the progression from stage I to stage II, from stage II to stage III, etc.

[0231] The term “regression,” as used in the context of regression of EMR2 -expressing cancer, includes the change of a cancer from a more severe to a less severe state. This could include a decrease in the number or severity of tumors, the degree of metastasis, the speed with which the cancer is growing or spreading, and the like. For example, “the regression of colon cancer” includes the regression of such a cancer from a more severe to a less severe state, such as the progression from stage III to stage II, from stage II to stage I, etc.

[0232] The term “stable” as used in the context of stable EMR2 -expressing cancer, is intended to describe a disease condition that is not, or has not, changed significantly enough over a clinically relevant period of time to be considered a progressing cancer or a regressing cancer.

[0233] The embodiments described herein are not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary.Multispecific Antibodies

[0234] Multispecific antibodies that bind to EMR2 and / or TRBV19, and multispecific binding fragments thereof are provided herein. Such antibodies or antibody fragments may allow for more specific targeting to particular subsets of cells as compared to antibodies targeting only one or two of these targets.

[0235] Bispecific antibodies

[0236] In some embodiments, provided herein are bispecific antibodies that bind to EMR2 and TRBV19, and bispecific binding fragments thereof. This can be achieved by, for example, making a molecule which comprises a first region binding specifically to EMR2, and a second binding region binding specifically to TRBV19. The antigen-binding regions can take any form that allows specific recognition of the target, for example the binding region may be or may include a heavy chain variable domain, an Fv (combination of a heavy chain variable domain and a light chain variable domain), an single-chain Fv (scFv), a stapled scFv (“spFv”), an Fab, a binding domain based on a fibronectin type III domain (such as from fibronectin, or based on a consensus of the type III domains from fibronectin, or from tenascin or based on a consensus of the type III domains from tenascin, such as the Centyrin molecules from Janssen Biotech, Inc., see e.g. W02010 / 051274 and W02010 / 093627). Accordingly, bispecific molecules comprising three different antigen-binding regions which bind EMR2 and TRBV 19, respectively, are provided.

[0237] In some embodiments, the EMR2 x TRBV19-multispecific antibody comprises a first heavy chain (HC1) and a light chain (LC) that pair to form a first antigen-binding site that specifically binds a first antigen and a second heavy chain (HC2) comprises a second antigenbinding site that specifically binds a second antigen. The HC1 and HC2 may each comprise a Fragment crystallizable (Fc) domain comprising a CH2-CH3 domain. In some embodiments, the EMR2 x TRBV19-bispecific antibody comprises a EMR2-specific arm comprising a first heavy chain (HC1) and a light chain (LC) that pair to form a first antigen-binding site that specifically binds EMR2, a second heavy chain (HC2) that comprises a second antigen-binding site that specifically binds TRBV19 (TRBV19). In some embodiments, the EMR2 x TRB VI 9-bispecific antibody comprises a EMR2-specific arm comprising a first heavy chain (HC1) and a light chain (LC) that pair to form a first antigen-binding site that specifically binds TRBV19 (V017), asecond heavy chain (HC2) that comprises a second antigen-binding site that specifically binds EMR2.

[0238] In some embodiments, the first antigen-binding site comprises a fragment antigenbinding (Fab) region. In some embodiments, the second antigen-binding site comprises a singlechain variable fragment (scFv) or a stapled scFv (spFv). In some embodiments, the first antigenbinding site comprises a scFv or a spFv. In some embodiments, the second antigen-binding site comprises a Fab. In some embodiments, the spFv comprises a C220S mutation.

[0239] In one embodiment, the EMR2-binding arm comprises a fragment antigen-binding (Fab) region, and the TRBV19-binding arm comprises a single-chain variable fragment (scFv) or spFv. In some embodiments, the spFv comprises a C220S mutation.

[0240] In one embodiment, the TRB V 19-binding arm comprises a fragment antigen-binding (Fab) region, and the EMR2-binding arm comprises a single-chain variable fragment (scFv) or spFv. In some embodiments, the spFv comprises a C220S mutation.

[0241] In some embodiments, the multispecific antibodies (e.g., bispecific antibodies) of the disclosure include antibodies having a full length antibody structure. “Full length antibody” as used herein refers to an antibody having two full length antibody heavy chains and two full length antibody light chains. A full length antibody heavy chain (HC) includes heavy chain variable and constant domains VH, CHI, CH2, and CH3. A full length antibody light chain (LC) includes light chain variable and constant domains VL and CL. The full length antibody may be lacking the C-terminal lysine (K) in either one or both heavy chains. The term “Fab-arm” or "half molecule" refers to one heavy chain-light chain pair that specifically binds an antigen. In some embodiments, one of the antigen-binding domains is a non-antibody based binding domain, e.g. a binding domain of based on a fibronectin type 3 domain, e.g. Centyrin.

[0242] EMR2-binding arm

[0243] The multispecific antibodies (e.g., bispecific antibodies) described herein comprise an antigen-binding site specific for EMR2. In some embodiments, the EMR2-binding arm binds human EMR2. In some embodiments, the EMR2-binding arm binds human EMR2 and cynomolgus monkey EMR2. In some embodiments, the EMR2-binding arm binds human EMR2 but not to cynomolgus monkey EMR2. In some embodiments, the EMR2-binding arm binds bind to an epitope including one or more residues from the EMR2 extracellular domain (ECD). In some embodiments, the EMR2-binding arm binds to one or more residues of a polypeptidehaving the amino acid sequence of SEQ ID NO: 215 (G-protein-coupled receptor autoproteolysis inducing (GAIN) domain and / or GPS motif of epidermal-growth-factor-like modulecontaining mucin-like hormone receptor 2 (EMR2)). In some embodiments, the EMR2-binding arm binds to residues D261-Q478 of human EMR2. In some embodiments, the EMR2-binding arm binds to the GAIN domain comprising the amino acid sequence SEQ ID NO: 215. In some embodiments, the EMR2-binding arm binds to the GPS motif comprising the amino acid sequence SEQ ID NO: 216. In some embodiments, the EMR2-binding arm binds to SEQ ID NO: 217 or SEQ ID NO: 218. Such EMR2-binding arms may bind to EMR2 with an affinity of 5X10'7M or less, such as 1X10'7M or less, 5xlO'8M or less, 1X10'8M or less, 5xlO'9M or less, 1X10'9M, or 5x1 O'10M or less. In one embodiment, the EMR2-binding arm binds to the EMR2 with an affinity of about 1 xlO-11M to 1 xlO'9M. In one embodiment, the EMR2-binding arm binds to the EMR2 with an affinity of about 1 xlO-11M, about 2 xlO'nM, about 3 xlO'nM, about 4 xlO'nM, about 5 xlO-11M, about 6 xlO-11M, about 7 xlO'nM, about 8 xlO'nM, about 9x1 O'nM, 1 xlO'loM, about 2 xlO'loM, about 3 xlO'loM, about 4 xlO'loM, about 5 xlO'loM, about 6 xlO'loM, about 7 xlO'loM, about 8 xlO'loM, about 9xlO'loM or about 1X10'9M. In some embodiments, the EMR2-binding arm binds to EMR2 with a dissociation constant (KD) between about 0.01 nM to about 5 nM. In some embodiments, the EMR2-binding arm binds to EMR2 with an ECso between about 0.1 nM to about 15 nM.

[0244] Table 1-1 to 1-5 and Table 1-6 provide a summary of examples of some EMR2-specific antibodies described herein:

[0245] Table 1-1. CDR sequences (AbM) of exemplary mAbs generated against human EMR2

[0246] Table 1-2. CDR sequences (KABAT) of exemplary mAbs generated against human EMR2

[0247] Table 1-3. CDR sequences (CHOTHIA) of exemplary mAbs generated against human EMR2

[0248] Table 1-4. CDR sequences (IMGT) of exemplary mAbs generated against human EMR2

[0249] Table 1-5. CDR sequences (CONTACT) of exemplary mAbs generated against human EMR2

[0250] Table 1-6. VH and VL sequences of exemplary mAbs generated against human EMR2

[0251] In some embodiments, the EMR2-binding arm comprises a heavy chain variable domain comprising a CDR1 , a CDR2, and a CDR3 of any one of the antibodies described in Tables 1-1 to 1-5. In some embodiments, the EMR2-binding arm comprises a light chain variable region comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Tables 1-1 to 1-5. In some embodiments, the EMR2-binding arm comprises a heavy chainvariable domain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Tables 1-1 to 1-5 and a light chain variable region comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Tables 1-1 to 1-5. In some embodiments, the EMR2-binding arm competes for binding to EMR2 with an antibody or an antigen-binding comprising a heavy chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Tables 1-1 to 1-5 and a light chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Tables 1-1 to 1-5.

[0252] In some embodiments, the EMR2-binding arm comprises a heavy chain variable domain of any one of the antibodies described in Table 1-6. In some embodiments, the EMR2- binding arm comprises a light chain variable region of any one of the antibodies described in Table 1-6. In some embodiments, the EMR2-binding arm comprises a heavy chain variable domain of any one of the antibodies described in Table 1-6 and a light chain variable region of any one of the antibodies described in Table 1-6. In some embodiments, the EMR2-binding arm competes for binding to EMR2 with an antibody or an antigen-binding comprising a heavy chain variable domain of any one of the antibodies described in Table 1-6 and a light chain variable domain of any one of the antibodies described in Table 1-6.

[0253] In some embodiments, the EMR2-binding arm comprises a heavy chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Tables 1-1 to 1-5. In some embodiments, the EMR2-binding arm comprises a light chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Tables 1-1 to 1-5. In some embodiments, the EMR2-binding arm comprises a heavy chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Tables 1-1 to 1-5 and a light chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Tables 1-1 to 1-5.

[0254] In some embodiments, the EMR2-binding arm comprises a heavy chain comprising a heavy chain variable domain of any one of the antibodies described in Table 1-6. In some embodiments, the EMR2-binding arm comprises a light chain comprising a light chain variable domain of any one of the antibodies described in Table 1-6. In some embodiments, the EMR2- binding arm comprises a heavy chain comprising a heavy chain variable domain of any one of the antibodies described in Table 1-6 and a light chain comprising a light chain variable domain of any one of the antibodies described in Table 1-6.

[0255] In some embodiments, the antibody or the antigen-binding fragment thereof, comprises: a) a heavy chain complementarity determining region (HCDR) 1 , a HCDR2 and a HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 63, and a light chain complementarity determining region (LCDR) 1, a LCDR2 and a LCDR3 of the light chain variable region (VL) of SEQ ID NO: 64; b) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 95, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 96; c) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 127, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 128; or d) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 191, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 192. In some embodiments, the antibody or the antigen-binding fragment thereof, comprises a HCDR1 , a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 95, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 96.

[0256] In some embodiments, the antibody or the antigen-binding fragment thereof, comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of: a) SEQ ID NO: 33, 34, 35, 48, 49, and 50, respectively; b) SEQ ID NO: 36, 37, 38, 51, 52, and 53, respectively; c) SEQ ID NO: 39, 40, 41, 54, 55, and 56, respectively; d) SEQ ID NO: 42, 43, 44, 57, amino acid sequence EVS, and SEQ ID NO: 59, respectively; e) SEQ ID NO: 45, 46, 47, 60, 61, and 62, respectively; f) SEQ ID NO: 65, 66, 67, 80, 81, and 82, respectively; g) SEQ ID NO: 68, 69, 70, 83, 84, and 85, respectively; h) SEQ ID NO: 71, 72, 73, 86, 87, and 88, respectively; i) SEQ ID NO: 74, 75, 76, 89, amino acid sequence AAI, and SEQ ID NO: 91, respectively; j) SEQ ID NO: 77, 78, 79, 92, 93, and 94, respectively; k) SEQ ID NO: 97, 98, 99, 112, 113, and 114, respectively; 1) SEQ ID NO: 100, 101, 102, 115, 116, and 117, respectively; m) SEQ ID NO: 103, 104, 105, 118, 119, and 120, respectively; n) SEQ ID NO: 106, 107, 108, 121, 122, and 123, respectively; o) SEQ ID NO: 109, 110, 111, 124, 125, and 126, respectively; p) SEQ ID NO: 161, 162, 163, 176, 177, and 178, respectively; q) SEQ ID NO: 164, 165, 166, 179, 180, and 181, respectively; r) SEQ ID NO: 167, 168, 169, 182, 18, and 184, respectively; s) SEQ ID NO: 170, 171, 172, 185, amino acid sequence EVS, and SEQ ID NO: 187, respectively; or t) SEQ ID NO: 173, 174, 175, 188, 189, and 190, respectively. In some embodiments, the antibody or the antigen-binding fragment thereof, comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NO: 65, 66, 67, 80, 81, and 82, respectively; SEQ ID NO: 68, 69, 70, 83, 84, and 85, respectively; SEQ ID NO: 71, 72, 73, 86, 87, and 88,respectively; SEQ ID NO: 74, 75, 76, 89, amino acid sequence AAI, and SEQ ID NO: 91, respectively; or SEQ ID NO: 77, 78, 79, 92, 93, and 94, respectively

[0257] In some embodiments, the antibody or an antigen-binding fragment thereof, comprises: a) the HC1 comprises the amino acid sequence of SEQ ID NO: 194 and the LC1 comprises the amino acid sequence of SEQ ID NO: 195; b) the HC1 comprises the amino acid sequence of SEQ ID NO: 196 and the LC1 comprises the amino acid sequence of SEQ ID NO: 197; or c) the HC1 comprises the amino acid sequence of SEQ ID NO: 198 and the LC1 comprises the amino acid sequence of SEQ ID NO: 199. In some embodiments, the antibody or an antigen-binding fragment thereof, comprises an HC1 comprising the amino acid sequence of SEQ ID NO: 198 and a LC1 comprising the amino acid sequence of SEQ ID NO: 199.

[0258] In some embodiments, the antibody or an antigen-binding fragment thereof, comprises a variable heavy chain region (VH) comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 63; b) the VH of SEQ ID NO: 95; c) the VH of SEQ ID NO: 127; or to d) the VH of SEQ ID NO: 191. In some embodiments, the antibody or an antigen-binding fragment thereof, comprises a variable heavy chain region (VH) comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the VH of SEQ ID NO: 95.

[0259] In some embodiments, the antibody or the antigen-binding fragment thereof, comprises or further comprises a VL comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 64; b) the VL of SEQ ID NO: 96; c) the VL of SEQ ID NO: 128; or to d) the VL of SEQ ID NO: 192. In some embodiments, the antibody or the antigen-binding fragment thereof, comprises or further comprises a VL comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the VL of SEQ ID NO: 96.

[0260] In some embodiments, the antibody or the antigen-binding fragment thereof, comprises or further comprises the antibody or the antigen-binding fragment thereof, comprises: a) the VH of SEQ ID NO: 63 and the VL of SEQ ID NO: 64; b) the VH of SEQ ID NO: 95 and the VL ofSEQ ID NO: 96; c) the VH of SEQ ID NO: 127 and the VL of SEQ ID NO: 128; or d) the VH of SEQ ID NO: 191 and the VL of SEQ ID NO: 192. In some embodiments, the antibody or the antigen-binding fragment thereof, comprises or further comprises the antibody or the antigenbinding fragment thereof, comprises the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 96.

[0261] In some embodiments, the present disclosure provides a polynucleotide comprising a sequence encoding an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 63; b) the VH of SEQ ID NO: 95; c) the VH of SEQ ID NO: 127; or d) the VH of SEQ ID NO: 191. In some embodiments, the present disclosure provides an isolated polynucleotide comprising a sequence encoding an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: the VH of SEQ ID NO: 95.

[0262] In some embodiments, the present disclosure provides a polynucleotide comprising a sequence encoding an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 64; b) the VL of SEQ ID NO: 96; c) the VL of SEQ ID NO: 128; or d) the VL of SEQ ID NO: 192. In some embodiments, the present disclosure provides an isolated polynucleotide comprising a sequence encoding an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the VL of SEQ ID NO: 96.

[0263] In some embodiments, the present disclosure provides an isolated polynucleotide comprising a sequence encoding: a) the amino acid sequence of SEQ ID NO: 63 and / or the amino acid sequence of SEQ ID NO: 64; b) the amino acid sequence of SEQ ID NO: 95 and / or the amino acid sequence of SEQ ID NO: 96; c) the amino acid sequence of SEQ ID NO: 127 and / or the amino acid sequence of SEQ ID NO: 128; or d) the amino acid sequence of SEQ ID NO: 191 and / or the amino acid sequence of SEQ ID NO: 192. In some embodiments, the present disclosure provides an isolated polynucleotide comprising a sequence encoding: the amino acid sequence of SEQ ID NO: 95 and / or the amino acid sequence of SEQ ID NO: 96.In some embodiments, the polynucleotide comprises a sequence encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 194, 196, 198, or 202. In someembodiments, the polynucleotide comprises a sequence encoding a light chain comprising the amino acid sequence of SEQ ID NO: 195, 197, or 199. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding: a) the amino acid sequence of SEQ ID NO: 194 and / or SEQ ID NO: 195; b) the amino acid sequence of SEQ ID NO: 196 and / or SEQ ID NO: 197; c) the amino acid sequence of SEQ ID NO: 198 and / or the amino acid sequence of SEQ ID NO: 199; or d) the amino acid sequence of SEQ ID NO: 202. In some embodiments, the isolated polynucleotide comprises a sequence encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 198. In some embodiments, the isolated polynucleotide comprises a sequence encoding a light chain comprising the amino acid sequence of SEQ ID NO: 199. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding: the amino acid sequence of SEQ ID NO: 198 and / or the amino acid sequence of SEQ ID NO: 199.

[0264] The EMR2-binding arm may be derived from any species by recombinant means. For example, the EMR2 antigen-binding region may be derived from mouse, rat, goat, horse, swine, bovine, chicken, rabbit, camelid, donkey, human, or chimeric versions thereof. For use in administration to humans, non-human derived antigen-binding fragments may be genetically or structurally altered to be less antigenic upon administration to a human patient. In some embodiments, the EMR2-binding arm comprises antigen-binding fragments which is chimeric.

[0265] In some embodiments, the EMR2-binding arm comprises humanized antigen-binding fragments. Humanized antigen-binding fragments may be derived from chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, scFv, spFv, Fab, Fab’, F(ab’)2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies or antigen-binding fragments are human immunoglobulins (recipient antibody) or antigen-binding fragments in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In general, the humanized antibody antigen-binding fragments will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. The humanized antibody antigen-binding fragments mayinclude at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0266] TRB V 19-binding arm

[0267] The multispecific antibodies (e.g., bispecific antibodies) described herein may comprise an antigen-binding site specific for TRB V 19. In some embodiments, the TRB V 19-binding arm binds human TRBV 19. In some embodiments, TRB V 19-binding arm binds human TRB V 19 and cynomolgus monkey TRBV 19, preferably the extracellular domain thereof. In some embodiments, TRBV 19-binding arm binds human TRBV 19 but not to cynomolgus monkey TRBV19.

[0268] In some embodiments, the TRB VI 9-binding arm comprises a heavy chain CDR1, CDR2, and CDR3 derived from an antibody clone as described in Tables 2-1 to 2-5. In some embodiments, the TRBV 19-binding arm comprises a light chain CDR1 , CDR2, and CDR3 derived from an antibody clone as described in Tables 2-1 to 2-5. In some embodiments, the TRBV 19-binding arm comprises heavy chain CDR1 , CDR2, and CDR3 and light chain CDR1 , CDR2, and CDR3 derived from an antibody clone as described in Tables 2-1 to 2-5.

[0269] In some exemplary embodiments, the TRBV 19-binding arm comprises a heavy chain variable domain derived from an antibody clone as described in Table 2-6. In some exemplary embodiments, the TRB VI 9-binding arm comprises heavy chain variable domain and light chain variable domain derived from an antibody clone as described in Table 2-6.

[0270] Tables 2-1 to 2-5 and Table 2-6 provide a summary of examples of some TRBV19- specific antibodies described herein:

[0271] Table 2-1. CDR sequences (AbM) of exemplary mAbs generated against human TRBV19

[0272] Table 2-2. CDR sequences (KABAT) of exemplary mAbs generated against human TRBV19

[0273] Table 2-3. CDR sequences (CHOTHIA) of exemplary mAbs generated against human TRBV19

[0274] Table 2-4. CDR sequences (IMGT) of exemplary mAbs generated against human TRBV19

[0275] Table 2-5. CDR sequences (CONTACT) of exemplary mAbs generated against human TRBV19

[0276] Table 2-6. VH and VL sequences of exemplary mAbs generated against human TRBV19

[0277] In some embodiments, the TRB VI 9-binding arm comprises a heavy chain variable domain comprising a CDR1 , a CDR2, and a CDR3 of any one of the antibodies described in Tables 2-1 to 2-5. In some embodiments, the TRBV19-binding arm comprises a light chain variable domain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Tables 2-1 to 2-5. In some embodiments, the TRB VI 9-binding arm comprises a heavy chain variable domain comprising a CDR1 , a CDR2, and a CDR3 of any one of the antibodies described in Table 2a and a light chain variable domain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Tables 2-1 to 2-5. In some embodiments, the TRB VI 9-binding arm competes for binding to TRB VI 9 with an antibody or an antigenbinding comprising a heavy chain comprising a CDR1 , a CDR2, and a CDR3 of any one of the antibodies described in Tables 2-1 to 2-5 and a light chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Tables 2-1 to 2-5.

[0278] In some embodiments, the TRB VI 9-binding arm comprises a heavy chain variable domain of any one of the antibodies described in Table 2-6. In some embodiments, the TRB VI 9- binding arm comprises a light chain variable region of any one of the antibodies described in Table 2b. In some embodiments, the TRB VI 9-binding arm comprises a heavy chain variable domain of any one of the antibodies described in Table 2-6 and a light chain variable region of any one of the antibodies described in Table 2-6. In some embodiments, the TRB VI 9-binding arm competes for binding to TRBV19 with an antibody or an antigen-binding comprising a heavy chain variable domain of any one of the antibodies described in Table 2-6 and a light chain variable domain of any one of the antibodies described in Table 2-6.

[0279] In some embodiments, the TRB VI 9-binding arm comprises a heavy chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Tables 2-1 to 2-5. In some embodiments, the TRB VI 9-binding arm comprises a light chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Tables 2-1 to 2-5. In some embodiments, the TRB VI 9-binding arm comprises a heavy chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Tables 2-1 to 2-5 and a light chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Tables 2-1 to 2-5.

[0280] In some embodiments, the TRB VI 9-binding arm comprises a heavy chain comprising a heavy chain variable domain of any one of the antibodies described in Table 2-6. In some embodiments, the TRB VI 9-binding arm comprises a light chain comprising a light chain variable domain of any one of the antibodies described in Table 2-6. In some embodiments, the TRB VI 9-binding arm comprises a heavy chain comprising a heavy chain variable domain of any one of the antibodies described in Table 2-6 and a light chain comprising a light chain variable domain of any one of the antibodies described in Table 2-6.In some embodiments, the antibody or the antigen-binding fragment thereof, comprises: a) a heavy chain complementarity determining region (HCDR) 1 , a HCDR2 and a HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 31 and a light chain complementarity determining region (LCDR) 1, a LCDR2 and a LCDR3 of the light chain variable region (VL) of SEQ ID NO: 32; or b) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 159 and a LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 160. In some embodiments, the antibody or the antigen-binding fragment thereof, comprises: a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 159 and a LCDR1 , LCDR2, and LCDR3 of the VL of SEQ ID NO: 160.

[0281] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of: a) SEQ ID NOs: 1, 2, 3, 16, 17, and 18, respectively; b) SEQ ID NOs: 4, 5, 6, 19, 20, and 21, respectively; c) SEQ ID NOs: 7, 8, 9, 22, 23, and 24, respectively; d) SEQ ID NOs: 10, 11, 12, 25, amino acid sequence KVS, and SEQ ID NO: 27, respectively; e) SEQ ID NOs: 13, 14, 15, 28, 29, and 30, respectively; f) SEQ ID NOs: 129, 130, 131, 144, 145, and 146, respectively; g) SEQ ID NOs: 132, 133, 134, 147, 148, and 149, respectively; h) SEQ ID NOs:135, 136, 137, 150, 151, and 152, respectively; i) SEQ ID NOs: 138, 139, 140, 153, 154, and 155, respectively; or j) SEQ ID NOs: 141, 142, 143, 156, 157, and 158, respectively. In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 129, 130, 131, 144, 145, and 146, respectively; SEQ ID NOs: 132, 133, 134, 147, 148, and 149, respectively; SEQ ID NOs: 135, 136, 137, 150, 151, and 152, respectively; SEQ ID NOs: 138, 139, 140, 153, 154, and 155, respectively; or SEQ ID NOs: 141, 142, 143, 156, 157, and 158, respectively.

[0282] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 31; or b) the VH of SEQ ID NO: 159. In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: the VH of SEQ ID NO: 159.

[0283] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, comprises or further comprises a light chain variable region (VL) comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 32; or b) the VL of SEQ ID NO: 160. In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, comprises or further comprises a VL comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: the VL of SEQ ID NO: 160.

[0284] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, comprises: a) the VH of SEQ ID NO: 31 and the VL of SEQ ID NO: 32; or b) the VH of SEQ ID NO: 159 and the VL of SEQ ID NO: 160. In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, comprises: the VH of SEQ ID NO: 159 and the VL of SEQ ID NO: 160.

[0285] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, comprises: a) a heavy chain complementarity determining region (HCDR) 1, a HCDR2 and a HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 63, and a light chain complementarity determining region (LCDR) 1 , a LCDR2 and a LCDR3 of the light chain variable region (VL) of SEQ ID NO: 64; b) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 95, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 96; c) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 127, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 128; or d) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 191, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 192. In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, comprises: a) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 95, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 96.

[0286] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, comprises a HC1 comprises the amino acid sequence of SEQ ID NO: 193 or 200. In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, comprises a HC1 comprises the amino acid sequence of SEQ ID NO: 193.

[0287] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, comprises a LC1 comprises the amino acid sequence of SEQ ID NO: 201.

[0288] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, comprises: a) the HC1 comprises the amino acid sequence of SEQ ID NO: 193; or b) the HC1 comprises the amino acid sequence of SEQ ID NO: 200 and the LC1 comprises the amino acid sequence of SEQ ID NO: 201. In some embodiments, the HC2 comprises the amino acid sequence of SEQ ID NO: 194, 196, 198, or 202. In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, comprises the HC1 comprises the amino acid sequence of SEQ ID NO: 193. In some embodiments, the HC2 comprises the amino acid sequence of SEQ ID NO: 198. In some embodiments, the LC2 comprises the amino acid sequence of SEQ ID NO: 195, 197, or 199. In some embodiments, the LC2 comprises the amino acid sequence of SEQ ID NO: 199. In some embodiments, a) the HC2 comprises the amino acid sequence of SEQ ID NO: 194 and the LC2 comprises the amino acid sequence of SEQ ID NO: 195; b) the HC2 comprises the amino acid sequence of SEQ ID NO: 196 and the LC1 comprises the amino acid sequence of SEQ ID NO: 197; c) the HC2 comprises the amino acid sequence ofSEQ ID NO: 198 and the LC1 comprises the amino acid sequence of SEQ ID NO: 199; or d) the HC2 comprises the amino acid sequence of SEQ ID NO: 202. In some embodiments, the HC2 comprises the amino acid sequence of SEQ ID NO: 198 and the LC1 comprises the amino acid sequence of SEQ ID NO: 199. In some embodiments, a) the HC1 comprises the amino acid sequence of SEQ ID NO: 193, the HC2 comprises the amino acid sequence of SEQ ID NO: 194, and the LC2 comprises the amino acid sequence of SEQ ID NO 195; b) the HC1 comprises the amino acid sequence of SEQ ID NO: 193, the HC2 comprises the amino acid sequence of SEQ ID NO: 196, and the LC2 comprises the amino acid sequence of SEQ ID NO: 197; c) the HC1 comprises the amino acid sequence of SEQ ID NO: 193, the HC2 comprises the amino acid sequence of SEQ ID NO: 198, and the LC2 comprises the amino acid sequence of SEQ ID NO: 199; or d) the HC1 comprises the amino acid sequence of SEQ ID NO: 200, the LC1 comprises the amino acid sequence of SEQ ID NO: 201, and the HC2 comprises the amino acid sequence of SEQ ID NO: 202. In some embodiments, the HC1 comprises the amino acid sequence of SEQ ID NO: 193, the HC2 comprises the amino acid sequence of SEQ ID NO: 198, and the LC2 comprises the amino acid sequence of SEQ ID NO: 199.

[0289] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by an isolated polynucleotide, wherein the isolated polynucleotide comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 63; b) the VH of SEQ ID NO: 95; c) the VH of SEQ ID NO: 127; or d) the VH of SEQ ID NO: 191. In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by a polynucleotide, wherein the polynucleotide comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: the VH of SEQ ID NO: 95.

[0290] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by a polynucleotide, wherein the polynucleotide comprises or further comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 64; b) theVL of SEQ ID NO: 96; c) the VL of SEQ ID NO: 128; or d) the VL of SEQ ID NO: 192. In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by a polynucleotide, wherein the polynucleotide comprises or further comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: the VL of SEQ ID NO: 96.

[0291] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by an isolated polynucleotide, wherein the isolated polynucleotide comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 31; or b) the VH of SEQ ID NO: 159. In some embodiments, the bispecific antibody or the bispecific antigenbinding fragment thereof, is encoded by an isolated polynucleotide, wherein the isolated polynucleotide comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the VH of SEQ ID NO: 159.

[0292] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by an isolated polynucleotide, wherein the isolated polynucleotide comprises or further comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 32; or b) the VL of SEQ ID NO: 160. In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by an isolated polynucleotide, wherein the isolated polynucleotide comprises or further comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: the VL of SEQ ID NO: 160.

[0293] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by an isolated polynucleotide, wherein the isolated polynucleotide comprises a sequence encoding a first antigen-binding site that specifically binds TRBV19, the sequencecomprising a nucleotide sequence encoding: a) the amino acid sequence of SEQ ID NO: 31 and / or the amino acid sequence of SEQ ID NO: 32; or b) the amino acid sequence of SEQ ID NO: 159 and / or the amino acid sequence of SEQ ID NO: 160. In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by an isolated polynucleotide, wherein the isolated polynucleotide comprises a sequence encoding a first antigen-binding site that specifically binds TRBV19, the sequence comprising a nucleotide sequence encoding: the amino acid sequence of SEQ ID NO: 159 and / or the amino acid sequence of SEQ ID NO: 160.

[0294] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by an isolated polynucleotide, wherein the isolated polynucleotide comprises a sequence encoding a second antigen-binding site that specifically binds EMR2, the sequence comprising a nucleotide sequence encoding: a) the amino acid sequence of SEQ ID NO: 63 and / or the amino acid sequence of SEQ ID NO: 64; b) the amino acid sequence of SEQ ID NO: 95 and / or the amino acid sequence of SEQ ID NO: 96; c) the amino acid sequence of SEQ ID NO: 127 and / or the amino acid sequence of SEQ ID NO: 128; or d) the amino acid sequence of SEQ ID NO: 191 and / or the amino acid sequence of SEQ ID NO: 192. In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by a polynucleotide, wherein the polynucleotide comprises a sequence encoding a second antigenbinding site that specifically binds EMR2, the sequence comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 95 and / or the amino acid sequence of SEQ ID NO: 96.

[0295] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by an isolated polynucleotide, wherein the isolated polynucleotide comprises a sequence encoding an HC1 comprising the amino acid sequence of SEQ ID NO: 193 or 200. In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by an isolated polynucleotide, wherein the isolated polynucleotide comprises a sequence encoding an HC1 comprising the amino acid sequence of SEQ ID NO: 193.

[0296] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by a polynucleotide, wherein the polynucleotide comprises a sequence encoding a LC1 comprising the amino acid sequence of SEQ ID NO: 201.

[0297] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by an isolated polynucleotide, wherein the isolated polynucleotide comprises a nucleotide sequence encoding: a) the amino acid sequence of SEQ ID NO: 193; or b) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 201. In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by an isolated polynucleotide, wherein the isolated polynucleotide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 193.

[0298] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by an isolated polynucleotide, wherein the isolated polynucleotide comprises a sequence encoding an HC2 comprising the nucleotide sequence of SEQ ID NO: 194, 196, 198, or 202. In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by an isolated polynucleotide, wherein the isolated polynucleotide comprises a sequence encoding an HC2 comprising the nucleotide sequence of SEQ ID NO: 198.

[0299] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by an polynucleotide, wherein the isolated polynucleotide comprises a sequence encoding a LC2 comprising the nucleotide sequence of SEQ ID NO: 195, 197, or 199. In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by an polynucleotide, wherein the isolated polynucleotide comprises a sequence encoding a LC2 comprising the nucleotide sequence of SEQ ID NO: 199.

[0300] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by an isolated polynucleotide, wherein the isolated polynucleotide comprises a sequence encoding: a) the amino acid sequence of SEQ ID NO: 194 and / or the amino acid sequence of SEQ ID NO: 195; b) the amino acid sequence of SEQ ID NO: 196 and / or the amino acid sequence of SEQ ID NO: 197; c) the amino acid sequence of SEQ ID NO: 198 and / or the amino acid sequence of SEQ ID NO: 199; or d) the amino acid sequence of SEQ ID NO: 202. In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by an isolated polynucleotide, wherein the isolated polynucleotide comprises a sequence encoding: the amino acid sequence of SEQ ID NO: 198 and / or the amino acid sequence of SEQ ID NO: 199.

[0301] In some embodiments, the bispecific antibody or the bispecific antigen-binding fragment thereof, is encoded by an isolated polynucleotide, wherein the isolated polynucleotide comprisesa sequence encoding: a) the amino acid sequence of SEQ ID NO: 193, the amino acid sequence of SEQ ID NO: 194 and the amino acid of SEQ ID NO: 195; b) the amino acid sequence of SEQ ID NO: 193, the amino acid sequence of SEQ ID NO: 196 and the amino acid sequence of SEQ ID NO: 197; c) the amino acid sequence of SEQ ID NO: 193, the amino acid sequence of SEQ ID NO: 198 and the amino acid sequence of SEQ ID NO: 199; or d) the amino acid sequence of SEQ ID NO: 200, the amino acid sequence of SEQ ID NO: 201, and the amino acid sequence of SEQ ID NO: 202. In some embodiments, the bispecific antibody or the bispecific antigenbinding fragment thereof, is encoded by an isolated polynucleotide, wherein the isolated polynucleotide comprises a sequence encoding: the amino acid sequence of SEQ ID NO: 193, the amino acid sequence of SEQ ID NO: 198 and the amino acid sequence of SEQ ID NO: 199

[0302] The TRB VI 9-binding arm may be derived from any species by recombinant means. For example, the TRBV19 antigen-binding region may be derived from mouse, rat, goat, horse, swine, bovine, chicken, rabbit, camelid, donkey, human, or chimeric versions thereof. For use in administration to humans, non-human derived antigen-binding fragments may be genetically or structurally altered to be less antigenic upon administration to a human patient. In some embodiments, the TRBV 19-binding arm comprises antigen-binding fragments which is chimeric.

[0303] In some embodiments, the TRB VI 9-binding arm comprises humanized antigen-binding fragments. Humanized antigen-binding fragments may be derived from chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, scFv, spFv, Fab, Fab’, F(ab’)2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies or antigen-binding fragments are human immunoglobulins (recipient antibody) or antigen-binding fragments in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In general, the humanized antibody antigen-binding fragments will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. The humanized antibody antigen-binding fragments may include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0304] In some embodiments, the multispecific antibodies described herein may adopt any format which has been described in the art for multispecific antibodies. In some embodiments, the multispecific antibody comprises a bispecific antibody which is a diabody, a cross-body, or a bispecific antibody obtained via a controlled Fab arm exchange as those described in the present disclosure.

[0305] In some embodiments, the multispecific antibodies include IgG-like molecules with complementary CH3 domains to force heterodimerization; recombinant IgG-like dual targeting molecules, wherein the two sides of the molecule each contain the Fab fragment or part of the Fab fragment of at least two different antibodies; IgG fusion molecules, wherein full length IgG antibodies are fused to an extra Fab fragment or parts of Fab fragment; Fc fusion molecules, wherein single chain Fv molecules or spFv or stabilized diabodies are fused to heavy-chain constant-domains, Fc-regions or parts thereof; Fab fusion molecules, wherein different Fab- fragments are fused together; scFv-, spFv-, and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) wherein different single chain Fv molecules or spFv molecules or different diabodies or different heavy-chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.

[0306] In some embodiments, IgG-like molecules with complementary CH3 domains molecules include the Triomab / Quadroma (Trion Pharma / Fresenius Biotech), the Knobs-into- Holes (Genentech), CrossMAbs (Roche) and the electrostatically-matched (Amgen), the LUZ-Y (Genentech), the Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), the Biclonic (Merus), the DuoBody (Genmab A / S), and other asymmetric mutations (e.g., Zymeworks).

[0307] In some embodiments, recombinant IgG-like dual targeting molecules include Dual Targeting (DT)-Ig (GSK / Domantis), Two-in-one Antibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star) and CovX-body (CovX / Pfizer).

[0308] In some embodiments, IgG fusion molecules include Dual Variable Domain (DVD)-Ig (Abbott), IgG-like Bispecific (InnClone / Eli Lilly), Ts2Ab (Medlmmune / AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idee) and TvAb (Roche).

[0309] In some embodiments, Fc fusion molecules include ScFv / Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), Dual AffinityRetargeting Technology (Fc-DART) (MacroGenics) and Dual(ScFv).sub.2-Fab (National Research Center for Antibody Medicine— China).

[0310] In some embodiments, Fab fusion bispecific antibodies include F(ab)2 (Medarex / AMGEN), Dual- Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech). ScFv-, diabody- based and domain antibodies include but are not limited to Bispecific T Cell Engager (BiTE) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) and COMBODY (Epigen Biotech), dual targeting nanobodies (Ablynx), dual targeting heavy chain only domain antibodies.

[0311] Full length multispecific antibodies of the present disclosure may be generated for example using Fab arm exchange (or half molecule exchange) between two mono specific bivalent antibodies by introducing substitutions at the heavy chain CH3 interface in each half molecule to favor heterodimer formation of two antibody half molecules having distinct specificity either in vitro in cell-free environment or using co-expression. The Fab arm exchange reaction is the result of a disulfide-bond isomerization reaction and dissociation-association of CH3 domains. The heavy-chain disulfide bonds in the hinge regions of the parent mono specific antibodies are reduced. The resulting free cysteines of one of the parent monospecific antibodies form an inter heavy-chain disulfide bond with cysteine residues of a second parent mono specific antibody molecule and simultaneously CH3 domains of the parent antibodies release and reform by dissociation-association. The CH3 domains of the Fab arms may be engineered to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody having two Fab arms or half molecules which each bind a distinct epitope, e.g., an epitope on EMR2 and an epitope on TRBV19.

[0312] "Homodimerization" as used herein refers to an interaction of two heavy chains having identical CH3 amino acid sequences. "Homodimer" as used herein refers to an antibody having two heavy chains with identical CH3 amino acid sequences.

[0313] "Heterodimerization" as used herein refers to an interaction of two heavy chains having non-identical CH3 amino acid sequences. "Heterodimer" as used herein refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.

[0314] The "knob-in-hole" strategy (see, e.g., PCT Int. Publ. No. WO 2006 / 028936) may be used to generate full length multispecific antibodies. Briefly, selected amino acids forming the interface of the CH3 domains in human IgG can be mutated at positions affecting CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into a heavy chain of an antibody specifically binding a first antigen and an amino acid with a large side chain (knob) is introduced into a heavy chain of an antibody specifically binding a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction of the heavy chain with a "hole" with the heavy chain with a "knob". Exemplary CH3 substitution pairs forming a knob and a hole are (expressed as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S and T366W / T366S L368 A_Y407V.

[0315] In some embodiments of the multispecific antibody or the multispecific binding fragment described herein, the Fc domain of the first heavy chain (HC1) comprise mutations T366S, L368A and Y407V and the Fc domain of the second heavy chain (HC2) comprises mutation T366W. In some embodiments, the Fc domain of the second heavy chain (HC2) comprise mutations T366S, L368A and Y407V and the Fc domain of the first heavy chain (HC1) comprises mutation T366W.

[0316] Other strategies such as promoting heavy chain heterodimerization using electrostatic interactions by substituting positively charged residues at one CH3 surface and negatively charged residues at a second CH3 surface may be used, as described in, e.g., US Pat. Publ. No. US2010 / 0015133; US Pat. Publ. No. US2009 / 0182127; US Pat. Publ. No. US2010 / 028637 or US Pat. Publ. No. US2011 / 0123532. In other strategies, heterodimerization may be promoted by the following substitutions (expressed as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): L351 Y_F405 AY407 V / T394W, T366I_K392M_T394W / F405 A_Y407V, T366L K392M_T394W / F405 A_Y407V, L351 Y_Y407A / T366A_K409F, L351Y_Y407A / T366V K409F Y407A / T366A_K409F, or T350V_L351Y_F405A Y407V / T350V_T366L_K392L_T394W as described in, e.g, U.S. Pat. Publ. No. US2012 / 0149876 or U.S. Pat. Publ. No. US2013 / 0195849 (Zymeworks).

[0317] In addition to methods described above, multispecific antibodies of the disclosure may be generated in vitro in a cell-free environment by introducing asymmetrical mutations in the CH3 regions of two mono specific homodimeric antibodies and forming the multispecific heterodimeric antibody from two parent monospecific homodimeric antibodies in reducing conditions to allow disulfide bond isomerization according to methods described in, e.g., Int. Pat. Publ. No. W02011 / 131746. In the methods, the first monospecific bivalent antibody (e.g., anti- EMR2 antibody) and the second monospecific bivalent antibody (e.g., anti- TRBV19 antibody) are engineered to have certain substitutions at the CH3 domain that promotes heterodimer stability; the antibodies are incubated together under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization; thereby generating the multispecific antibody by Fab arm exchange. The incubation conditions may optimally be restored to non-reducing conditions. Exemplary reducing agents that may be used are 2- mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris (2- carboxy ethyl) phosphine (TCEP), L-cysteine and beta-mercaptoethanol, preferably a reducing agent selected from the group consisting of: 2-mercaptoethylamine, dithiothreitol and tris (2- carboxy ethyl) phosphine. For example, incubation for at least 90 min at a temperature of at least 20° C in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH from 5-8, for example at pH of 7.0 or at pH of 7.4 may be used.

[0318] In some embodiments, the multispecific antibodies or antigen-binding fragments are IgG, or derivatives thereof. The IgG class is divided in four isotypes: IgGl, IgG2, IgG3 and IgG4 in humans. They share more than 95% homology in the amino acid sequences of the Fc regions but show major differences in the amino acid composition and structure of the hinge region. The Fc region mediates effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to Fc receptors (FcyRs) on the surface of immune effector cells such as natural killers and macrophages, leading to the phagocytosis or lysis of the targeted cells. In CDC, the antibodies kill the targeted cells by triggering the complement cascade at the cell surface. The antibodies described herein include antibodies with the described features of the variable domains in combination with any of the IgG isotypes, including modified versions in which the Fc sequence has been modified to effect different effector functions.

[0319] For many applications of therapeutic antibodies, Fc-mediated effector functions are not part of the mechanism of action. These Fc-mediated effector functions can be detrimental and potentially pose a safety risk by causing off-mechanism toxicity. Modifying effector functions can be achieved by engineering the Fc regions to reduce their binding to FcyRs or the complement factors. The binding of IgGto the activating (FcyRI, FcyRIIa, FcyRIIIa and FcyRIIIb) and inhibitory (FcyRIIb) FcyRs or the first component of complement (Clq) depends on residues located in the hinge region and the CH2 domain. Mutations have been introduced in IgGl, IgG2 and IgG4 to reduce or silence Fc functionalities. The antibodies described herein may include these modifications.

[0320] In one embodiment, the antibody comprises an Fc region with one or more of the following properties: (a) reduced effector function when compared to the parent Fc; (b) reduced affinity to FcyRI, FcyRIIa, FcyRIIb, FcyRIIIb and / or FcyRIIIa, (c) reduced affinity to FcyRI (d) reduced affinity to FcyRIIa (e) reduced affinity to FcyRIIb, (f) reduced affinity to FcyRIIIb or (g) reduced affinity to FcyRIIIa.

[0321] In some embodiments, the antibodies or antigen-binding fragments are IgG, or derivatives thereof, e.g., IgGl, IgG2, IgG3, and IgG4 isotypes. In some embodiments wherein the antibody has an IgGl isotype, the antibody contains L234A, L235A, D265S and / or K409R substitution(s) in its Fc region. In some embodiments wherein the antibody has an IgG4 isotype, the antibody contains S228P, L234A, and L235A substitutions in its Fc region. The antibodies described herein may include these modifications.

[0322] In some embodiments, the Fc domains of HC1 and / or HC2 of a multispecific antibody described herein each comprise one or more mutations selected from L234A, L235A, and D265S. In some embodiments, the Fc domains of HC1 and HC2 each comprise mutations L234A, L235A, and D265S.

[0323] In some embodiments, the Fc domains of HC1 or HC2 of a multispecific antibody described herein further comprises one or more mutations which reduce Fc binding to protein A. In some embodiments, the Fc domains of HC1 or HC2 comprises mutations H435R and / or Y436F. In some embodiments, the Fc domain of HC1 comprises mutations H435R and / or Y436F. In some embodiments, the Fc domain of HC2 comprises mutations H435R and / or Y436F.

[0324] In some embodiments, the HC1 comprises, from the N- to C-terminus, a heavy chain variable domain (VH) associated with the first antigen-binding site, a CHI domain, the Fc domain, a linker, and the third antigen-binding site.

[0325] In some embodiments, the HC2 comprises, from the N-to C-terminus, the second antigen-binding site, the Fc domain, a linker, and the third antigen-binding site.

[0326] In various embodiments, the scFv or spFv used in multispecific antibodies described herein comprises, from the N- to C-terminus, a VH, a linker and a VL in the format VH-L-VL or a VL, a linker and a VH in the format VL-L-VH. In some embodiments, the scFv or spFV comprises, from the N- to C-terminus, a VL, a linker and a VH in the format VL-L-VH. In some embodiments, the scFv or spFv comprises, from the N- to C-terminus, a VH, a linker and a VH in the format VL-L-VH.

[0327] Linkers used in the present disclosure may be about 5-50 amino acids long. In some embodiments, the linker is about 10-40 amino acids long. In some embodiments, the linker is about 10-35 amino acids long. In some embodiments, the linker is about 10-30 amino acids long. In some embodiments, the linker is about 10-25 amino acids long. In some embodiments, the linker is about 10-20 amino acids long. In some embodiments, the linker is about 15-20 amino acids long. In some embodiments, the linker is 6 amino acids long. In some embodiments, the linker is 7 amino acids long. In some embodiments, the linker is 8 amino acids long. In some embodiments, the linker is 9 amino acids long. In some embodiments, the linker is 10 amino acids long. In some embodiments, the linker is 11 amino acids long. In some embodiments, the linker is 12 amino acids long. In some embodiments, the linker is 13 amino acids long. In some embodiments, the linker is 14 amino acids long. In some embodiments, the linker is 15 amino acids long. In some embodiments, the linker is 16 amino acids long. In some embodiments, the linker is 17 amino acids long. In some embodiments, the linker is 18 amino acids long. In some embodiments, the linker is 19 amino acids long. In some embodiments, the linker is 20 amino acids long. In some embodiments, the linker is 21 amino acids long. In some embodiments, the linker is 22 amino acids long. In some embodiments, the linker is 23 amino acids long. In some embodiments, the linker is 24 amino acids long. In some embodiments, the linker is 25 amino acids long. In some embodiments, the linker is 26 amino acids long. In some embodiments, the linker is 27 amino acids long. In some embodiments, the linker is 28 amino acids long. In some embodiments, the linker is 29 amino acids long. In some embodiments, thelinker is 30 amino acids long. In some embodiments, the linker is 31 amino acids long. In some embodiments, the linker is 32 amino acids long. In some embodiments, the linker is 33 amino acids long. In some embodiments, the linker is 34 amino acids long. In some embodiments, the linker is 35 amino acids long. In some embodiments, the linker is 36 amino acids long. In some embodiments, the linker is 37 amino acids long. In some embodiments, the linker is 38 amino acids long. In some embodiments, the linker is 39 amino acids long. In some embodiments, the linker is 40 amino acids long. Exemplary linkers that may be used are Gly rich linkers, Gly and Ser containing linkers, Gly and Ala containing linkers, Ala and Ser containing linkers, and other flexible linkers.

[0328] In some embodiments, the linker comprises SEQ ID NO: 221.

[0329] In some embodiments, a bispecific antibody, or a bispecific antigen-binding fragment of the present disclosure comprises a HC1 / LC1 and / or LC2 / HC2 of any one of the antibodies described in Table 3.

[0330] Table 3 provides a summary of examples of some EMR2 x TRBV19 bispecific antibodies described herein:

[0331] Table 3. Exemplary EMR2 x TRBV19 bispecific antibodies

[0332] In some embodiments, the antibody or the antigen-binding fragment thereof, comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 194, 196, 198, or 202. In some embodiments, the antibody or the antigen-binding fragment thereof, comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 198.

[0333] In some embodiments, the antibody or the antigen-binding fragment thereof, comprises a light chain comprises the amino acid sequence of SEQ ID NO: 195, 197 or 199. In some embodiments, the antibody or the antigen-binding fragment thereof, comprises a light chain comprises the amino acid sequence of SEQ ID NO: 199.

[0334] In some embodiments, the antibody or the antigen-binding fragment thereof, comprises: a) the HC1 comprising the amino acid sequence of SEQ ID NO: 194 and the LC1 comprising the amino acid sequence of SEQ ID NO: 195; b) the HC1 comprising the amino acid sequence of SEQ ID NO: 196 and the LC1 comprising the amino acid sequence of SEQ ID NO: 197; or c) the HC1 comprising the amino acid sequence of SEQ ID NO: 198 and the LC1 comprising the amino acid sequence of SEQ ID NO: 199. In some embodiments, the antibody or the antigen-binding fragment thereof, comprises: the HC1 comprising the amino acid sequence of SEQ ID NO: 198 and the LC1 comprising the amino acid sequence of SEQ ID NO: 199.

[0335] In some embodiments, the present disclosure provides an isolated polynucleotide comprising a sequence encoding the amino acid sequence of SEQ ID NO: 194, 196, 198, or 202.

[0336] In some embodiments, the present disclosure provides an isolated polynucleotide comprising a sequence encoding the amino acid sequence of SEQ ID NO: 198.

[0337] In some embodiments, the present disclosure provides an isolated polynucleotide comprising a sequence encoding the amino acid sequence of SEQ ID NO: 195, 197 or 199.

[0338] In some embodiments, the present disclosure provides an isolated polynucleotide comprising a sequence encoding the amino acid sequence of SEQ ID NO: 199.

[0339] In some embodiments, the present disclosure provides an isolated polynucleotide comprising a sequence encoding a) the amino acid sequence of SEQ ID NO: 194 and / or the amino acid sequence of SEQ ID NO: 195; b) the amino acid sequence of SEQ ID NO: 196 and / or the amino acid sequence of SEQ ID NO: 197; or c) the amino acid sequence of SEQ ID NO: 198 and / or the amino acid sequence of SEQ ID NO: 199. In some embodiments, the present disclosure provides an isolated polynucleotide comprising a sequence encoding the amino acid sequence of SEQ ID NO: 198 and / or the amino acid sequence of SEQ ID NO: 199.

[0340] In addition to the described multispecific antibodies or antigen-binding fragments, also provided are polynucleotide sequences capable of encoding the described multispecific antibodies or antigen-binding fragments. Vectors comprising the described polynucleotides are also provided, as are cells expressing the multispecific antibodies or antigen-binding fragmentsprovided herein. Also described are cells capable of expressing the disclosed vectors. These cells may be mammalian cells (such as 293F cells, CHO cells), insect cells (such as Sf7 cells), yeast cells, plant cells, or bacteria cells (such as E. coli). The described antibodies may also be produced by hybridoma cells. The described antibodies may also be recombinantly produced.

[0341] Polynucleotides encoding recombinant antigen-binding proteins also are within the scope of the disclosure. In some embodiments, the polynucleotides described (and the peptides they encode) include a leader sequence. Any leader sequence known in the art may be employed. The leader sequence may include, but is not limited to, a restriction site or a translation start site.

[0342] The multispecific antibodies or antigen-binding fragments described herein include variants having single or multiple amino acid substitutions, deletions, or additions that retain the biological properties (e.g., binding affinity or immune effector activity) of the described multispecific antibodies or antigen-binding fragments. In the context of the present disclosure the following notations are, unless otherwise indicated, used to describe a mutation; i) substitution of an amino acid in a given position is written as e.g. K409R which means a substitution of a Lysine in position 409 with an Arginine; and ii) for specific variants the specific three or one letter codes are used, including the codes Xaa and X to indicate any amino acid residue. Thus, the substitution of Arginine for Lysine in position 409 is designated as: K409R, or the substitution of any amino acid residue for Lysine in position 409 is designated as K409X. In case of deletion of Lysine in position 409 it is indicated by K409*. The skilled person may produce variants having single or multiple amino acid substitutions, deletions, or additions.

[0343] These variants may include: (a) variants in which one or more amino acid residues are substituted with conservative or nonconservative amino acids, (b) variants in which one or more amino acids are added to or deleted from the polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which the polypeptide is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the polypeptide, such as, for example, an epitope for an antibody, a polyhistidine sequence, a biotin moiety and the like. Antibodies or antigen-binding fragments described herein may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or nonconserved positions. In other embodiments, amino acid residues at nonconserved positionsare substituted with conservative or nonconservative residues. The techniques for obtaining these variants, including genetic (deletions, mutations, etc.), chemical, and enzymatic techniques, are known to persons having ordinary skill in the art.

[0344] The multispecific antibodies or antigen-binding fragments described herein may embody several antibody isotypes, such as IgM, IgD, IgG, IgA and IgE. In some embodiments, the antibody isotype is IgG. In some embodiments the antibody isotype is IgGl, IgG2, IgG3, or IgG4 isotype, preferably IgGl or IgG4 isotype. In some embodiments, the antibody isotype is IgGl or IgG4. Antibody or antigen-binding fragment thereof, specificity is largely determined by the amino acid sequence, and arrangement, of the CDRs. Therefore, the CDRs of one isotype may be transferred to another isotype without altering antigen specificity. Alternatively, techniques have been established to cause hybridomas to switch from producing one antibody isotype to another (isotype switching) without altering antigen specificity. Accordingly, such antibody isotypes are within the scope of the described antibodies or antigen-binding fragments.

[0345] Also provided are vectors comprising the polynucleotides described herein. The vectors can be expression vectors. Recombinant expression vectors containing a sequence encoding a polypeptide of interest are thus contemplated as within the scope of this disclosure. The expression vector may contain one or more additional sequences such as but not limited to regulatory sequences (e.g., promoter, enhancer), a selection marker, and a polyadenylation signal. Vectors for transforming a wide variety of host cells are well known and include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), as well as other bacterial, yeast and viral vectors. Viral vectors can include an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpes simplex virus vector, and a poxvirus vector.

[0346] Recombinant expression vectors within the scope of the description include synthetic, genomic, or cDNA-derived nucleic acid fragments that encode at least one recombinant protein which may be operably linked to suitable regulatory elements. Such regulatory elements may include a transcriptional promoter, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. Expression vectors, especially mammalian expression vectors, may also include one or more nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, other 5' or 3' flanking nontranscribed sequences, 5' or 3' nontranslated sequences(such as necessary ribosome binding sites), a polyadenylation site, splice donor and acceptor sites, or transcriptional termination sequences. An origin of replication that confers the ability to replicate in a host may also be incorporated.

[0347] The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells may be provided by viral sources. Exemplary vectors may be constructed as described by Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

[0348] In some embodiments, the multispecific antibody- or antigen-binding fragment-coding sequence is placed under control of a powerful constitutive promoter, such as the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin, human muscle creatine, and others. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use with the described embodiments. Such viral promoters include without limitation, Cytomegalovirus (CMV) immediate early promoter, the early and late promoters of SV40, the Mouse Mammary Tumor Virus (MMTV) promoter, the long terminal repeats (LTRs) of Maloney leukemia virus, Human Immunodeficiency Virus (HIV), Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV), and other retroviruses, and the thymidine kinase promoter of Herpes Simplex Virus. In one embodiment, the multispecific antibody or the antigen-binding fragment thereof, coding sequence is placed under control of an inducible promoter such as the metallothionein promoter, tetracycline-inducible promoter, doxycycline-inducible promoter, promoters that contain one or more interferon-stimulated response elements (ISRE) such as protein kinase R 2',5'-oligoadenylate synthetases, Mx genes, AD ARI, and the like.

[0349] Vectors described herein may contain one or more Internal Ribosome Entry Site(s) (IRES). Inclusion of an IRES sequence into fusion vectors may be beneficial for enhancing expression of some proteins. In some embodiments the vector system will include one or more polyadenylation sites (e.g., SV40), which may be upstream or downstream of any of the aforementioned nucleic acid sequences. Vector components may be contiguously linked, or arranged in a manner that provides optimal spacing for expressing the gene products (i.e., by the introduction of “spacer” nucleotides between the ORFs), or positioned in another way. Regulatory elements, such as the IRES motif, may also be arranged to provide optimal spacing for expression.

[0350] The vectors may comprise selection markers, which are well known in the art. Selection markers include positive and negative selection markers, for example, antibiotic resistance genes (e.g., neomycin resistance gene, a hygromycin resistance gene, a kanamycin resistance gene, a tetracycline resistance gene, a penicillin resistance gene, a puromycin resistance gene, a blasticidin resistance gene), glutamate synthase genes, HSV-TK, HSV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase gene for 6- methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). A nucleic acid sequence encoding a selection marker or the cloning site may be upstream or downstream of a nucleic acid sequence encoding a polypeptide of interest or cloning site.

[0351] The vectors described herein may be used to transform various cells with the genes encoding the described antibodies or antigen-binding fragments. For example, the vectors may be used to generate multispecific antibody or an antigen-binding fragment-producing cells. Thus, another aspect features host cells transformed with vectors comprising a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof, that specifically binds EMR2 and / or TRBV19, such as the antibodies or antigen-binding fragments described and exemplified herein.

[0352] Numerous techniques are known in the art for the introduction of foreign genes into cells and may be used to construct the recombinant cells for purposes of carrying out the described methods, in accordance with the various embodiments described and exemplified herein. The technique used should provide for the stable transfer of the heterologous gene sequence to the host cell, such that the heterologous gene sequence is heritable and expressible by the cell progeny, and so that the necessary development and physiological functions of the recipient cells are not disrupted. Techniques which may be used include but are not limited to chromosome transfer (e.g., cell fusion, chromosome mediated gene transfer, micro cell mediated gene transfer), physical methods (e.g., transfection, spheroplast fusion, micro injection, electroporation, liposome carrier), viral vector transfer (e.g., recombinant DNA viruses, recombinant RNA viruses) and the like (described in Cline, 29 Pharmac. Ther. 69-92 (1985)). Calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts with mammalian cells may also be used to transform cells.

[0353] Cells suitable for use in the expression of the multispecific antibodies or antigenbinding fragments described herein are preferably eukaryotic cells, more preferably cells ofplant, rodent, or human origin, for example but not limited to NSO, CHO, CH0K1, perC.6, Tk- tsl3, BHK, HEK293 cells, COS-7, T98G, CV-l / EBNA, L cells, C127, 3T3, HeLa, NS1, Sp2 / 0 myeloma cells, and BHK cell lines, among others. In addition, expression of antibodies may be accomplished using hybridoma cells. Methods for producing hybridomas are well established in the art.

[0354] Cells transformed with expression vectors described herein may be selected or screened for recombinant expression of the antibodies or antigen-binding fragments described herein. Recombinant-positive cells are expanded and screened for subclones exhibiting a desired phenotype, such as high level expression, enhanced growth properties, or the ability to yield proteins with desired biochemical characteristics, for example, due to protein modification or altered post-translational modifications. These phenotypes may be due to inherent properties of a given subclone or to mutation. Mutations may be effected through the use of chemicals, UV- wavelength light, radiation, viruses, insertional mutagens, inhibition of DNA mismatch repair, or a combination of such methods.

[0355] Exemplary Monospecific Antibodies

[0356] In some embodiments, the disclosure provides an antibody or an antigen-binding fragment thereof, that specifically binds the GAIN domain and / or GPS motif of epidermal- growth-factor-like module-containing mucin-like hormone receptor 2 (EMR2).

[0357] In some embodiments, the GAIN domain comprises amino acid residues D261-Q478 of human EMR2. In some embodiments, the GAIN domain comprises the amino acid sequence SEQ ID NO: 215.

[0358] In some embodiments, the GPS motif comprises the amino acid sequence SEQ ID NO: 216.

[0359] In some embodiments, the antibody or the antigen-binding fragment thereof, binds to an epitope comprising SEQ ID NO: 217 or SEQ ID NO: 218.

[0360] In some embodiments, the antibody or the antigen-binding fragment thereof, binds to human EMR2 with a dissociation constant (KD) between about 0.01 nM to about 5 nM. In some embodiments, the antibody or the antigen-binding fragment thereof, binds to human EMR2 with an ECso between about 0.1 nM to about 15 nM.

[0361] In some embodiments, the antibody or the antigen-binding fragment thereof, is or comprises a, a Fab fragment, a F(ab')2 fragment, F(ab)'3 fragments, a single-chain variablefragment (scFv), a bis-scFv, a (scFv)2, a stapled scFv (spFv), a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a single heavy chain antibody, a camelid antibody, a shark antibody, or a chemically modified derivative thereof.

[0362] In some embodiments, the antibody or the antigen-binding fragment thereof, comprises a Fab. In some embodiments, the antibody or the antigen-binding fragment thereof, further comprises a CHI domain.

[0363] In some embodiments, the antibody or the antigen-binding fragment thereof, does not comprise a CHI domain. In some embodiments, the antibody or the antigen-binding fragment thereof, comprises a scFv or a spFv. In some embodiments, the scFv or spFv comprises a signal sequence, a heavy chain variable sequence, a GS-Linker, and a light chain variable sequence.

[0364] In some embodiments, the antibody or the antigen-binding fragment thereof, further comprises an Fc domain. In some embodiments, the Fc domain of the antibody or the antigenbinding fragment thereof, is an IgA, an IgG, an IgE, or an IgM. In some embodiments, the Fc domain of the antibody or the antigen-binding fragment thereof, is an IgG. In some embodiments, the IgG is IgGl or IgG4.

[0365] In some embodiments, the Fc domain comprises one or more different mutations which promote heterodimerization. In some embodiments, the Fc domain comprises mutations T366S, L368A and Y407V (EU numbering) or mutation T366W (EU numbering).

[0366] In some embodiments, the Fc domains of HC1 and / or HC2 further comprise one or more mutations which reduce Fc binding to a Fey receptor. In some embodiments, the Fey receptor is FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, and / or FcyRIIIB. In some embodiments, the Fc domain comprises one or more mutations selected from L234A, L235A, and D265S (EU numbering). In some embodiments, the Fc domain comprises mutations L234A, L235A, and D265S (EU numbering).

[0367] In some embodiments, the Fc domain further comprises one or more mutations which reduce Fc binding to protein A. In some embodiments, the Fc domain comprises mutations H435R and / or Y436F (EU numbering). In some embodiments, the Fc domain comprises mutations H435R and Y436F (EU numbering).

[0368] In some embodiments, the antibody or the antigen-binding fragment thereof, comprises a humanized antibody or an antigen binding fragment thereof, a human antibody or an antigenbinding fragment thereof, a murine antibody or an antigen binding fragment thereof, a chimeric antibody or an antigen binding fragment thereof, a monospecific antibody or a monospecific antigen binding fragment thereof, a bispecific antibody or a bispecific antigen binding fragment thereof, a multispecific antibody or a multispecific antigen binding fragment thereof.

[0369] In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, comprises the amino acid sequences of the EMR2-binding arm as described herein. In any of the foregoing embodiments, the antibody or the antigen-binding fragment thereof, is encoded by a polynucleotide comprising a nucleotide sequence that encodes the amino acid sequences of the EMR2-binding arm as described herein.

[0370] Therapeutic composition and methods of treatment using multispecific antibodies and multispecific antigen-binding fragments thereof and / or monospecific antibodies and monospecific antigen-binding fragments thereof

[0371] The multispecific antibodies discussed above, for example the EMR2 x TRBV19 bispecific antibodies and the EMR2 monospecific antibodies discussed above, are useful in therapy. In particular, the multispecific antibodies are useful in treating cancer. Also provided herein are therapeutic compositions for the treatment of a hyperproliferative disorder in a mammal which comprises a therapeutically effective amount of a multispecific antibody or a multispecific antigen-binding fragment described herein or a monospecific antibody or a monospecific antigen-binding fragment as described herein and a pharmaceutically acceptable carrier. In some embodiments, the bispecific antibody is a EMR2 x TRBV19 bispecific antibody as described herein, or a EMR2 x TRB V 19-bispecific antigen-binding fragment thereof. In some embodiments, the monospecific antibody is a EMR2 monospecific antibody as described herein, or a EMR2-monospecific antigen binding fragment thereof. In one embodiment, said pharmaceutical composition is for the treatment of a EMR2-expressing cancer, including (but not limited to) hematological cancers such as, e.g., myeloid malignancies. In one embodiment said pharmaceutical composition is for the treatment of a EMR2-expressing cancer, including (but not limited to) the following: AML, CML, or myelodysplastic neoplasms (MDS).

[0372] The pharmaceutical compositions provided herein comprise: a) an effective amount of a multispecific antibody or the antibody fragment of the present disclosure, and b) a pharmaceutically acceptable excipient and / or carrier, which may be inert or physiologically active. In some embodiments, the bispecific antibody is a EMR2 x TRBV19 bispecific antibodyas described herein, or a EMR2 x TRB V 19-bispecific antigen-binding fragment thereof. In some embodiments, the monospecific antibody is a EMR2 monospecific antibody as described herein, or a EMR2-monospecific antigen binding fragment thereof.

[0373] Also provided herein are methods for inhibiting cytotoxicity of a cancer cell or redirecting immune or T cells against cancer cells expressing EMR2. Any of the multispecific antibodies or antibody fragments of the disclosure may be used therapeutically. For example, in one embodiment the EMR2 x TRBV 19-multispecific antibody or the EMR2 monospecific antibody may be used therapeutically to treat cancer in a subject. The method for inhibiting cytotoxicity of a cancer cell or redirecting immune or T cells against cancer cells can be practiced in vitro, in vivo, or ex vivo.

[0374] In an exemplary embodiment, multispecific antibodies or antibody fragments of the disclosure are used for the treatment of a hyperproliferative disorder in a mammal. In a further exemplary embodiment, one of the pharmaceutical compositions disclosed above, and which contains a multispecific antibody or an antibody fragment of the disclosure, is used for the treatment of a hyperproliferative disorder in a mammal. In one embodiment, the disorder is a cancer, e.g., an EMR2-expressing cancer. In particular, the EMR2-expressing cancer is a hematological cancer, e.g., myeloid malignancies. In one embodiment said pharmaceutical composition is for the treatment of a EMR2-expressing cancer, including (but not limited to) the following: AML, CML, or MDS. In exemplary embodiments, the multispecific antibody is a EMR2 x TRB VI 9-multispecific antibody as described herein, or a multispecific antigen-binding fragment thereof, and more preferably a EMR2 x TRBV19-bispecific antibody as described herein, or a EMR2 x TRBV 19-bispecific antigen-binding fragment thereof. In some embodiments, the monospecific antibody is a EMR2 monospecific antibody as described herein, or a EMR2-monospecific antigen binding fragment thereof.

[0375] Accordingly, the pharmaceutical compositions of the disclosure are useful in the treatment or prevention of a variety of cancers, e.g., EMR2-expressing cancers. In particular, the EMR2-expressing cancer is a hematological cancer, e.g., myeloid malignancies. In one embodiment said pharmaceutical composition is for the treatment of a EMR2-expressing cancer, including (but not limited to) the following: AML, CML, or MDS.

[0376] Similarly, further provided herein is a method for redirecting a T cell to EMR2- expressing cancer cells in a subject in need thereof, said method comprising administering tosaid subject a therapeutically effective amount of the bispecific antibody or the bispecific antigen-binding fragment, with an effective amount of a multispecific antibody or the antibody fragment of the present disclosure, either alone or in combination with other cytotoxic or therapeutic agents. In exemplary embodiments, the multispecific antibody is a EMR2 x TRBV19-multispecific antibody as described herein, or a multispecific antigen-binding fragment thereof, and more preferably a EMR2 x TRB VI 9-bispecific antibody as described herein, or a EMR2 x TRB V 19-bispecific antigen-binding fragment thereof. In some embodiments, the monospecific antibody is a EMR2 monospecific antibody as described herein, or a EMR2- monospecific antigen binding fragment thereof.

[0377] In some embodiments, the multispecific antibody or the multispecific antigen-binding fragment, polynucleotide, vector, pharmaceutical composition, or host cell is administered to the subject. In some embodiments, the administration of the multispecific antibody or the multispecific antigen-binding fragment, polynucleotide, vector, pharmaceutical composition, or host cell is ex vivo.

[0378] Examples of in vitro uses include treatments of autologous bone marrow prior to their transplant into the same patient in order to kill diseased or malignant cells; and prevent graft- versus-host-disease (GVHD); treatments of cell cultures in order to kill all cells except for desired variants that do not express the target antigen; or to kill variants that express undesired antigen. The conditions of non-clinical in vitro use are readily determined by one of ordinary skill in the art.

[0379] Examples of clinical ex vivo use are to remove tumor cells from bone marrow prior to autologous transplantation in cancer treatment. Treatment can be carried out as follows. Bone marrow is harvested from the patient or other individual and then incubated in medium containing serum to which is added the cytotoxic agent of the disclosure. Concentrations range from about 10 pM to 1 pM, for about 30 min to about 48 hours at about 37 °C. The exact conditions of concentration and time of incubation, i.e., the dose, are readily determined by one of ordinary skill in the art. After incubation, the bone marrow cells are washed with medium containing serum and returned to the patient by i.v. infusion according to known methods. In circumstances where the patient receives other treatment such as a course of ablative chemotherapy or total-body irradiation between the time of harvest of the marrow and reinfusionof the treated cells, the treated marrow cells are stored frozen in liquid nitrogen using standard medical equipment.

[0380] For clinical in vivo use, a therapeutically effective amount of the multispecific antibody or the antigen-binding fragment is administered to a subject in need thereof. For example, the EMR2 x TRBV19-multispecific antibodies and multispecific antigen-binding fragments thereof or the EMR2-specific antibodies and antigen-binding fragments thereof may be useful in the treatment of a cancer in a subject in need thereof. In some embodiments, the cancer is AML, CML, or MDS. In exemplary embodiments, the multispecific antibody is a EMR2 x TRBV19- multispecific antibody as described herein, or a multispecific antigen-binding fragment thereof, and more preferably a EMR2 x TRB V 19-bispecific antibody as described herein, or a EMR2 x TRBV 19-bispecific antigen-binding fragment thereof. In some embodiments, the monospecific antibody is a EMR2-monospecific antibody as described herein, or a EMR2-monospecific antigen binding fragment thereof. In some embodiments, the subject is a mammal, preferably a human. In some embodiments, the multispecific antibody or the antigen-binding fragment will be administered as a solution that has been tested for sterility.

[0381] In some embodiments, the methods describe herein further comprise administering a second therapeutic agent. In some embodiments, the second therapeutic agent is a surgery, chemotherapy, androgen deprivation therapy, radiation, or any combination thereof.

[0382] In one embodiment, a method for treating a disorder involving cells expressing EMR2 and / or TRBV19 in a subject, which method comprises administration of a therapeutically effective amount of a multispecific antibody or the fragment, such as a EMR2 x TRB VI 9 multispecific antibody described herein, and radiotherapy to a subject in need thereof is provided. In one embodiment is provided a method for treating or preventing cancer, which method comprises administration of a therapeutically effective amount of a multispecific antibody or the fragment, such as a EMR2 x TRB VI 9 antibody described herein, and radiotherapy to a subject in need thereof. Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals to a patient is provided. The source of radiation may be either external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be used in practicing such methods include, e.g., radium, cesium- 137, iridium-192, americium- 241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, actinium-225, and indium- 111.

[0383] EXAMPLES

[0384] The following examples are provided to supplement the prior disclosure and to provide a better understanding of the subject matter described herein. These examples should not be considered to limit the described subject matter. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be apparent to persons skilled in the art and are to be included within, and can be made without departing from, the true scope of the invention.

[0385] Materials and Methods

[0386] EMR2 Recombinant proteins

[0387] Human EMR2 protein is composed of a large extracellular N-terminal domain and a Class B GPCR 7-transmembrane (7TM) C-terminal portion. Located in the ECD proximal to the cell membrane resides a GPS motif that contains a protease site that is cleaved to activate the formation of a non-covalent active receptor complex of the large N-terminal domain and the GPCR-7TM domain halves. For antibody discovery purposes, the present efforts were focused on the antibody accessible extracellular N-terminal domain specifically the GAIN domain and the GPS motif proximal to the cell membrane to avoid cross reactivity to CD97, another EGF- TM7 receptor family member. CD97 / AGRE5 (Uniprot P48960), is 97% similar in sequence to EMR2 in the first 260 amino acids where the 5 EGF-like tandem sequences reside in the N- terminal ECD. The EMR2 GAIN domain portion, however is only 49.7% identical to the CD97 GAIN domain which would facilitate generating antibodies specific for EMR2 and not to CD97.

[0388] Two human EMR2 GAIN domain constructs were designed with and without the GPS motif. The first construct, EMR2W4, contains the GAIN domain plus GPS motif (EMR2 D261- T540, UniprotKB Q9UHX3 numbering), was expressed with an Avitag at the C-terminus followed by a 6xHis tag (SEQ ID NO: 226). The second construct EMR2W6 was designed without the GPS (EMR2 D261-Q478) and tagged with the Avitag and 6xHis (SEQ ID NO: 226) at the C-terminus.

[0389] The EMR2 constructs were transiently expressed in HEK293-6E or Expi293 cells and purified by utilizing the C-terminal 6xHis tag (SEQ ID NO: 226) followed by size exclusionchromatography. The purified proteins were used as immunization antigens to generate antibodies against the EMR2 GAIN domain. These antibodies were used to derive the EMR2 binding arm of the bispecific antibodies of the present disclosure.

[0390] EMR2 Cell Lines

[0391] Full length human EMR2 with its native signal sequence was overexpressed in NSO cells.

[0392] An N-terminal ECD truncated EMR2, human EMR2 GAIN + GPCR-7TM, (D261 - N823, EMR2W13) with a non-native signal sequence was also overexpressed in NSO cells.

[0393] Vb 17 proteins

[0394] The first TCR Val0.2-Vbl7 knob in hole Fc protein (B17W38) was designed based on the crystal structure PDB 2VLR and used to test binding of the Vbl7 arm of the bispecific antibodies of the present disclosure.

[0395] Because the B17W38 Vbl7 chain demonstrated ragged processing of the signal sequence, a second construct Bl 7W40 was produced. Changes were introduced to obtain correct processing of the signal sequence and to facilitate simpler mass spectrometry analysis of the purified protein. These changes included using a different signal sequence, removal of the N- terminal valine and a Ser to Cys mutation was made. Also in the Vai 0.2 chain, an extra threonine was introduced at the N-terminus.

[0396] EMR2 and related protein sequences

[0397] huEMR2 GAIN domain + GPS, D261-T540 UmprotKB Q9UHX3 with C-terminal Avi / 6x His tag (“6x His tag” disclosed as SEQ ID NO: 226), EMR2W4 (SEQ ID NO: 203)

[0398] MARKSALLALALLLLGFPGAWGDMTFSTWTPPPGVHSQTLSRFFDKVQDLGRD YKPGLANNTIQSILQALDELLEAPGDLETLPRLQQHCVASHLLDGLEDVLRGLSKNLSNG LLNFSYPAGTELSLEVQKQVDRSVTLRQNQAVMQLDWNQAQKSGDPGPSWGLVSIPG MGKLLAEAPLVLEPEKQMLLHETHQGLLQDGSPILLSDVISAFLSNNDTQNLSSPVTFTF SHRSVIPRQKVLCVFWEHGQNGCGHWATTGCSTIGTRDTSTICRCTHLSSFAVLMAHYD VQEEDPVLTVITGGGGSGLNDIFEAQKIEWHEGGGGSHHHHHHG

[0399] huEMR2 GAIN domain, D261-Q478 UmprotKB Q9UHX3 with C-terminal Avi / 6x His tag (“6x His tag” disclosed as SEQ ID NO: 226), EMR2W6 (SEQ ID NO: 204)

[0400] MARKSALLALALLLLGFPGAWGDMTFSTWTPPPGVHSQTLSRFFDKVQDLGRD YKPGLANNTIQSILQALDELLEAPGDLETLPRLQQHCVASHLLDGLEDVLRGLSKNLSNG LLNFSYPAGTELSLEVQKQVDRSVTLRQNQAVMQLDWNQAQKSGDPGPSWGLVSIPG MGKLLAEAPLVLEPEKQMLLHETHQGLLQDGSPILLSDVISAFLSNNDTQNLSSPVTFTF SHRSVIPRQGGGGSGLNDIFEAQKIEWHEGGGGSHHHHHHG

[0401] huEMR2 full length, M1-N823 UmprotKB Q9UHX3-1, EMR2W12 (SEQ ID NO: 205)

[0402] MGGRVFLVFLAFCVWLTLPGAETQDSRGCARWCPQDSSCVNATACRCNPGFSS FSEIITTPMETCDDINECATLSKVSCGKFSDCWNTEGSYDCVCSPGYEPVSGAKTFKNESENTCQDVDECQQNPRLCKSYGTCVNTLGSYTCQCLPGFKLKPEDPKLCTDVNECTSGQ NPCHSSTHCLNNVGSYQCRCRPGWQPIPGSPNGPNNTVCEDVDECSSGQHQCDSSTVCF NTVGSYSCRCRPGWKPRHGIPNNQKDTVCEDMTFSTWTPPPGVHSQTLSRFFDKVQDL GRDYKPGLANNTIQSILQALDELLEAPGDLETLPRLQQHCVASHLLDGLEDVLRGLSKN LSNGLLNFSYPAGTELSLEVQKQVDRSVTLRQNQAVMQLDWNQAQKSGDPGPSWGL VSIPGMGKLLAEAPLVLEPEKQMLLHETHQGLLQDGSPILLSDVISAFLSNNDTQNLSSPVTFTFSHRSVIPRQKVLCVFWEHGQNGCGHWATTGCSTIGTRDTSTICRCTHLSSFAVL MAHYDVQEEDPVLTVITYMGLSVSLLCLLLAALTFLLCKAIQNTSTSLHLQLSLCLFLAH LLFLVAIDQTGHKVLCSIIAGTLHYLYLATLTWMLLEALYLFLTARNLTWNYSSINRFM KKLMFPVGYGVPAVTVAISAASRPHLYGTPSRCWLQPEKGFIWGFLGPVCAIFSVNLVL FLVTLWILKNRLSSLNSEVSTLRNTRMLAFKATAQLFILGCTWCLGILQVGPAARVMAY LFTIINSLQGVFIFLVYCLLSQQVREQYGKWSKGIRKLKTESEMHTLSSSAKADTSKPSTVN

[0403] huEMR2 GAIN + GPCR 7TM, D261-N823 UmprotKB Q9UHX3, EMR2W13 (SEQ ID NO: 206)

[0404] MARKSALLALALLLLGFPGAWGDMTFSTWTPPPGVHSQTLSRFFDKVQDLGRD YKPGLANNTIQSILQALDELLEAPGDLETLPRLQQHCVASHLLDGLEDVLRGLSKNLSNGLLNFSYPAGTELSLEVQKQVDRSVTLRQNQAVMQLDWNQAQKSGDPGPSWGLVSIPG MGKLLAEAPLVLEPEKQMLLHETHQGLLQDGSPILLSDVISAFLSNNDTQNLSSPVTFTFSHRSVIPRQKVLCVFWEHGQNGC GHWATTGCSTIGTRDTSTICRCTHLSSFAVLMAHYD VQEEDPVLTVITYMGLSVSLLCLLLAALTFLLCKAIQNTSTSLHLQLSLCLFLAHLLFLVA IDQTGHKVLCSIIAGTLHYLYLATLTWMLLEALYLFLTARNLTWNYSSINRFMKKLMF PVGYGVPAVTVAISAASRPHLYGTPSRCWLQPEKGFIWGFLGPVCAIFSVNLVLFLVTL WILKNRLSSLNSEVSTLRNTRMLAFKATAQLFILGCTWCLGILQVGPAARVMAYLFTIIN SLQGVFIFLVYCLLSQQVREQYGKWSKGIRKLKTESEMHTLSSSAKADTSKPSTVN

[0405] TCR Val0.2 x Vbl7 -KiHFc (knob-in-hole Fc), B17W38 Val0.2 -knob-in-hole Fc chain 1 (SEQ ID NO: 211)

[0406] MAWVWTLLFLMAAAQSIQAQLLEQSPQFLSIQEGENLTVYCNSSSVFSSLQWYR QEPGEGPVLLVTWTGGEVKKLKRLTFQFGDARKDSSLHITAAQPGDTGLYLCAGAGSQGNLIFGKGTKLSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYI TDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSEPKSCDKTHTCPPC PAPELLGGPSVFLFPPI<PI<DTLMISRTPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI< TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CT<VSNI<ALPAPIEI<TISI<AI<GQPREPQ VYVYPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFAL VSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0407] Vbl7 -knob-in-hole Fc chain 2 (SEQ ID NO: 212)

[0408] MAWVWTLLFLMAAAQSIQAVDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAM YWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLC AS S SRS S YEQ YFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGF YPD HVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYSLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADEPKSCDKTHTCPPCPAPELLGGPSVFLFPPI<PI<DTLMISRTPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNST YRVVSVLTVLHQDWLNGI<EYI<CT<VSNI<ALPAPIEI<TISI<AI<GQPREPQVYVLPPSREE MTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0409] TCR Val0.2 x Vbl7 -KiHFc (knob-in-hole Fc), B17W40 Val0.2-knob-in-hole Fc Chain 1 (SEQ ID NO: 213)

[0410] MAWVWTLLFLMAAAQSIQATQLLEQSPQFLSIQEGENLTVYCNSSSVFSSLQWY RQEPGEGPVLLVTWTGGEVKKLKRLTFQFGDARKDSSLHITAAQPGDTGLYLCAGAGS QGNLIFGKGTKLSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYI TDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSEPKSCDKTHTCPPC PAPELLGGPSVFLFPPI<PI<DTLMISRTPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI< TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAPIEI<TISI<AI<GQPREPQ VYVYPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFAL VSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0411] Vbl7-knob-in-hole-Fc Chain 2 (SEQ ID NO: 214)

[0412] MARI<SALLALALLLLFGPAWADGGITQSPI<YLFRI<EGQNVTLSCEQNLNHDAM YWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLC AS S SRS S YEQ YFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPD HVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQ VQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADEPKSCDKTHTCPPCPAPELLGGPSVF LFPPI<PI<DTLMISRTPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNST YRVVSVLTVLHQDWLNGI<EYI<CT<VSNI<ALPAPIEI<TISI<AI<GQPREPQVYVLPPSREE MTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0413] Single underlined sequences are the signal sequences for the expressed proteins, protein tags are double underlined, linker sequences are wave underlined, and Avitag is dotted underlined.

[0414] Immunization

[0415] Twenty-four (24) Ablexis mice were immunized with Human EMR2 alone or combined with Rhesus EMR2 using 4 different protocols (AB298, AB299, AB300, AB301).Prior to injection, Human and Rhesus EMR2 GAIN Avi 6xHis (SEQ ID NO: 226) were produced at Syngene (batches EMR2W4.001 and EMR2W8.001). AB298, AB299 receivedCFA / Sigma adjuvant and AB300, AB301 received CL413 adjuvant. The mice received weeklyRIMMS + IP injections for 5 weeks and final boost on Day 42. The final boost contained lOugHuman EMR2 and 50ug mouse CD40 monoclonal antibody. Sera was collected at Day 36 and assessed for circulating IgG specific antibodies to human EMR2. Titers were determined via cell based FACS with OCL AML-3 and OCL AML-5 cells.

[0416] Hybridoma Fusion

[0417] On day 45 and day 46 of the immunization schedule, lymph nodes and spleen were harvested from the mice for B lymphocytes fusion. Lymph nodes and spleens were homogenized by group into single-cell suspensions and fused with FO multiple myeloma cell line for hybridoma generation. A total of four separate fusions were performed as follows: Fusion 1 (lymph nodes) and fusion 2 (spleen) from mouse #’s 1, 5, 6, 9, 11, 12, 15, 17, 18. Fusion 3 (lymph nodes) and fusion 4 (spleen) from mouse #’s 2, 14, 19, 20, 21, 22, 23. The hybridoma cells were then plated and cultured for 7-14 days.

[0418] scFv conversion of EMR2 hybridomas (EMR2-00030, EMR2-00031)

[0419] This is a summary of the process used to generate and identify the EMR2 binder EMR2B57 which was used on EMR2xVbl7 EMVBB27. This binder was generated from ‘direct PCR’ HTP scFv screening based approach. cDNA from V-gene recovery was used to pair the VH / VL wells directly with the scFv linker and then the generated scFv pairs were tested in E. coli-based expression and screening platform.

[0420] Four 96- well plates were used for the EMR2 scFv conversion from V-region cloning group. In total, there were 358 unique scFv PCR wells and screened -2300 colonies. Identified 511 samples from the 2300 colonies as positive binders and sent these off to sequencing. Upon receiving sequencing results, SL performed alignments and clustering to identify 156 unique scFv consensus sequences. These sequences then moved into thermal stability screening to identify rank.

[0421] Summary

[0422] Total sequences: 512

[0423] HC: 491

[0424] LC: 493

[0425] Assembled scFv 477

[0426] Clusters by CDR: 156

[0427] Hybridoma Screening

[0428] Hybridoma supernatants were screened by cell MSD on OCI-AML-3 cells (AML cell line with high EMR2-expression). Primary hits were defined as samples giving assay signal greater than 9.33 times the negative control average. A total of 616 samples meeting this criterion were subsequently scaled up and re-screened by FACS and ELISA. FACs assay was done on primary EMR2-expressing OCI-AML-3, OCI-AML-5, SIG-M5 (very low expression)and negative CARNAVAL cells. Samples were also screened by ELISA for human or mouse light chains. A total of 348 hits met the desired binding profile (binding to OCI- AML-3 cells >5x S:B by flow). Selected hits from the combined confirmatory ELISA and FACS screen are expanded in 48-well plate format. From these hits, frozen cell stocks, cell lysates and culture supernatant were made. The selected hybridoma lysates were sent to the molecular biology team for variable region cloning. The recovered variable regions were cloned into a human IgGl vector and recombinantly expressed. The human IgGl antibodies were tested by flow cytometry for cell binding to OCI3 cells with negative counter screen against Carnaval cells. 92 clones meeting the criteria were sequenced, expressed, and tested for additional characterization.

[0429] A total of 134 mAbs were recovered and recombinantly expressed.

[0430] EMR2-00061

[0431] 128 out of 134 EMR2 tested samples showed S / B>5 on OciAml-3 cells at 50nM or 1 OnM concentration

[0432] EMR2-00064

[0433] Biophysical testing: Affinity kinetics and Thermal stability was completed for 128 mAbs using hu EMR2 on Biacore 8K+

[0434] EMR2 Affinity

[0435] *51 % of IgG mAbs and 98% VHHs bound Hu-EMR2.

[0436] Thermostability

[0437] In both mAb panels, roughly 30% of binders have TM1 >69°C.

[0438] 30% (19 of 65) of EMR2 binders from affinity screen have a TM1 >69°C.

[0439] 8% of the anti-EMR2 VHH binders were >55°C.

[0440] 8% (5 of 61) of binders from affinity screen have a TM1 >55°C.

[0441] The Fab arm of molecule EMVBB8 was derived from Hybridoma clone GDB# EMR2HB26SC1147J74C09 (CL002044637).

[0442] The Fab arm of EMVBB7 was derived from hybridoma clone GDB# EMR2HB25SC1147_026G09 (CL002031617).

[0443] The Fab arm of EMVBB6 was derived from hybridoma clone GDB# EMR2SB1 SCI 147_207P22(CL002297775).

[0444] The scFv arm of EMVBB27 was generated from a direct PCR high-throughput scFv screening based approach. cDNA from V-gene recovery was used to pair the VH / VL wellsdirectly with the scFv linker and then the generated scFv pairs were tested in an E. coli-based expression and screening platform.

[0445] T-Cell Engagement Cytotoxicity Assay using EMR2xTRBV19 leads

[0446] EMR2-positive AML cell line 0CI-AML3, were incubated with EMR2xTRBV19 Abs with either pan human T-cells for 72 h at 37 °C at the effector to target (E:T) ratio of 10: 1 or with healthy human peripheral blood mononuclear cells (PBMCs) with E:T ration of 5: 1 for 96 h at 37 °C. Cell cytotoxicity was measured at by flow cytometry.

[0447] Example 1. Target Validation

[0448] Targeted immunotherapy in AML remains a challenge due to the heterogenous nature of the AML cancer cells (blasts) and the lack of AML-specific antigens. While several immunotherapies targeting CD33, CD 123, or CLL1, including chimeric antigen receptor (CAR)- T and CAR-NK cell therapies, are being evaluated clinically, challenges have been observed with CD3 TCE therapies due to lack of efficacy and tolerability as well as an unacceptable safety profile. EMR2xTRBV19 TCEs represent a treatment that may address unmet medical needs of patients with AML whose disease no longer responds to SoC. Bispecific TRBV19 antibodies could provide potent cytotoxicity to cancer cells by engaging a small population of cytotoxic T cells while avoiding pan-T-cell activation that is known to cause CRS.

[0449] TRBV19+ T cell

[0450] During T-cell development, T-cell receptor (TCR) diversity results from variable diversity joining (VDJ)-domain recombination, leading to unique TCR Va and VP features in specific T-cell clones. The TCR0 locus comprises a cluster of 52 functional VP gene segments, a single D gene segment, thirteen J gene segments, and 2 TRBC genes. There are 24 families of VP gene segments in humans. Consequently, each TCR VP family is used by only 0.58% to 10.84% of the TCR repertoire (van der Geest KS, Abdulahad WH, Horst G, et al. Quantifying distribution of flow cytometric TCR-VP usage with economic statistics. PLoS One. 2015;10(4):e0125373). Extensive repertoire analysis has shown preferential skewing of distinct TCR VP genes for selective expansion of T cells in certain diseases such as VP6 and Vpi5 in rheumatoid arthritis (Jenkins RN, Nikaein A, Zimmermann A, Meek K, Lipsky PE. T cell receptor V beta gene bias in rheumatoid arthritis. J Clin Invest. 1993;92(6):2688-2701), VP8 in IgA nephropathy (Muro K, Yamagata K, Kobayashi M, Hirayama K, Koyama A. Usage of T cellreceptor variable segments of the beta-chain in IgA nephropathy. Nephron. 2002;92(l): 56-63), and V05, V08, V015, V016, and V018 in sarcoidosis (Forman JD, Klein JT, Silver RF, Liu MC, Greenlee BM, Moller DR. Selective activation and accumulation of oligoclonal V beta-specific T cells in active pulmonary sarcoidosis. J Clin Invest. 1994;94(4): 1533-1542).

[0451] Similarly, after influenza A infection, Ml 58-66 peptide is presented in the context of major histocompatibility complex (MHC)-allele human leukocyte antigen (HLA)-A*0201 and the resulting cytotoxic T-lymphocytes (CTLs) predominantly express TRBV19 (Lawson TM, Man S, Williams S, Boon AC, Zambon M, Borysiewicz LK. Influenza A antigen exposure selects dominant Vbetal7+ TCR in human CD8+ cytotoxic T cell responses. Int Immunol.2001 ; 13(11 ): 1373-1381). Expansion of CD8+TRBV19+correlates with M158-66-specific lysis while TRBV19 depletion from PBMCs abrogates CTL response to the influenza infection (Lehner PJ, Wang EC, Moss PA, et al. Human HLA-A0201 -restricted cytotoxic T lymphocyte recognition of influenza A is dominated by T cells bearing the V beta 17 gene segment. J Exp Med. 1995; 181 (1): 79-91). Upon clearance of infection, a highly focused memory T-cell repertoire - wherein TRBV19 cells prevail - provides efficient recall response in future exposures. Since influenza A is a common viral infection of humans and most adults have serological evidence of previous influenza A exposure, the frequency of TRBV19+T cells was relatively stable in the peripheral blood of healthy donors and AML patients with a mean value of 5.7% and 6.7%, respectively (FIG. 1).

[0452] The strong cytotoxic potential and retained memory phenotype of TRBV19+T cells make them an attractive candidate for immune-cell-engaging approaches. Leveraging a small and relatively stable population of T cells with strong cytotoxic potential would potentially reduce the risk of CRS while inducing potent antitumor efficacy over time.

[0453] EMR2 as Tumor-associated Antigen

[0454] The adhesion G-protein-coupled receptors (aGPCRs) constitute an evolutionarily membrane protein family with emerging roles in many important biological processes. They are uniquely characterized by the chimeric composition of a large extracellular domain (ECD) and a 7-pass transmembrane (7TM) region. The aGPCRs are further classified into subfamilies based on the nature of their N-terminal domains (i.e., lectin-like, Ig-like, epidermal growth factor [EGF]-like, or cadherin-like motifs). One subfamily, the adhesion G-protein-coupled receptor E (ADGRE) family (i.e., EMR1, 2, 3, 4, and CD97) is characterized by the presence of severaltandem EGF-like domains in their N-terminus (McKnight AJ, Gordon S. EGF-TM7: a novel subfamily of seven-transmembrane-region leukocyte cell-surface molecules. Immunol Today. 1996;17(6):283-287). Within this family, the EGF domains of EMR2 and CD97 demonstrate the most homology, differing by only 6 amino acids (Lin HH, Stacey M, Hamann J, Gordon S, McKnight AJ. Human EMR2, a novel EGF-TM7 molecule on chromosome 19pl 3.1 , is closely related to CD97. Genomics. 2000;67(2): 188-200), whereas EMR2 and EMR3 are the only members that lack mouse orthologs.

[0455] A defining feature of EMR2, similar to aGPCRs, is the presence of a GAIN domain, which is capable of self-catalytic cleavage, resulting in the generation of an extracellular N- terminal fragment and a 7TM C-terminal fragment that is involved in the cellular adhesion and signaling functions (FIG. 2) (Huang YS, Chiang NY, Chang GW, Lin HH. Membraneassociation of EMR2 / ADGRE2-NTF is regulated by site-specific N-glycosylation. Sci Rep. 2018;8(l):4532).

[0456] EMR2 expression is restricted to myeloid cells including mature monocytes, macrophages, and BDCA-3+myeloid dendritic cells, whereas minimal expression is found on granulocytes (Kwakkenbos MJ, Chang GW, Lin HH, et al. The human EGF-TM7 family member EMR2 is a heterodimeric receptor expressed on myeloid cells. J Leukoc Biol. 2002;71(5):854-862). Expression is highly regulated during monocyte / macrophage differentiation (Chang GW, Davies JQ, Stacey M, et al. CD312, the human adhesion-GPCR EMR2, is differentially expressed during differentiation, maturation, and activation of myeloid cells. Biochem Biophys Res Commun. 2007;353(l): 133-138; Boyden SE, Desai A, Cruse G, et al. Vibratory urticaria associated with a missense variant in ADGRE2. N Engl J Med.2016;374(7):656-663). Unlike CD97, no expression of EMR2 is reported on resting or activated lymphocytes.

[0457] Extensive studies of EMR2 are hampered by the lack of mouse orthologues. Functionally, EMR2 has also been implicated in autoimmune disease and neutrophil function. EMR2+macrophages and dendritic cells are increased in the synovium of patients with rheumatoid arthritis (Kop EN, Kwakkenbos MJ, Teske GJ, et al. Identification of the epidermal growth factor-TM7 receptor EMR2 and its ligand dermatan sulfate in rheumatoid synovial tissue. Arthritis Rheum. 2005;52(2):442-450). Additionally, EMR2 is implicated in neutrophil function by mediating activation and cytokine secretion in the presence of lipopolysaccharide and IL- 10(Chang GW, Davies JQ, Stacey M, et al. CD312, the human adhesion-GPCR EMR2, is differentially expressed during differentiation, maturation, and activation of myeloid cells. Biochem Biophys Res Commun. 2007;353(l): 133-138). Circulating neutrophils in patients with systemic inflammation also exhibit elevated EMR2 expression (Chen TY, Hwang TL, Lin CY, et al. EMR2 receptor ligation modulates cytokine secretion profiles and cell survival of lipopolysaccharide-treated neutrophils. Chang Gung Med J. 2011;34(5):468-477.). Additionally, foamy macrophages in atherosclerotic vessels and splenocytes in patients with Gaucher’s disease express EMR2 (van Eijk M, Aust G, Brouwer MS, et al. Differential expression of the EGF-TM7 family members CD97 and EMR2 in lipid-laden macrophages in atherosclerosis, multiple sclerosis and Gaucher disease. Immunol Lett. 2010;129(2):64-71).

[0458] Several reports have identified EMR2 in human neoplasms (Aust G, Steinert M, Schutz A, et al. CD97, but not its closely related EGF-TM7 family member EMR2, is expressed on gastric, pancreatic, and esophageal carcinomas. Am J Clin Pathol. 2002;118(5):699-707). EMR2 mRNA expression in AML was reported to be high when compared to normal cells (FIG. 15).

[0459] In an extensive AML surfaceome dataset, EMR2 was found to be expressed at low levels in the gut, ovary, and spleen, and the fluorescence-activated cell sorting (FACS) analyses in that study detected EMR2 in -93% of cells in AML patient samples (Perna F, Berman SH, Soni RK, et al. Integrating proteomics and transcriptomics for systematic combinatorial chimeric antigen receptor therapy of AML. Cancer Cell. 2017;32(4):506-519.e5). To confirm the protein expression of EMR2 and receptor density, FACS analysis was performed on AML cell lines (Table 4) and primary AML patient samples as well as the different healthy hematopoietic cells (FIG. 4). In addition to the myeloid populations, low / limited EMR2 expression was detected on lymphocytes, corresponding to mean values of 1.4% on B cells, 3.6% on NK cells, and 1% on T cells (CD4+and CD8+T cells). Furthermore, receptor densities of the positive populations of lymphocytes (B, T, and NK cells) were found in a range of 100 to 200 receptors, representing 3% of that on monocytes (4,959 receptors / cell).

[0460] Table 4. Calculated receptor counts for EMR2 on different myeloid cell lines.

[0461] EMR2, epidermal-growth-factor-like module-containing mucin-like hormone receptor-like 2; FAB, French- American-British.

[0462] Receptor density of EMR2 was measured in different malignant myeloid cell lines. FAB classification of leukemia subtypes is depicted when available.

[0463] Alternatively spliced transcripts (transmembrane and soluble) of EMR2 were detected in colorectal carcinoma cell lines due to partial or complete deletion of Exon 13 of the canonical transcript that leads to the soluble isoform (Lin HH, Stacey M, Yona S, Chang GW. GPS proteolytic cleavage of adhesion-GPCRs. Adv Exp Med Biol. 2010;706:49-58). Physiological soluble EMR2 levels were assessed in sera of healthy donors (n=9) and AML patients (n=10) by enzyme-linked immunosorbent assay (ELISA) detecting the ECD domain. Soluble EMR2 concentrations were low and comparable between healthy donors and AML patients, ranging from 1.08 pM (based on reported ECD size of 65 kDa [9]) to 61.54 pM with a median of 5.00 pM in healthy donors and ranging from 1.08 pM to 59.97 pM with a median of 4.03 pM in AML patients (FIG. 5).

[0464] Overall, the high expression of EMR2 on AML blast and the distinctive features of TRBV19+T cells together provide a novel opportunity for immunotherapy.

[0465] Putative Target Liabilities

[0466] Due to expression on myeloid progenitors and downstream myeloid cells, AML- targeted therapies have been associated with hematological adverse events such as myelosuppression and cytopenias (Maakaron JE, Rogosheske J, Long M, Bachanova V, Mims AS. CD33 -targeted therapies: beating the disease or beaten to death? J Clin Pharmacol.2021 ;61 (1): 7-17; Uckun FM, Lin TL, Mims AS, et al. A clinical phase IB study of the CD3xCD123 bispecific antibody APVO436 in patients with relapsed / refractory acute myeloid leukemia or myelodysplastic syndrome. Cancers (Basel). 2021 ; 13(16):4113). However preclinical data presented below indicates a therapeutic window between targeting AML cells and healthy myeloid cells with the bispecific molecules of the present disclosure.

[0467] Example 2. Therapeutic Profile

[0468] The bispecific molecules of the present example are an IgGl bispecific antibody that simultaneously binds to the 0 subunit (TRBV19, herein called TRBV19) of TCR (Uniprot ID: A0A5B3) on T cells and to EMR2 on AML cells. The antibody features the AAS mutations in the constant region to abolish interaction with Fc receptors and heterodimerization is enhanced using the knobs-into- holes platform mutations (Ridgway JB, Presta LG, Carter P. ‘Knobs-into- holes’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. 1996;9(7): 617-621). The molecule comprises an anti-TRBV19 spFv fused onto the N-terminus of the ‘knob’ Fc region (i.e., T366W). The ‘hole’ chain (i.e., T366S, L368A, Y407V) features an anti-EMR2 Fab at the N-terminus of the Fc. The hole chain also contains the ‘RF’ mutations (i.e., H435R, Y436F) to disrupt protein A binding of monomeric and homodimerized hole chains (Tustian AD, Endicott C, Adams B, Mattila J, Bak H. Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity. MAbs. 2016;8(4): 828-838). The bispecific was developed to evaluate the therapeutic potential of tumortargeting EMR2 and of TRBV19 for T-cell engagement.

[0469] Intrinsic Design Properties of the bispecific antibodies

[0470] Table 5. Biophysical assessment and results.

[0471] AAS, L234A, L235A, D265S; AUC, analytical ultracentrifugation; aHIC, analytical hydrophobic interaction chromatography; B17W38, ValO.2-TRBV19-Fc fusion protein; cIEF, capillary isoelectric focusing; cyno, cynomolgus monkey; Cys, cysteine; EMR2, epidermal-growth-factor-like module-containing mucin-like hormone receptor-like 2; DSC, differential scanning calorimetry; EMR2W14, Cyno EMR2 GAIN domain protein; EMR2W4, Human EMR2 GAIN domain protein; Fc, fragment crystallizable; FcRn, neonatal Fc receptor; FDS, fluorescence detection system; gly, glycosylation; HDX-MS, hydrogen-deuterium exchange mass spectrometry; hu, human; Ig, immunoglobulin; KD, equilibrium dissociation constant; mAb, monoclonal antibody; MS, mass spectrometry; N / A, not assessed; pl, isoelectric point; ref, reference; SEC, size-exclusion chromatography; SPR, surface plasmon resonance; Tm, melting temperature; TMP, target medicinal product; Ton, temperature of onset of melting.

[0472] Table 6. Chemical and PTM stability in forced degradation conditions.Assay Release Physio- pH 8.5 pH 5.0 Chem Ox Thermal logicalIntact mass 127,380.1 127,381.6 127,382.3 127,381.7 127,476 127,379.8(Da)% Monomer 99.4 98.7 97.4 98.4 97.9 98.4(aSEC)% Purity 100 / 99.5 98.6 / 99.3 94.4 / 96.9 98.8 / 99.4 94.3 / 99.6 99 / 99.4Clipping None None None None Trace None(intact mass) clipping(EMR2 SPR 100 100 97 98 100 98[M] / % active)Binding 9.4X10’8 / l.OxlO’7 / 8.9xl0’8 / 9.7X10’8 / 9.3 X10’8 / 9.4X10’8 / (TRBV19 SPR 100 106 101 101 104 106[M] / % active)CDR oxidation <1.0% <1.0% <1.0% <1.0% <1.0% <1.0%CDR 1.7% N33 1.5% N56 1.8% N35 2.2% N35 2.1% N35Deamidation 1.9% N35 LCDR 1 of HCDR2 of LCDR1 of LCDR1 of LCDR1 ofLCDR1 of LC2 HC2 LC2 LC2 LC2LC2 3.0% N35 4.3% N33 1.4% N92LCDR1 of LCDR1 of LCDR3 ofLC2 LC2 LC23.7% N35 LCDR1 of LC2CDR 2.1% D185 2.1% D185 2.0% D185 1.9% D185 2.0% D185 inIsomerization HCDR2 of HCDR2 of HCDR2 of HCDR2 of HCDR2 of2.1% D185 HC1 HC1 HC1 HC1 HC1HCDR2 of 1.5% D99 1.4% D99 2.0% D99 1.3% D99 2.1% D99HC1 HCDR3 of HCDR3 of HCDR3 of HCDR3 of HCDR3 of1.2% D99 HC2 HC2 HC2 HC2 HC2HCDR3 of HC2

[0473] aSEC, analytical size-exclusion chromatography; CDR, complementarity determining region; Chem Ox, chemical oxidation; DSF, differential scanning fluorimetry; EMR2, epidermal-growth-factor-like module-containing mucin- like hormone receptor-like 2; GXII, capillary electrophoresis; HC, heavy chain; HCDR, heavy-chain complementarity determining region; KD, equilibrium dissociation constant; LC, light chain; LCDR, light-chain complementarity determining region; N / A, not assessed; NR / R, non-reduced / reduced; PTM, post-translational modification; SPR, surface plasmon resonance; Tm, melting temperature; Ton, temperature of onset of melting.

[0474] EMR2 Target Arm and TRBV19 Arm 37°C Binding Characterization of the bispecific

[0475] Flow cytometry was used to measure EMR2 arm binding affinity of the bispecific to AML cell lines MOLM-13, 0CLAML2, 0CLAML3, and 0CLAML5 that endogenously express EMR2 at varying receptor densities. Bispecific antibody binding to OCI-LY10, a B-cell lymphoma line that does not express EMR2, was also tested to confirm target specificity. Cell lines were administered increasing concentrations of a bispecific antibody, EMR2xNull, and TRBV19xNull for 1 hour at 37°C. A bispecific antibody showed concentration-dependent binding with an EC50 range of 27 to 55 nM on all EMR2-expressing cell lines (Table 7, FIG. 6). The TRBV19xNull negative control had no specific binding to any of the 5 tumor cell lines tested.

[0476] Table 7. EMR2 arm binding affinity to AML cell lines.MOLM-13 OCI-AML2 OCI-AML3 OCI-AML5 OCI-LY10 Binding EC7, (nM) ± SEM 53±20 _ 55±54 _ 48±30 _ 27±7 _ NDAML, acute myeloid leukemia; EC50, 50% effective concentration; EMR2, epidermal-growth-factor-like module- containing mucin-like hormone receptor-like 2; ND, not detected; SEM, standard error of the mean.

[0477] Bispecific Antibody kinetic binding to OCI-AML3 cells

[0478] Flow cytometry was used to assess stability of EMR2 binding of a bispecific antibody to the OCI-AML3 cell line, which endogenously expresses EMR2. When OCI-AML3 cells were administered a bispecific antibody for 1, 3, 5, and 24 hours at 37°C, stable binding was observed at all concentrations tested (3, 30, and 300 nM; FIG. 7). TRBV19xNull, which lacks the EMR2 binding arm present in the bispecific antibody, displayed no binding to OCI-AML3 cells at any concentration or timepoint.

[0479] Bispecific antibody binding to TRBV19+ T cells

[0480] Flow cytometry was used to measure specific binding of the TRBV19 arm of the bispecific antibody to TRBV19+T cells from 6 different healthy human pan-T-cell donors. When pan-T cells were administered increasing concentrations of the bispecific antibody at 1 hour, 37°C, concentration- dependent binding was observed (FIG. 8, Table 8). The EC50 value of detection of TRBV19+cells for the bispecific antibody was 7.8 ± 0.9 nM. Specific binding to TRBV19+cells was not detected when stained with EMR2xNull, which lacks the TRBV19-spFv arm.

[0481] Table 8. EMR2 arm binding affinity to AML cell lines.Parameter Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 Donor 6ECso (nM) 7.8 9.7 6.2 10.6 9.5 4.9EC50, 50% effective concentration.Data from 6 pan-T-cell donors. Values are calculated as log (agonist) versus variable slope (4 parameters).

[0482] The bispecific antibody displayed good intrinsic biophysical properties.

[0483] The bispecific antibody as described in the example is a fully human bispecific monoclonal antibody targeting the TRBV19 TCR with one binding arm and tumor cell-surface antigen EMR2 on the other binding arm. The bispecific antibody showed good intrinsic biophysical properties and bound to all tested EMR2-expressing cell lines. The bispecific antibody showed stable tumor cell binding profiles over 24 hours. The bispecific antibody also showed binding to a small subpopulation of primary human T cells expressing TRBV19 on the cell surface.

[0484] Example 3. Cytotoxicity Assays for the EMR2xTRBV 19 Abs

[0485] The EMR2 antibodies were assessed for cell cytotoxicity of OCI-AML3 AML cell line in T-cell or PBMC cytotoxicity assays. The results from one T-cell donor or one PBMC donor are shown in FIG. 9 A (T-cell cytotoxicity assay) and 9B (PBMC cytotoxicity assay), where a dose range of EMR2xTRBV19 antibodies was used.

[0486] Example 4. Human IgGl Glm(17), Kappa bispecific knobs-into-holes antibody targeting TCR TRBV19 and ERM2

[0487] The present disclosure provides an immunoglobulin (Ig) G1 bispecific antibodies that simultaneously bind to the T cell receptor (TCR) TRBV19 on T lymphocytes cells, and to EMR2 (Adhesion G protein-coupled receptor E2, Uniprot ID: Q9UHX3) on tumor cells. The exemplary antibody of the present disclosure features mutations of L234A, L235A, and D265S (AAS) in the constant region (Fc) to abolish interaction with Fc receptors and heterodimerization is enhanced using the knobs-into-holes platform mutations (Ridgway, J. B., Presta, L. G. & Carter, P. ‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng 9, 617-621 (1996)). The molecule comprises an anti-TRBV19 spFv fused onto the N-terminus of the “Knob” Fc region (T366W). The “hole” chain (T366S, L368A, Y407V) features an anti-ERM2 Fab at the N-terminus. The hole chain also contains the “RF” mutations (H435R, Y436F) to disrupt protein A binding of monomeric and homodimerized hole chains (Tustian, A. D., Endicott, C., Adams, B., Mattila, J. & Bak, H. Development of purificationprocesses for fully human bispecific antibodies based upon modification of protein A binding avidity. MAbs 8, 828-838 (2016)).

[0488] Source of Coding Sequence

[0489] The bispecific was generated by co-expression of the anti-TRBV19 spFv-Fc “knob” Heavy Chain (HC1) with the anti-EMR2 Fab Heavy Chain containing the “hole” and RF mutations (HC2) and paired with the EMR2 Light Chain 2 (LC2).

[0490] The anti-TCR Vbl7 variable region VR000071196 was derived from murine clone E17.5F3.15.13 obtained from Beckman Coulter. The parental murine variable sequences were humanized and a deamidation site was mitigated. Briefly, the murine complementarity determination regions (CDRs) according to the AbM definitions were grafted into the human IGHV4-61*02-IGHJ6-01 and IGKVl-39*01-IGKJ2-01 human germlines. A human / murine binary library of VH variants in the frameworks (sequential positions 41, 45, 49, 68, 72 and 98) were generated and screened against Vbl7+cells and the best binder with two back mutations (V72R and R98S) was selected as the humanized variant. The parental Light Chain CDR1 contained a deamidation PTM-risk NG sequence motif at positions 33(N)-34(G), and this risk was eliminated by mutation of G34R after all possible mutations in both N and G positions were tested. The Vbl7 binder Bl 7B852-G34R was formatted as spFv in the LH orientation (light chain-linker-heavy chain) as described in Boucher et al. (Boucher, L.E, Prinslow, E.G, et al. "Stapling" scFv for multispecific biotherapeutics of superior properties. mAbs 15(1): 2195517. (2023)) and fused to the N-terminus of Fc for the final molecule.The anti-ERM2 variable region VR000049625 featured in the bispecific is derived from the human IgGl , Kappa antibody named EMR2B454, discovered by immunizing transgenic humanized mice [Ablexis] with recombinant EMR2. The anti-EMR2 binder was formatted as a Fab at the N-terminus.

[0491] Generation of the expression plasmids used to generate the manufacturing cell line was performed as previously known.

[0492] Amino Acid Sequence of an Exemplary Bispecific Molecule

[0493] The amino acid sequence for exemplary bispecific Heavy Chain 1, Heavy Chain 2, and Light Chain 2 are shown in FIG.10A-10C. The sequences were confirmed by peptide mapping and mass spectrometry. The complementarity-determining regions (CDRs) using AbM definition are shown in bold. Amino acid sequences of exemplary bispecific antibodies are also shown inFIGs. 30-33. Amino acid sequences of exemplary Kappa, Lambda, CH and CHI regions are shown in FIG. 34.

[0494] The Asp residue at position 1 of exemplary bispecific Heavy Chain 1 , Glu at position 1 of exemplary bispecific Heavy Chain 2, and Glu residue at position 1 of exemplary bispecific Light Chain 2 constitute the N-termini of the mature chains.

[0495] The following mutations (Eu numbering) were introduced:

[0496] L234A L235A D265S (AAS) to eliminate binding to Fc gamma receptors FcyRI, FcyRIIA, FcyRIIb, and FcyRIIIa (Fc receptor silencing).

[0497] With the purpose of enabling formation of the bispecific antibody, T366W (knob) was introduced into Heavy Chain 1, and T366S L368A Y407V (hole) was introduced into the Heavy Chain 2 (Ridgway, J. B., Presta, L. G. & Carter, P. 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng 9, 617-621 (1996)).

[0498] H435R Y436F (RF) mutation set introduced to disrupt protein A binding of monomeric and homodimerized HC2 / LC2 chains (Tustian, A. D., Endicott, C., Adams, B., Mattila, J. & Bak, H. Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity. MAbs 8, 828-838 (2016)).

[0499] C220S in Heavy Chain 1 introduced to prevent unpaired cysteine in the scFv binder.

[0500] Example 5. In Vitro Pharmacology Studies

[0501] Bispecific Antibody T-cell-mediated Cytotoxicity Assays

[0502] The activity of the bispecific antibody in T-cell-mediated cytotoxicity was evaluated using coculture assays of healthy-donor pan-T cells and a panel of EMR2+ AML cell lines with different expression levels (i.e., 0CI-AML3, 0CI-AML5, and 0CI-AML2) or an EMR2- cell line (i.e., OCI-LY10). Cytotoxicity to cancer cells was assessed at 3 and 6 days using flow cytometry. The bispecific antibody was able to elicit potent cytotoxicity to all EMR2+ cell lines at both time points (FIG. 11). No cytotoxicity was induced in presence of the TAA-negative cell line or with the NullxTRBV19 negative control.

[0503] Bispecific antibody mediated TRBV19+ T-cell Activation in Vitro

[0504] To assess the T-cell activation and expansion profile upon treatment, the expression of a late activation marker (i.e., CD25) was measured on T cells. T-cell activation and expansion (~2- to 3 -fold in pan-T cells at Day 6) were induced in the presence of the bispecific antibodyonly when incubated with EMR2+and not in the presence of the EMR2- cell lines, demonstrating antigen specificity of its activity (FIGs. 12A-12B). While potent cytotoxicity could be readily observed at Day 3 (FIG. 11), minimal T-cell activation and expansion were detected at the same timepoint. The effect of the bispecific antibody was both dose and time dependent, leading to a greater induction of T-cell activation and expansion at Day 6. The negative control NullxTRBV19 antibody did not induce significant T-cell activation in any of the cell lines tested.

[0505] When looking more specifically within the TRBV19+and TRBV19- T-cell subpopulations, it could be demonstrated that the bispecific antibody-induced expansion and activation was highly restricted to the TRBV19+T cells (FIGs. 13A-13C). No significant expression of the CD25 activation marker could be detected within the TRBV19- T cells at Day 3 for all cell lines tested. Minimal levels of T-cell activation were observed at the top doses of the bispecific antibody within TRBV19- T cells at Day 6, which could potentially be due to allogeneic reaction after a long coculture time leading to non-specific T-cell activation.

[0506] Measurement of bispecific antibody-induced Cytokine Production in a Flow-cytometrybased Assay

[0507] To better understand the TRBV19+T-cell profile upon activation, a flow-cytometrybased assay was set up to look at the intracellular production of interferon (IFN)-y, IL-2, and TNF-a at different timepoints. 0CI-AML3 cells and pan-T cells (n=6 different healthy donors) were cocultured at an effector-to-target (E:T) ratio of 10: 1 (relative E:T ratio for TRBV19+T cells of 0.5: 1) and analyzed by flow cytometry after 48 and 72 hours of incubation with the bispecific antibody or the NullxTRBV19 control. The comparator antibody A was assessed in parallel. The percentage of cytotoxicity as well as cytokine-producing T cells and the intensity of the signal (i.e., mean fluorescence intensity [MFI]) for each cytokine was evaluated. While potent cytotoxicity was observed readily at 48 hours, the percentages of granzyme-B-expressing cells was minimal in the total T-cell population (FIG. 14A). This was specifically detected in the TRBV19+population and not in the TRBV19- population. Further evaluation of cytokine expression (IFN-y, IL-2, and TNF-a) showed similar effects, where minimal expression was detected within the total T-cell population that was specific for the TRBV19+ population (Figure 14B-14D). IL-6 expression on pan-T cells and TRBV19+ cells was low, corresponding to a maximum of -20% expression at the highest concentrations. It is noteworthy that the intensity of cytokine expression on single-cell level within the TRBV19+ population engaged by the bispecificantibody was slightly higher than the signal detected within pan- T cells engaged with the control comparator antibody A (FIGs. 15A-15D). Similar data were observed at 72 hours. These data could indicate the potential of the TRBV19+ T cells to induce a potent response despite representing only -5% of pan-T cells, further validating the hypothesis that TRBV19+ T cells are characterized by a memory / primed phenotype.

[0508] Bispecific Antibody PBMC-mediated Cytotoxicity Assays

[0509] To characterize the kinetics of the bispecific antibody in a more physiological setting, a time course PBMC-based cytotoxicity assay was optimized to determine cancer cell cytotoxicity as well as T-cell activation. PBMCs from a total of 5 healthy donors were cocultured at a ratio of 5: 1 with the 0CI-AML3 cell line as target cells. Treatment with a dose titration of the bispecific antibody or NullxTRBV19 antibodies was performed at 24, 48, 72, 96, and 120 hours. Cell cytotoxicity and T-cell activation analysis were assessed by flow cytometry. The bispecific antibody showed dose- and time-dependent induction of cytotoxicity against 0CI-AML3 cells where maximum cytotoxicity was reached between 72 and 96 hours (FIGs. 16A-16F, Table 9). T-cell activation analysis showed a maximum of -30% pan-T-cell activation at 120 hours as measured by the expression of the late activation marker CD25. Among the TRBV19+T cells, maximum specific T-cell activation was detected between 72 and 96 hours, corelating with the cytotoxicity data. No impact on cancer cell cytotoxicity nor T-cell activation was observed in presence of the NullxTRBV19 control. As a reference, the control comparator antibody A antibody was included in the assay. While maximum cytotoxicity was reached at earlier timepoints with the control comparator antibody A, the bispecific antibody led to comparable cytotoxicity over time (FIGs. 17A-17C). Additionally, significantly higher overall pan-T-cell activation was observed as early as 48 hours (Table 10).

[0510] Table 9. Maximum values and EC50 values of cytotoxicity and T-cell activation from PBMC cytotoxicity assays.24 hours 48 hours 72 hours 96 hours 120 hoursCytotoxicityMax value (%) 3.2 60.7 86.2 96.6 95.7ECso (nM) 0.02 0.01 0.004 0.003 0.004Pan-T-cell activationMax value (%) 6.7 8.2 13.4 23.0 31.5ECso (nM) 0.02 0.01 0.01 0.01 0.01EC50, 50% effective concentration; max, maximum; PBMC, peripheral blood mononuclear cell.Data from 5 healthy donors were pooled and represented as mean. A nonlinear regressionmodel was used for EC50 and max mean value estimation across the donors. The model was fit using log-transformed data in GraphPad Prism 10.

[0511] Table 10. Maximum values and EC50 values of cytotoxicity and T-cell activation from PBMC cytotoxicity assays.24 hours 48 hours 72 hours 96 hours 120 hoursCytotoxicityBispecrfic Max value (%) 3.2 60.7 86.2 96.6 96.7Antibody ECso tnM) 0.02 0.01 0.004 0.003 0.004Control Max value (%) 33.5 81.2 95.4 95.8 94.2 comparator antibody AECso tnM) 0.03 0.02 0.01 0.01 0.01Pan-T-cell activationBispecrfic Max value (%) 6.7 8.2 13.4 23.0 31.5Antibody ECso tnM) 0.02 0.01 0.01 0.01 0.01Control Max value (%) 51.3 74.2 85.4 89.0 85.5 comparator antibody AECso tnM) 0.12 0.06 0.04 0.03 0.03

[0512] CD, cluster of differentiation; EC50, 50% effective concentration; EMR2, epidermal-growth-factor-like module- containing mucin-like hormone receptor-like 2; max, maximum; PBMC, peripheral blood mononuclear cell.

[0513] Data from 5 healthy donors were pooled and represented as mean. A nonlinear regression model was used for EC50 and max mean value estimation across the donors. The model was fit using log -transformed data in GraphPad Prism 10.

[0514] Cytokine Release Profiling of the Bispecific Antibody

[0515] To characterize the cytokine release profile of the bispecific antibody, cytokine levels were measured using the Meso Scale Discovery (MSD) ELISA Proinflammatory Panel 1 in the supernatants of the PBMC assays. Supernatants were collected at each time point and data was normalized to untreated control wells. Similarly, the control comparator antibody A was used in parallel as a reference for cytokine production. Overall, the kinetics of cytokine secretion were different between the bispecific antibody and control comparator antibody A. For most cytokines, peak cytokine production was detected between 72 and 96 hours for the bispecific antibody (FIG. 18) while for the control comparator antibody A, maximum productions were readily detected at 48 hours (FIG. 19). Moreover, there was a significant difference in the highest cytokine concentrations between both antibodies, where the bispecific antibody led to significantly lower production of IL- 10, IL- 10, and TNF-a (Table 11 and Table 12). While IL-6 expression on T cells was low as indicated above, high production levels were observed at 72hours in the PBMC assays, potentially indicating that IL-6 could be secreted by other immune cells or the cancer cells. This is in line with previous data showing that monocytes and AML leukemic blasts release IL-6. Interestingly, single-cell RNA sequencing (RNAseq) as well as bulk RNAseq indicated that 0CLAML3 cell lines express some levels of IL-6 and IFN-y at baseline and after TCE treatment. Altogether these data further validated the hypothesis that the bispecific antibody could lead to a lower risk of T-cell-mediated CRS by selectively engaging and activating a specific T-cell subpopulation.

[0516] Table 11. Bispecific Antibody-induced inflammatory cytokines in the presence of OCL AML3 cells: EC50 and max cytokine release values.Cytokine Max value (pg / mL) - time point ECso (nM)IFN-y 77,827 - 96 hours 0.03IL-ip 162 - 72 hours 0.02IL- 10 1,199 - 72 hours 0.02IL-2 2,463 - 72 hours 0.08IL-6 2,189 - 120 hours 0.01TNF-a 368.7 - 72 hours 0.07

[0517] EC50, 50% effective concentration; IFN, interferon; IL, interleukin; max, maximum; MSD, Meso Scale Discovery; NE, not estimable; PBMC, peripheral blood mononuclear cell; SEM, standard error of the mean; TNF, tumor necrosis factor.

[0518] Supernatant were collected at the different time points and analyzed for inflammatory cytokines using MSD Proinflammatory kit. Graphing of data was done in GraphPad Prism 10. Data from 5 different donors were pooled and represented as mean ± SEM.

[0519] Table 12. Inflammatory cytokines induced by the control comparator antibody A in the presence of OCI-AML3 cells: EC50 and max cytokine release values.Cytokine Max value (pg / mL) - time point ECso (nM)IFN-y 88,580 - 48 hours 0.02IL-ip 362.5 - 72 hours 0.1IL-10 3,825 - 48 hours 0.07IL-2 3,269 - 48 hours 0.2IL-6 1,868 - 120 hours 0.01TNF-a 1,254 - 48 hours 0.3

[0520] CD, cluster of differentiation; EC50, 50% effective concentration; EMR2, epidermal-growth-factor-like module- containing mucin-like hormone receptor-like 2; IFN, interferon; IL, interleukin; max, maximum; MSD, Meso Scale Discovery; SEM, standard error of the mean; TNF, tumor necrosis factor.Supernatant were collected at the different time points and analyzed for inflammatory cytokines using MSD Proinflammatory kit. Graphing of data was done in GraphPad Prism 10. Data from 5 different donors were pooled and represented as mean ± SEM

[0521] Bispecific antibody-mediated Cytotoxicity on Primary AML BM Blast

[0522] To further assess the cytotoxic potential of the bispecific antibody in a relevant AML setting, cytotoxicity assays were performed using AML BM samples as target cells. Since the viability of primary AML BM cells is compromised at 48 hours in vitro, the assay was performed for 24 hours at a relative E:T ratio of 2: 1 with pan-T cells as effectors, the bispecific antibody promoted a dose-dependent reduction of blasts in the tested AML BM donors (n=3) (FIGs. 20A-20D). While no pan-T-cell activation was detected, specific activation of TRBV19+T cells was observed. No activation was detected in the TRBV19- T cells. The NullxTRBV19 control did not show appreciable cancer cell cytotoxicity nor T-cell activation.

[0523] Activity of the Bispecific Antibody on Healthy HSPCs and Myeloid Cells

[0524] Similar to other targets pursued in AML, EMR2 is also expressed on healthy myeloid cells (FIG. 4). To determine the activity of the bispecific antibody on cancer cells versus healthy HSPCs, colony-forming unit (CFU) assays were performed with healthy CD34+HSPCs. A coculture assay of pan-T cells and CD34+cells or OCI-AML3 cells was set up. The inhibition of colony formation of 0CI-AML3 cells induced by the bispecific antibody could be readily seen at doses between 0.01 and 0.1 nM. At the same concentrations, no significant effects were observed on the clonogenic potential of CD34+HSPCs (FIG. 21 A). While increasing concentrations of the bispecific antibody (up to 100 nM) did not cause >~50% reduction in the CD34+HSPC colony formation, ~85% inhibition of OCI-AML3 colonies was observed.

[0525] To evaluate the effect of the bispecific antibody treatment on healthy monocytes, PBMC cytotoxicity assays with 0CI-AML3 cocultures were performed as described above and cytotoxicity of monocytes was assessed. At 72 hours, the bispecific antibody induced >85% cytotoxicity on 0CI-AML3 cells while monocyte cytotoxicity showed a plateau effect at 60% (FIG. 21B). In contrast, comparable cytotoxicity to cancer cells and monocytes was observed with the control comparator A antibody (FIG. 22).

[0526] Example 6. In Vivo Pharmacology Studies

[0527] The in vivo antitumor activity of the bispecific antibody was evaluated in 2 disseminated EMR2+AML models labelled with luciferase (luc): M0LM-13-luc and OCL AML3-luc. Tumor-bearing female NSG (i.e., non-obese diabetic [NOD] severe combined immunodeficiency [scid] gamma or NOD.Cg PrkdcscldIl-2rgtmlwj1 / SzJ) mice were humanized with either CD3+pan-T cells or isolated TRBV19+T cells from healthy donors.

[0528] The tolerability of the bispecific antibody could not be assessed with respect to EMR2 or TRBV19 binding to host tissues due to the lack of cross-reactivity to corresponding mouse antigens; the engrafted human T cells did bind the bispecific antibody. Engraftment of human T cells can lead to body weight loss due to eventual graft-versus-host disease (GvHD), however treatment with the bispecific antibody did not result in significant body weight loss as compared to the Dulbecco’s phosphate-buffered saline (DPBS)-treated control group. Disseminated AML xenograft models including MOLM-13 and 0CI-AML3 generally home to the BM of the hind limb as well as the spinal column ultimately leading to hind limb paralysis. Animals were monitored daily for negative clinical signs related to excessive tumor burden and monitored for body weight loss twice per week. When individual animals exhibited negative clinical signs or reached >20% body weight loss as compared to initial body weights, they were removed from the study and humanely euthanized. Whole-body in vivo imaging was performed twice weekly for the MOLM-13 disseminated model and weekly for the 0CI-AML3 disseminated model according to rate of disease progression.

[0529] Efficacy of the bispecific antibody in M0LM-13-luc Established Disseminated Model in T-cell-humanized Mice

[0530] M0LM-13-luc cells (1 *105) were injected IV on Day 0. Mice (n=10 / group) were randomized by bioluminescence intensity (BLI) and humanized with either pan CD3+T cells at 2 different concentrations (l *107or 4*107) or TRBV19+T cells at 2 different concentrations (2 x 106or 1 x 107) per mouse on Day 3 to evaluate the efficacy of the bispecific antibody under varied conditions of T-cell humanization. Starting on Day 4, mice were intraperitoneally (IP) dosed with the bispecific antibody twice weekly at 0.05 and 1 mg / kg, or DPBS for a total of 10 doses.

[0531] Significant antitumor efficacy was observed with the bispecific antibody treatment at 1 mg / kg in mice humanized with all the various conditions of T cells as assessed by growth rate over time (p<0.0001), as compared to the DPBS-treated control group (humanized with 1 xlO7pan CD3+T cells), with 99% A tumor growth inhibition (TGI) in groups humanized with pan CD3+T cells, and 91% and 98% ATGI in groups humanized with 2xl06or 1 xlO7TRBV19+T cells, respectively, on Day 18 post tumor implantation (FIG. 23 A and FIG. 23B). Significant antitumor efficacy was also observed in mice treated with the bispecific antibody at 0.05 mg / kg humanized with 1 xlO7TRBV19+cells as assessed by growth rate over time (pO.0001), with99% ATGI on Day 18 post tumor implantation. Treatment with the bispecific antibody at 1 mg / kg elicited 8 and 2 complete responses (CR) in the groups humanized with 4 / 107and 1 / 107pan CD3+T cells, respectively, and 2 CRs in the group humanized with 1 x 107TRBV19+T cells (FIG. 23D). Treatment with the bispecific antibody at 0.05 mg / kg of mice humanized with 1 xlO7TRBV 19+T cells resulted in 1 CR.

[0532] The bispecific antibody at 1 mg / kg in mice humanized with 4xl07or 1 xlO7pan CD3+T cells resulted in median survival values of >46 and 44 days, respectively, compared to the median survival of 19 days in the DPBS-treated control group resulting in a biologically significant percent increased life span (ILS; i.e., >25%) of >142% and 132%, respectively (FIG. 23C). the bispecific antibody at 1 mg / kg in mice humanized with 2xl06or 1 xlO7TRBV19+T cells resulted in median survival values of 24 and 37 days, respectively, resulting in a biologically significant 26% and 92% ILS, respectively, compared to the DPBS control. The bispecific antibody at 0.05 mg / kg in mice humanized with 1 xlO7TRBV19+T cells resulted in median survival of 39 days, resulting in a biologically significant 103% ILS compared to the DPBS control. Statistical differences in survival curves were observed with the bispecific antibody treatment as compared to DPBS (p<0.0001).

[0533] It was expected that mice humanized with 4xl07pan CD3+T cells or 2xl06TRBV19+T cells would have similar antitumor responses due to being humanized with theoretically equivalent numbers of TRBV19+T cells; however, greater TGI and CRs were observed with 4xl07pan CD3+T cells (FIG. 23D). Presence of different T-cell subtypes may have contributed to a greater antitumor response in mice humanized with 4xl07pan CD3+T cells, which is more physiologically relevant. Alternatively, 2xl06T cells might not be a sufficient number of T cells to humanize the mice.

[0534] The observed AML-derived bioluminescence signals demonstrated a characteristic distribution pattern of M0LM-13-luc cells, homing to the hind limbs (ventral image, FIG. 24) and spinal column (dorsal image, data not shown) of the mice. Treatment with the bispecific antibody reduced tumor burden visually, with a more pronounced effect in the groups receiving higher numbers of T-cell humanization.

[0535] Efficacy of the bispecific antibody in OCI-AML3-luc Established Disseminated Model in T-cell-humanized Mice

[0536] The ability of the bispecific antibody to control AML disseminated disease was evaluated in a second xenograft model, i.e., OCI-AML3-luc. In Study ONC2023-210, mice bearing established OCI-AML3-luc xenografts were IP dosed with DPBS or the bispecific antibody twice weekly at 0.01, 0.1, and 1 mg / kg for a total of 14 doses (n=10 / group). Mice were humanized with either 2*107pan CD3+or 1 *107TRBV19+T cells. As a reference, mice humanized with 2 / 107pan CD3+T cells were treated with the control comparator antibody A at 1 mg / kg (FIGs. 25A and 25B).

[0537] In mice humanized with 1 x 107TRBV19+T cells, significant antitumor efficacy was observed with the bispecific antibody treatment at all tested doses (0.01, 0.1, and 1 mg / kg) assessed by change in mean tumor burden on Day 33 post tumor implantation (p<0.001), as compared to the DPBS-treated group with 100% ATGI in all treated groups (FIGs. 26A and 26B). At Day 33, partial regression was observed in 2 mice in the 0.01 mg / kg group and in 4 mice in both the 0.1 and 1 mg / kg treatment groups. In mice humanized with 2xl07pan CD3+T cells (corresponding to -I xlO6TRBV19+T cells), significant antitumor efficacy was observed with the bispecific antibody 2 treatment at all tested doses (0.01, 0.1, and 1 mg / kg) on Day 33 (p<0.001), as compared to the DPBS-treated group, with 94%, 95%, and 97% ATGI, respectively (FIG. 26A).

[0538] The bispecific antibody at all tested doses (0.01, 0.1, and 1 mg / kg) in mice humanized with 1 xlO7TRBV19+T cells resulted in median survival of >56 days compared to the median survival of 34 days in the DPBS-treated control group resulting in a biologically significant ILS (i.e., >25%) of >65% (FIG. 26C). In mice humanized with 2xl07pan CD3+T cells, treatment with the bispecific antibody at 0.01 mg / kg resulted in a median survival of 48 days with a biologically significant 41% ILS, while treatment with 0.1 and 1 mg / kg the bispecific antibody resulted in a median survival of 55 days, resulting in a biologically significant 62% ILS, compared to DPBS control. Statistical differences in survival curves were observed with the bispecific antibody treatment as compared to DPBS (p<0.0001).

[0539] The observed AML-derived bioluminescence signals demonstrated a characteristic distribution pattern of OCI-AML3-luc cells, homing with the highest degree to the spinal column (dorsal image, FIG. 27) and also to the hind limbs. Treatment with the bispecific antibody reduced tumor burden visually, with a more pronounced effect in the groups receiving higher numbers of T-cell humanization. Although the bispecific antibody reduced or eliminated tumorburden in the primary locations (spine and hind limb), BLI was also observed in secondary tumor locations due to homing of the cancer cells in immune-privileged sites such as the ovaries, lymph nodes, and central nervous system, areas not typically associated with AML. BLI imaging coupled with micro-computed tomography analysis performed on Day 32 showed that the bispecific antibody completely eradicated primary disease as evidenced by the lack of signal in the spinal cord of the treated mice compared to DPBS-treated group. Further analysis on Day 57 continued to show lack of tumor burden in the spinal cord and hind limbs of the mice while BLI signals were detected in the lymph nodes, ovaries, and SC tissue, highlighting the efficacy of the bispecific antibody in eradicating primary AML progression.

[0540] Taken together, the present in vitro and in vivo results document the bispecific antibody’s ability to induce potent and TAA-specific cytotoxicity, while leading to low levels of T-cell activation and cytokine release.

[0541] In vitro, the bispecific antibody led to cytotoxicity in a panel of AML cancer cell models showing different levels of EMR2 surface expression. No impact on cancer cell viability of TAA-negative cell lines was reported. T-cell activation was exclusively observed within the TRBV19+T-cell subpopulation, leading to low levels of pan-T-cell activation. Most importantly, selective engagement and activation of TRBV19+T cells was associated with low levels of cytokine production, further validating the hypothesis of lowering the risk of CRS with specific TRBV19+T-cell engagement.

[0542] In vivo, treatment with the bispecific antibody demonstrated robust efficacy in 2 different AML models (i.e., M0LM-13-luc and OCI-AML3-luc). The results from the present in vivo studies demonstrated that the bispecific antibody can inhibit AML progression leading to a significant increase in the animal’s life span.

[0543] Overall, the present results are in line with the present hypothesis of the TRBV19+platform aiming at selectively recruiting a small population of TRBV19+T cells by the bispecific antibody, to develop a TCE with an improved therapeutic window and a potential for less CRS.

[0544] Example 7. Potential for On-target / Off-tumor Efficacy

[0545] Although EMR2 expression is predominantly observed in myeloid lineages (monocytes / macrophages / dendritic cells / mast cells) across various tissues, the target is also expressed in healthy HSCs and to low / limited levels in granulocytes (basophils, eosinophils, and neutrophils), immature NK cells, B cells, and T cells. The potential for on-target effects of the bispecificantibody on HSCs, granulocytes (basophils and neutrophils), and on immature NK cells was evaluated to determine on-target / off-tumor toxicity of the bispecific antibody.

[0546] Normal Tissue Expression of EMR2 in Humans

[0547] With TCE antibodies, on-target / off-tumor toxicity has been identified as a risk in cases where expression of the targeted TAA(s) is not restricted to the tumor. Normal human expression data in the public domain on EMR2-related immune cell and tissue expression were reviewed. Propriety databases including DICE database, FANTOM5, Blueprint, and bulk RNA-Seq showed EMR2 expression is enriched in HSCs, progenitor cells, and myeloid lineage cells, and weakly or not expressed in lymphoid lineage cells. Peer-reviewed literature shows that EMR2 expression is enriched in myeloid cells, primarily monocytic and to a lesser extent granulocytic and lineages, including mast cells, and absent or very weakly expressed by the lymphoid cell lines Ramos (B-lymphoblastic) and Jurkat (T-lymphoblastic), respectively. Based on internal data as well as publicly available databases, low level of EMR2 expression was observed in small subset of B cells, NK cells and T celK

[0548] In addition, EMR2 target expression profiling by immunohistochemistry (IHC) was performed using normal human formalin-fixed, paraffin-embedded (FFPE) tissue samples and human mast cell pellet. The IHC assay relied on the mouse monoclonal anti-EMR2 antibody clone [2A1] (ThermoFisher, MA5-28205; 8) that was qualified for FFPE matrix on suitable controls. Assay development included confirmation of specificity of this reagent to EMR2 (ADGRE2) based on no cross-reactivity to CD97 (ADGRE5). EMR2 IHC positive labeling was confirmed in the human mast cell pellet and, in tissues, predominantly observed in mononuclear leukocytes with morphology most consistent with myeloid lineages (monocytes / macrophages / dendritic cells / mast cells) across various tissues: BM, colon and small intestine (lamina propria), gallbladder (lamina propria and muscle layer), liver (macrophages and Kupffer cells), spleen (predominantly leukocyte in the red pulp), lymph nodes, tonsil, appendix, lung (subepithelium and interstitium), skin (dermis), kidney (interstitium and glomeruli), and interstitium of ovary and pancreas. Granulocytes (neutrophils and eosinophils) were weakly labeled across tissues; neutrophils labeled in muscle layer in one of the three gallbladders. EMR2 IHC was negative in cerebellum, cerebrum, and meninges.

[0549] Whole-blood Neutrophil, Monocyte, NK Cell, and Basophil Activation Assay

[0550] A flow-cytometry-based human whole-blood assay was performed to assess if the bispecific antibody has a functional impact on peripheral neutrophils, monocytes, and / or NK cells. Preliminary results demonstrated that the bispecific antibody, but not a NullxTRBV19 negative control antibody, resulted in a moderate level of activation of monocytes and neutrophils, and to a lesser extent NK cells, after an overnight culture, albeit with variability in responses between donors. Further characterization of whether leukocyte activation is a result of direct, or indirect target engagement will be determined.

[0551] To assess the bispecific antibody results in activation of peripheral blood basophils, a flow cytometric assay was conducted in human whole blood. No basophil cell activation was detected (n=3 donors, t <40 minutes), indicating a low risk of anaphylactoid reactions due to direct target engagement on basophils.

[0552] Effect on HSPCs

[0553] To assess whether the bispecific antibody has an effect on the ability of HSPCs to proliferate and differentiate appropriately, in vitro CFU cell assays were conducted by setting up a coculture assay of pan-T cells and CD34+cells or 0CI-AML3 cells with the bispecific antibody at 0.001 to 100 nM. Results demonstrated that the bispecific antibody impacted clonogenic potential of HSPCs (>1 nM), however in this in vitro system there was a greater inhibition of the colony formation potential of OCI-AML3 cancer cells (>0.01 nM).

[0554] Potential for Off-target Binding

[0555] Off-target Binding by Retrogenix Screen

[0556] The potential for off-target binding of the bispecific antibody was evaluated in a human cell microarray platform (Retrogenix, Charles River Laboratories) to determine binding specificity. The antibody was screened for binding against fixed, transfected human HEK293 cells, individually expressing 6,105 full-length human plasma membrane proteins, secreted proteins, and cell surface-tethered secreted proteins plus a further 400 human heterodimers. The bispecific antibody was demonstrated to bind specifically to its primary target, EMR2, with strong intensity, and did not show any off-target interactions. Of note, TRBV19 was not in the library screen, and thus binding to TRBV19 could not be demonstrated for the bispecific antibody with this methodology. However, the bispecific antibody was shown herein to bind to TRBV19+cells by flow cytometry. These results indicate a low risk for off-target mediated toxicity for the bispecific antibody.

[0557] Example 8. Binding of the bispecific antibody to TRBV19-positive T cells.

[0558] Flow cytometry was used to measure specific binding of the TRBV19 arm of bispecific EMR2xTRB V 19 leads to TRB V 19-positive T cells from 2 different healthy human pan-T cell donors. When pan T cells were administered increasing concentrations of bispecific antibody for 1 hour at 37°C, concentration-dependent binding was observed. The EC50 (nM) of detection of TRBV19-positive cells was between 8.03 to 14.46 nM. (Fig. 29B).

[0559] Flow cytometry was also used to measure EMR2 arm binding affinity of bispecific lead molecules to varying EMR2 expressing cell lines (0CI-AML5 and M0LM13). When these cell lines were administered increasing concentrations of bispecific antibodies at 1 hour, 37°C, concentration-dependent binding was observed. OCI-AML 5 EMR2-K0 cells were used, which had the EMR2 gene genetically deleted, no detectable binding was observed. (FIG. 29C).

[0560] Example 9. Chemical and Post-translational modification (PTM) in degradation conditions of EMVBB8

[0561] Experiments were performed to determine the binding of the bispecific antibody EMVBB8 in a high pH of 8.5 under forced degradation conditions. Initial binding experiments by SPR were performed using the listed conditions: CM4 sensorchip coupled with anti-human- Fab antibody. Antibodies were captured to the chip surface and a titration of recombinant TRBV19 concentration ranging from 140-17.5 nM, 3 -fold dilution was flown over the chip surface to assess kinetic profiles. Results for the SPR binding experiments are shown in Fig. 28. As indicated in the first set of binding experiments, there was a 19% decrease in Vbl7 binding at high pH (HPH) as shown in Fig. 28. However, experiments were repeated in the same assay format but using a recombinant TRBV19 concentration range of 350-43.75nM, 2-fold dilution and show that the binding of the EMR2 arm and the TRBV 19 arm bind with similar binding affinity at pH 8.5 and 5.0 using SPR (Table 6). The optimized antigen concentration range allowed for full saturation of the TRBV19 arm and data analysis software was able to accurately determine Rmax values used in % Activity calculations. This differed from the first set of conditions shown in Fig. 28 where software was approximating Rmax causing variation and error in reported Rmax and subsequently % Activity. Furthermore, the second set of data utilized a different sample batch, wherein the second batch of bispecific antibodies for Table 6 was prepared in lOmM Acetate, pH 5.5 for enhanced stability. The first batch of bispecific antibodies was prepared in phosphate buffered saline (PBS).

[0562] EMBODIMENTSThe disclosure provided herein also provides the following non-limiting embodiments:1. An antibody or an antigen-binding fragment thereof, that specifically binds the G-protein- coupled receptor auto-proteolysis inducing (GAIN) domain and / or GPCR proteolytic site (GPS) motif of epidermal-growth-factor-like module-containing mucin-like hormone receptor 2 (EMR2).2. The antibody or the antigen-binding fragment thereof, according to item 1 , wherein the GAIN domain comprises amino acid residues D261-Q478 of SEQ ID NO: 205.3. The antibody or the antigen-binding fragment thereof, according to item 1, wherein the GAIN domain comprises the amino acid sequence SEQ ID NO: 215.4. The antibody or the antigen-binding fragment thereof, according to any one of items 1-3, wherein the GPS motif comprises the amino acid sequence SEQ ID NO: 216.5. The antibody or the antigen-binding fragment thereof, according to any one of items 1-4, wherein the antibody, or the antigen-binding fragment thereof, binds to an epitope comprising the amino acid sequence of SEQ ID NO: 217 or SEQ ID NO: 218.6. The antibody or the antigen-binding fragment thereof, according to any one of items 1-5, wherein the antibody, or the antigen-binding fragment thereof, binds to human EMR2 with a dissociation constant (KD) between about 0.01 nM to about 5 nM.7. The antibody or the antigen-binding fragment thereof, according to any one of items 1-5, wherein the antibody, or the antigen-binding fragment thereof, binds to human EMR2 with a half maximal effective concentration EC50 between about 0.1 nM to about 15 nM.8. The antibody or the antigen-binding fragment thereof, according to any one of items 1-7, wherein the antibody, or the antigen-binding fragment thereof, is or comprises a, a Fab fragment, a F(ab')2 fragment, F(ab)'3 fragments, a scFv, a bis-scFv, a (scFv)2, a stapled scFv (spFv), a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Immunoglobulin New Antigen Receptor (Ig NAR), a single heavy chain antibody, a camelid antibody, a shark antibody, or a chemically modified derivative thereof.9. The antibody or the antigen-binding fragment thereof, according to any one of items 1-8, wherein the antibody, or the antigen-binding fragment thereof, comprises a Fab.10. The antibody or the antigen-binding fragment thereof, according to any one of items 1-9, wherein the antibody, or the antigen-binding fragment thereof, further comprises a first constant Ig domain of the heavy chain (CHI domain).11. The antibody or the antigen-binding fragment thereof, according to any one of items 1-8, wherein the antibody, or the antigen-binding fragment thereof, does not comprise a CHI domain.12. The antibody or the antigen-binding fragment thereof, according to any one of items 1-8 and 11, wherein the antibody, or the antigen-binding fragment thereof, comprises a scFv or a spFv.13. The antibody or the antigen-binding fragment thereof, according to item 12, wherein the scFv or spFv comprises a signal sequence, a heavy chain variable sequence, a GS-Linker, and a light chain variable sequence.14. The antibody or the antigen-binding fragment thereof, according to any one of items 1-8 further comprising an Fc domain.15. The antibody or the antigen-binding fragment thereof, according to item 14, wherein the Fc domain of the antibody or the antigen-binding fragment thereof, is an IgA, an IgG, an IgE, or an IgM.16. The antibody or the antigen-binding fragment thereof, according to item 14 or item 15, wherein the Fc domain of the antibody or the antigen-binding fragment thereof, is an IgG.17. The antibody or the antigen-binding fragment thereof, according to item 16, wherein the IgG is IgGl or IgG4.18. The antibody or the antigen-binding fragment thereof, according to any one of items 14-17, wherein the Fc domain comprises one or more different mutations which promote heterodimerization.19. The antibody or the antigen-binding fragment thereof, according to any one of items 14-18, wherein the Fc domain comprises mutations T366S, L368A and Y407V (EU numbering) or mutation T366W (EU numbering).20. The antibody or the antigen-binding fragment thereof, according to any one of items 14-19, wherein the Fc domains of HC1 and / or HC2 further comprise one or more mutations which reduce Fc binding to a Fey receptor.21. The antibody or the antigen-binding fragment thereof, according to item 20, wherein the Fey receptor is FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, and / or FcyRIIIB.22. The antibody or the antigen-binding fragment thereof, according to any one of items 14-21, wherein the Fc domain comprises one or more mutations selected from L234A, L235A, and D265S (EU numbering).23. The antibody or the antigen-binding fragment thereof, according to any one of items 14-22, wherein the Fc domain comprises mutations L234A, L235A, and D265S (EU numbering).24. The antibody or the antigen-binding fragment thereof, according to any one of claims 14-23, wherein the Fc domain further comprises one or more mutations which reduce Fc binding to protein A.25. The antibody or the antigen-binding fragment thereof, according to any one of items 14-24, wherein the Fc domain comprises mutations H435R and / or Y436F (EU numbering).26. The antibody or the antigen-binding fragment thereof, according to any one of items 14-25, wherein the Fc domain comprises mutations H435R and Y436F (EU numbering).27. The antibody or the antigen-binding fragment thereof, according to any one of items 14-26, wherein the antibody, or the antigen-binding fragment thereof, comprises a humanized antibody or an antigen binding fragment thereof, a human antibody or an antigen binding fragment thereof, a murine antibody or an antigen binding fragment thereof, a chimeric antibody or an antigen binding fragment thereof, a monospecific antibody or a monospecific antigen binding fragment thereof, a bispecific antibody or a bispecific antigen binding fragment thereof, a multispecific antibody or a multispecific antigen binding fragment thereof.28. The antibody or the antigen-binding fragment thereof, according to any one of items 1-27, wherein the antibody, or the antigen-binding fragment thereof, comprises: a) a heavy chain complementarity determining region (HCDR) 1 , a HCDR2 and a HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 63, and a light chain complementarity determining region (LCDR) 1, a LCDR2 and a LCDR3 of the light chain variable region (VL) of SEQ ID NO: 64; b) a HCDR1 , a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 95, and a LCDR1 , a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 96; c) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 127, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 128; or d) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 191, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 192.29. The antibody or the antigen-binding fragment thereof, according to any one of items 1-28, wherein the antibody, or the antigen-binding fragment thereof, comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of: a) SEQ ID NO: 33, 34, 35, 48, 49, and 50, respectively; b) SEQ ID NO: 36, 37, 38, 51, 52, and 53, respectively; c) SEQ ID NO: 39, 40, 41, 54, 55, and 56, respectively; d) SEQ ID NO: 42, 43, 44, 57, 58, and 59, respectively; e) SEQ ID NO: 45, 46, 47, 60, 61, and 62, respectively; f) SEQ ID NO: 65, 66, 67, 80, 81, and 82, respectively; g) SEQ ID NO: 68, 69, 70, 83, 84, and 85, respectively; h) SEQ ID NO: 71, 72, 73, 86, 87, and 88, respectively; i) SEQ ID NO: 74, 75, 76, 89, amino acid sequence AAI, and SEQ ID NO: 91, respectively; j) SEQ ID NO: 77, 78, 79, 92, 93, and 94, respectively; k) SEQ ID NO: 97, 98, 99, 112, 113, and 114, respectively; 1) SEQ ID NO: 100, 101, 102, 115, 116, and 117, respectively; m) SEQ ID NO: 103, 104, 105, 118, 119, and 120, respectively; n) SEQ ID NO: 106, 107, 108, 121, amino acid sequence DNN, and SEQ ID NO: 123, respectively;o) SEQ ID NO: 109, 110, 111, 124, 125, and 126, respectively; p) SEQ ID NO: 161, 162, 163, 176, 177, and 178, respectively; q) SEQ ID NO: 164, 165, 166, 179, 180, and 181, respectively; r) SEQ ID NO: 167, 168, 169, 182, 18, and 184, respectively; s) SEQ ID NO: 170, 171, 172, 185, amino acid sequence EVS, and SEQ ID NO: 187, respectively; or t) SEQ ID NO: 173, 174, 175, 188, 189, and 190, respectively.30. The antibody or the antigen-binding fragment thereof, according to any one of items 1-29, wherein the antibody, or the antigen-binding fragment thereof, comprises a variable heavy chain region (VH) comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 63; b) the VH of SEQ ID NO: 95; c) the VH of SEQ ID NO: 127; or d) the VH of SEQ ID NO: 191.31. The antibody or the antigen-binding fragment thereof, according to any one of items 1-30, wherein the antibody, or the antigen-binding fragment thereof, comprises or further comprises a variable light chain region (VL) comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 64; b) the VL of SEQ ID NO: 96; c) the VL of SEQ ID NO: 128; or d) the VL of SEQ ID NO: 192.32. The antibody or the antigen-binding fragment thereof, according to any one of items 1-31, wherein the antibody, or the antigen-binding fragment thereof, comprises: a) the VH of SEQ ID NO: 63 and the VL of SEQ ID NO: 64; b) the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 96; c) the VH of SEQ ID NO: 127 and the VL of SEQ ID NO: 128; or d) the VH of SEQ ID NO: 191 and the VL of SEQ ID NO: 192.33. The antibody or the antigen-binding fragment thereof, according to any one of items 1-32, wherein the antibody, or the antigen-binding fragment thereof, comprises a scFv or a spFV which comprises, from the N- to C-terminus, a VH, a linker (L) and a VL in the format VH-L- VL or a VL, a linker and a VH in the format VL-L-VH.34. The antibody or the antigen-binding fragment thereof, according to item 33, wherein the VH comprises the amino acid sequence of SEQ ID NO: 191, the VL comprises the amino acid sequence of SEQ ID NO: 192, and the linker comprises SEQ ID NO: 221.35. The antibody or the antigen-binding fragment thereof, according to item 33, wherein the linker comprises SEQ ID NO: 221.36. The antibody or the antigen-binding fragment thereof, according to any one of items 1-35, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 194, 196, 198, or 202.37. The antibody or the -binding fragment thereof, according to any one of items 1-36, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 195, 197 or 199.38. The antibody or the antigen-binding fragment thereof, according to item 36 or item 37, wherein: a) the HC1 comprises the amino acid sequence of SEQ ID NO: 194 and the LC1 comprises the amino acid sequence of SEQ ID NO: 195; b) the HC1 comprises the amino acid sequence of SEQ ID NO: 196 and the LC1 comprises the amino acid sequence of SEQ ID NO: 197; or c) the HC1 comprises the amino acid sequence of SEQ ID NO: 198 and the LC1 comprises the amino acid sequence of SEQ ID NO: 199.39. An antibody or an antigen-binding fragment thereof, that binds to the same epitope of EMR2 as the antibody or the antigen-binding fragment thereof, according to any one of items 27-37.40. An antibody or an antigen-binding fragment thereof, that competes for binding to the same epitope of EMR2 with the antibody or the antigen-binding fragment thereof, according to any one of items 28-39.41. The antibody or the antigen-binding fragment thereof, according to any one of items 1-40, wherein the antibody, or the antigen-binding fragment thereof, is a monospecific antibody.42. The antibody or the antigen-binding fragment thereof, according to any one of items 1-40, wherein the antibody, or the antigen-binding fragment thereof, is a bispecific antibody which specifically binds to EMR2 and to a second antigen.43. The antibody or the antigen-binding fragment thereof, according to item 42, wherein the second antigen is T-cell Receptor Variable 19 (TRBV19).44. An isolated polynucleotide encoding the antibody or the antigen-binding fragment thereof, according to any one of items 1-43.45. The isolated polynucleotide of item 44, wherein the polynucleotide comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 63; b) the VH of SEQ ID NO: 95; c) the VH of SEQ ID NO: 127; or d) the VH of SEQ ID NO: 191.46. The isolated polynucleotide of item 44 or item 45, wherein the polynucleotide comprises or further comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 64; b) the VL of SEQ ID NO: 96; c) the VL of SEQ ID NO: 128; or d) the VL of SEQ ID NO: 192.47. The isolated of any one of items 44-46, wherein the polynucleotide comprises a sequence comprising a nucleotide sequence encoding: a) the amino acid sequence of SEQ ID NO: 63 and / or SEQ ID NO: 64; b) the amino acid sequence of SEQ ID NO: 95 and / or SEQ ID NO: 96; c) the amino acid sequence of SEQ ID NO: 127 and / or SEQ ID NO: 128; or d) the amino acid sequence of SEQ ID NO: 191 and / or the SEQ ID NO: 192.48. The isolated polynucleotide according to item 46 or item 47, wherein the polynucleotide comprises a sequence encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 194, 196, 198, or 202.49. The isolated polynucleotide according to any one of items 44-48, wherein the polynucleotide comprises a sequence encoding a light chain comprising the amino acid sequence of SEQ ID NO: 195, 197, or 199.50. The isolated polynucleotide of claim any one of items 44-49, comprising a nucleotide sequence encoding: a) the amino acid sequence of SEQ ID NO: 194 and / or SEQ ID NO: 195; b) the amino acid sequence of SEQ ID NO: 196 and / or SEQ ID NO: 197; c) the amino acid sequence of SEQ ID NO: 198 and / or the amino acid sequence of SEQ ID NO: 199; or d) the amino acid sequence of SEQ ID NO: 202.51. A vector comprising the isolated polynucleotide according to any one of items 44-50.52. The vector according to item 51, wherein the isolated polynucleotide is operably linked to an expression control sequence.53. The vector according to item 51 or item 52, wherein the vector is a viral vector.54. The vector according to item 53, wherein the viral vector is selected from an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpes simplex virus vector, and a poxvirus vector.55. A pharmaceutical composition comprising (i) the antibody or the antigen-binding fragment thereof, according to any one of items 1-43, or the polynucleotide according to any one of items44-50, or the vector according to any one of items 51-54, and (ii) a pharmaceutically acceptable carrier or excipient.56. A host cell expressing the antibody or the antigen-binding fragment thereof, according to any one of items 1-43.57. The host cell according to item 56, wherein the cell is a hybridoma.58. The host cell according to item 56, wherein the antibody, or the antigen-binding fragment thereof, is recombinantly produced.59. A host cell comprising the isolated polynucleotide according to any one of claims 44-50 or the vector according to any one of items 51-54.60. A method for treating a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody or the antigenbinding fragment thereof, according to any one of items 1-43, or the isolated polynucleotide according to any one of items 44-50, or the vector according to any one of items 51-54, or the pharmaceutical composition of claim 55, or the host cell according to any one of items 56-59.61. A method for inducing cytotoxicity of a cancer cell or redirecting immune or T cells against cancer cells, the method comprising administering to the cell an effective amount of the antibody or the antigen-binding fragment thereof, according to any one of items 1-43, or the polynucleotide according to any one of items 44-50, or the vector according to any one of items 51-54, or the pharmaceutical composition of item 55, or the host cell according to any one of items 56-59, wherein the effective amount is sufficient to inhibit the growth or proliferation of the cancer cell.62. The method according to item 61, wherein the cancer cell is in a subject and the antibody or the binding fragment, polynucleotide, vector, pharmaceutical composition, or host cell is administered to the subject.63. The method according to item 61 or item 62, wherein the administration is conducted ex vivo.64. The method according to any one of items 61-63, wherein the cancer is an EMR2-expressing cancer.65. The method according to item 64, wherein the EMR2-expressing cancer is a hematological cancer.66. The method according to item 65, wherein the hematological cancer is a myeloid malignancy.67. The method according to item 66, wherein the hematological cancer is an acute myeloid leukemia (AML), a chronic myelogenous leukemia (CML), or a myelodysplastic neoplasm.68. The method according to any one of items 60-67 further comprising administering a second therapeutic agent.69. The method according to item 68, wherein the second therapeutic agent is a surgery, a chemotherapy, an androgen deprivation therapy, a radiation, or any combination thereof.70. The antibody according to any one of items 1-43 for use in the method according to any one of items 60-69.71. The polynucleotide according to any one of items 44-50 for use in the method according to any one of items 60-69.72. The vector according to any one of items 51-54 for use in the method according to any one of items 60-69.73. The pharmaceutical composition according to item 55 for use in the method according to any one of items 60-69.74. The host cell according to any one of items 56-59 for use in the method according to any one of items 60-69.75. A method for generating the antibody or the antigen-binding fragment thereof, according to any one of items 1-43, wherein the method comprises culturing the host cell according to any one of items 56-59 and isolating the antibody or the antigen-binding fragment thereof.76. A kit comprising (i) the antibody or the antigen-binding fragment thereof, according to any one of items 1-43 and / or the isolated polynucleotide according to any one of items 44-50, and / or the vector according to any one of items 51-54, and / or the pharmaceutical composition of item 55, and / or the host cell according to any one of items 56-59, and (ii) packaging for the same and / or instructions for use.77. A bispecific antibody or a bispecific antigen-binding fragment thereof, that specifically binds (i) TRBV19 with a first antigen-binding site and (ii) GAIN domain and / or GPS motif of EMR2 with a second antigen-binding site.78. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to item 77, comprising: a) a first heavy chain (HC1); b) optionally a first light chain (LC1); c) a secondheavy chain (HC2); and d) optionally a second light chain (LC2), wherein (i) the HC1 and the LC1 form a first antigen-binding site that specifically binds a first antigen, (ii) the HC2 and the LC2 form a second antigen-binding site that specifically binds a second antigen, (iii) the HC1 and HC2 each comprise a Fc domain comprising a CH2-CH3 domain; and wherein the first antigen is TRBV19, and the second antigen is EMR2.79. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to item 77 or item 78, wherein the second antigen-binding site specifically binds the GAIN domain and / or GPS motif of EMR2.80. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to item 79, wherein the GAIN domain comprises amino acid residues D261-Q478 of SEQ ID NO: 205.81. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to item 79, wherein the GAIN domain comprises the amino acid sequence SEQ ID NO: 215.82. The bispecific antibody or bispecific antigen-binding fragment thereof, according to any one of items 77-81, wherein the GPS motif comprises the amino acid sequence SEQ ID NO: 216.83. The bispecific antibody or bispecific antigen-binding fragment thereof, according to any one of items 77-82, wherein the antibody, or the antigen-binding fragment thereof, binds to an epitope comprising the amino acid sequence of SEQ ID NO: 217 or SEQ ID NO: 218.84. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-83, wherein the first antigen-binding site comprises a Fab or a scFv or a spFv.85. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-84, wherein the Fc of the first antigen-binding site comprises a CHI domain.86. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-85, wherein the second antigen-binding site comprises a Fab or a scFv or a spFv.87. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-86, wherein the Fc of the second antigen-binding site further comprises a CHI domain.88. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-87, wherein the first antigen-binding site comprises a Fab and the second antigen-binding site comprises a scFv or a spFv.89. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-88, wherein the first antigen-binding site comprises a scFv or a spFv and the second antigen-binding site comprises a Fab.90. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-89, wherein the Fc domain of the antibody or the antigen-binding fragment thereof, is an IgA, an IgG, an IgE, or an IgM.91. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to item90, wherein the Fc domain of the antibody or the antigen-binding fragment thereof, is an IgG.92. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to item91, wherein the IgG is IgGl or IgG4.93. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-92, wherein the Fc domains of HC1 and HC2 comprise one or more different mutations which promote heterodimerization.94. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-93, wherein the Fc domain of the HC1 comprises mutations T366S, L368A and Y407V (EU numbering) and the Fc domain of the HC2 comprises mutation T366W (EU numbering).95. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-94, wherein the Fc domain of the HC2 comprises mutations T366S, L368A and Y407V (EU numbering) and the Fc domain of the HC1 comprises mutation T366W (EU numbering).96. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-95, wherein the Fc domains of HC1 and / or HC2 further comprise one or more mutations which reduce Fc binding to a Fey receptor.97. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to item 96, wherein the Fey receptor is FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, and / or FcyRIIIB.98. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-97, wherein the Fc domains of HC1 and / or HC2 each comprise one or more mutations selected from L234A, L235A, and D265S (EU numbering).99. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-98, wherein the Fc domains of HC1 and HC2 each comprise mutations L234A, L235A, and D265S (EU numbering).100. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-99, wherein the Fc domains of HC1 or HC2 further comprises one or more mutations which reduce Fc binding to protein A.101. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-100, wherein the Fc domains of HC1 or HC2 comprise mutations H435R and / or Y436F (EU numbering).102. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 770101, wherein the Fc domain of HC1 comprises mutations H435R and Y436F (EU numbering).103. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-102, wherein the Fc domain of HC2 comprises mutations H435R and Y436F (EU numbering).104. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-103, wherein the HC1 or HC2 comprise mutation C220S (EU numbering).105. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-104, wherein the HC1 comprises mutation C220S (EU numbering).106. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-104, wherein the HC2 comprises mutation C220S (EU numbering).107. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-106, wherein the antibody, or the antigen-binding fragment thereof, is a humanized antibody or an antigen binding fragment thereof, a human antibody or an antigen binding fragment thereof, a murine antibody or an antigen binding fragment thereof, a chimeric antibody or an antigen binding fragment thereof, or a chemically modified derivative thereof.108. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-107, wherein the first antigen-binding site that specifically binds TRBV19 comprises: a) a HCDR1, a HCDR2 and a HCDR3 of the VH of SEQ ID NO: 31 and a LCDR1, a LCDR2 and a LCDR3 of the VL of SEQ ID NO: 32; or b) a HCDR1, a HCDR2, and a HCDR3of the VH of SEQ ID NO: 159 and a LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 160.109. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to item 108, wherein the first antigen-binding site that specifically binds TRBV19 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of: a) SEQ ID NOs: 1, 2, 3, 16, 17, and 18, respectively; b) SEQ ID NOs: 4, 5, 6, 19, 20, and 21, respectively; c) SEQ ID NOs: 7, 8, 9, 22, 23, and 24, respectively; d) SEQ ID NOs: 10, 11, 12, 25, amino acid sequence KVS, and SEQ ID NO: 27, respectively; e) SEQ ID NOs: 13, 14, 15, 28, 29, and 30, respectively; f) SEQ ID NOs: 129, 130, 131, 144, 145, and 146, respectively; g) SEQ ID NOs: 132, 133, 134, 147, 148, and 149, respectively; h) SEQ ID NOs: 135, 136, 137, 150, 151, and 152, respectively; i) SEQ ID NOs: 138, 139, 140, 153, amino acid sequence KVS, and SEQ ID NO: 155, respectively; or j) SEQ ID NOs: 141, 142, 143, 156, 157, and 158, respectively.110. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-109, wherein the first antigen-binding site that specifically binds TRBV19 comprises a VH comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: the VH of SEQ ID NO: 31; or the VH of SEQ ID NO: 159.111. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-110, wherein the first antigen-binding site that specifically binds TRBV19 comprises or further comprises a VL comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: the VL of SEQ ID NO: 32; or the VL of SEQ ID NO: 160.112. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-111, wherein the first antigen-binding site that specifically binds TRBV19 comprises: the VH of SEQ ID NO: 31 and the VL of SEQ ID NO: 32; or the VH of SEQ ID NO: 159 and the VL of SEQ ID NO: 160.113. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-112, wherein the second antigen-binding site that specifically binds EMR2 comprises: a) a HCDR1, a HCDR2 and a HCDR3 of the VH of SEQ ID NO: 63, and a LCDR1, a LCDR2 and a LCDR3 of the VL of SEQ ID NO: 64; b) a HCDR1, a HCDR2, and a HCDR3 ofthe VH of SEQ ID NO: 95, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 96; c) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 127, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 128; or d) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 191, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 192.114. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-113, wherein the second antigen-binding site that specifically binds EMR2 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of: a) SEQ ID NO: 33, 34, 35, 48, 49, and 50, respectively; b) SEQ ID NO: 36, 37, 38, 51, 52, and 53, respectively; c) SEQ ID NO: 39, 40, 41, 54, 55, and 56, respectively; d) SEQ ID NO: 42, 43, 44, 57, amino acid sequence EVS, and SEQ ID NO: 59, respectively; e) SEQ ID NO: 45, 46, 47, 60, 61, and 62, respectively; f) SEQ ID NO: 65, 66, 67, 80, 81, and 82, respectively; g) SEQ ID NO: 68, 69, 70, 83, 84, and 85, respectively; h) SEQ ID NO: 71, 72, 73, 86, 87, and 88, respectively; i) SEQ ID NO: 74, 75, 76, 89, amino acid sequence AAI, and SEQ ID NO: 91, respectively; j) SEQ ID NO: 77, 78, 79, 92, 93, and 94, respectively; k) SEQ ID NO: 97, 98, 99, 112, 113, and114. respectively; 1) SEQ ID NO: 100, 101, 102, 115, 116, and 117, respectively; m) SEQ ID NO: 103, 104, 105, 118, 119, and 120, respectively; n) SEQ ID NO: 106, 107, 108, 121, ammo acid sequence DNN, and SEQ ID NO: 123, respectively; o) SEQ ID NO: 109, 110, 111, 124, 125, and 126, respectively; p) SEQ ID NO: 161, 162, 163, 176, 177, and 178, respectively; q) SEQ ID NO: 164, 165, 166, 179, 180, and 181, respectively; r) SEQ ID NO: 167, 168, 169, 182, 18, and 184, respectively; s) SEQ ID NO: 170, 171, 172, 185, amino acid sequence EVS, and SEQ ID NO: 187, respectively; or t) SEQ ID NO: 173, 174, 175, 188, 189, and 190, respectively.115. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-114, wherein the bispecific antibody or the bispecific antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 63; b) the VH of SEQ ID NO: 95; c) the VH of SEQ ID NO: 127; or d) the VH of SEQ ID NO: 191.116. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-115, wherein the bispecific antibody or the bispecific antigen-binding fragment thereof, comprises or further comprises a VL comprising an amino acid sequence that is at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 64; b) the VL of SEQ ID NO: 96; c) the VL of SEQ ID NO: 128; or d) the VL of SEQ ID NO: 192.117. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-116, wherein the first antigen-binding site and / or the second antigen-binding site that specifically binds EMR2 comprises: a) the VH of SEQ ID NO: 63 and the VL of SEQ ID NO: 64; b) the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 96; c) the VH of SEQ ID NO: 127 and the VL of SEQ ID NO: 128; or d) the VH of SEQ ID NO: 191 and the VL of SEQ ID NO: 192.118. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-117, wherein the bispecific antibody or the bispecific antigen-binding fragment thereof, comprises a scFv or a spFV which scFv or spFV comprises, from the N- to C-terminus, a VH, a linker (L) and a VL in the format VH-L-VL or a VL, a linker and a VH in the format VL- L-VH.119. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to item 118, wherein the VH comprises the amino acid sequence of SEQ ID NO: 159, the VL comprises the amino acid sequence of SEQ ID NO: 160, and the L comprises SEQ ID NO: 221.120. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to item 118, wherein the VH comprises the amino acid sequence of SEQ ID NO: 191, the VL comprises the amino acid sequence of SEQ ID NO: 192, and the L comprises SEQ ID NO: 221.121. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to item 118, wherein the L comprises SEQ ID NO: 221.122. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-121, wherein the first antigen-binding site specifically binds to TRBV19 with a dissociation constant (KD) that is between about 15 nM to about 200 nM.123. The bispecific antibody or the bispecific antigen-binding fragment thereof, according to any one of items 77-121, wherein the first antig...

Claims

Claims1. An antibody, or an antigen-binding fragment thereof, that specifically binds the G- protein-coupled receptor auto-proteolysis inducing (GAIN) domain and / or the GPCR proteolytic site (GPS) motif of epidermal-growth-factor-like module-containing mucin-like hormone receptor 2 (EMR2).

2. The antibody, or the antigen-binding fragment thereof, according to claim 1, wherein the GAIN domain comprises amino acid residues D261-Q478 of SEQ ID NO: 205.

3. The antibody, or antigen-binding fragment thereof, according to claim 1, wherein the GAIN domain comprises the amino acid sequence of SEQ ID NO: 215.

4. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1-3, wherein the GPS motif comprises the amino acid sequence of SEQ ID NO: 216.

5. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1 -4, wherein the antibody or the antigen-binding fragment thereof, binds to an epitope comprising the amino acid sequence of SEQ ID NO: 217 or SEQ ID NO: 218.

6. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1-5, wherein the antibody, or the antigen-binding fragment thereof, binds to human EMR2 with a dissociation constant (KD) between about 0.01 nM to about 5 nM.

7. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1-5, wherein the antibody, or the antigen-binding fragment thereof, binds to human EMR2 with a half maximal effective concentration (ECso) between about 0.1 nM to about 15 nM.

8. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1 -7, wherein the antibody, or the antigen-binding fragment thereof, is or comprises a fragment antigen-binding (Fab), a F(ab')2 fragment, F(ab)'3 fragments, a single-chain variable fragment (scFv), a bis-scFv, a (scFv)2, a stapled scFv (spFv), a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Immunoglobulin New Antigen Receptor (Ig NAR), a single heavy chain antibody, a camelid antibody, a shark antibody, or a chemically modified derivative thereof.

9. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1-8, wherein the antibody, or the antigen-binding fragment thereof, comprises a Fab.

10. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1 -9, wherein the antibody, or the antigen-binding fragment thereof, further comprises a first constant Ig domain of the heavy chain (CHI domain).

11. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1-8, wherein the antibody, or the antigen-binding fragment thereof, does not comprise a CHI domain.

12. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1-8 and 11, wherein the antibody, or the antigen-binding fragment thereof, comprises a scFv or a spFv.

13. The antibody, or the antigen-binding fragment thereof, according to claim 12, wherein the scFv or the spFv comprises a signal sequence, a heavy chain variable sequence, a GS-Linker, and a light chain variable sequence.

14. The antibody or, the antigen-binding fragment thereof, according to any one of claims 1-8, further comprising a fragment crystallizable (Fc) domain.

15. The antibody, or the antigen-binding fragment thereof, according to claim 14, wherein the Fc domain of the antibody, or the antigen-binding fragment thereof, is an IgA, an IgG, an IgE, or an IgM.

16. The antibody, or the antigen-binding fragment thereof, according to claim 14 or claim 15, wherein the Fc domain of the antibody, or the antigen-binding fragment thereof, is an IgG.

17. The antibody, or the antigen-binding fragment thereof, according to claim 16, wherein the IgG is IgGl or IgG4.

18. The antibody, or the antigen-binding fragment thereof, according to any one of claims 14-17, wherein the Fc domain comprises one or more different mutations which promote heterodimerization.

19. The antibody, or the antigen-binding fragment thereof, according to any one of claims 14-17, wherein the Fc domain comprises mutations T366S, L368A, and Y407V (EU numbering) or mutation T366W (EU numbering).

20. The antibody, or the antigen-binding fragment thereof, according to any one of claims 14-19, wherein the Fc domains of a first heavy chain (HC1) and / or a second heavychain (HC2) further comprise one or more mutations which reduce Fc binding to a Fey receptor.

21. The antibody, or the antigen-binding fragment thereof, according to claim 20, wherein the Fey receptor is FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, and / or FcyRIIIB.

22. The antibody, or the antigen-binding fragment thereof, according to any one of claims 14-21, wherein the Fc domain comprises one or more mutations selected from L234A, L235A, and D265S (EU numbering).

23. The antibody, or the antigen-binding fragment thereof, according to any one of claims 14-22, wherein the Fc domain comprises mutations L234A, L235A, and D265S (EU numbering).

24. The antibody, or the antigen-binding fragment thereof, according to any one of claims 14-23, wherein the Fc domain further comprises one or more mutations which reduce Fc binding to protein A.

25. The antibody, or the antigen-binding fragment thereof, according to any one of claims 14-24, wherein the Fc domain comprises mutations H435R and / or Y436F (EU numbering).

26. The antibody, or the antigen-binding fragment thereof, according to any one of claims 14-25, wherein the Fc domain comprises mutations H435R and Y436F (EU numbering).

27. The antibody, or the antigen-binding fragment thereof, according to any one of claims 14-26, wherein the antibody, or the antigen-binding fragment thereof, comprises a humanized antibody, or an antigen binding fragment thereof, a human antibody, or an antigen binding fragment thereof, a murine antibody, or an antigen binding fragment thereof, a chimeric antibody, or an antigen binding fragment thereof, a monospecific antibody, or a monospecific antigen binding fragment thereof, a bispecific antibody, or a bispecific antigen binding fragment thereof, a multispecific antibody, or a multispecific antigen binding fragment thereof.

28. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1-27, wherein the antibody, or the antigen-binding fragment thereof, comprises: a) a heavy chain complementarity determining region (HCDR) 1, a HCDR2 and a HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 63, and a light chaincomplementarity determining region (LCDR) 1 , a LCDR2 and a LCDR3 of the light chain variable region (VL) of SEQ ID NO: 64; b) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 95, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 96; c) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 127, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 128; or d) a HCDR1 , a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 191 , and a LCDR1 , a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 192.

29. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1 -28, wherein the antibody, or the antigen-binding fragment thereof, comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of: a) SEQ ID NO: 33, 34, 35, 48, 49, and 50, respectively; b) SEQ ID NO: 36, 37, 38, 51, 52, and 53, respectively; c) SEQ ID NO: 39, 40, 41, 54, 55, and 56, respectively; d) SEQ ID NO: 42, 43, 44, 57, amino acid sequence EVS, and SEQ ID NO: 59, respectively; e) SEQ ID NO: 45, 46, 47, 60, 61, and 62, respectively; f) SEQ ID NO: 65, 66, 67, 80, 81, and 82, respectively; g) SEQ ID NO: 68, 69, 70, 83, 84, and 85, respectively; h) SEQ ID NO: 71, 72, 73, 86, 87, and 88, respectively; i) SEQ ID NO: 74, 75, 76, 89, amino acid sequence AAI, and SEQ ID NO: 91, respectively; j) SEQ ID NO: 77, 78, 79, 92, 93, and 94, respectively; k) SEQ ID NO: 97, 98, 99, 112, 113, and 114, respectively; l) SEQ ID NO: 100, 101, 102, 115, 116, and 117, respectively; m) SEQ ID NO: 103, 104, 105, 118, 119, and 120, respectively; n) SEQ ID NO: 106, 107, 108, 121, ammo acid sequence DNN, and SEQ ID NO: 123, respectively; o) SEQ ID NO: 109, 110, 111, 124, 125, and 126, respectively; p) SEQ ID NO: 161, 162, 163, 176, 177, and 178, respectively; q) SEQ ID NO: 164, 165, 166, 179, 180, and 181, respectively; r) SEQ ID NO: 167, 168, 169, 182, 18, and 184, respectively;s) SEQ ID NO: 170, 171, 172, 185, amino acid sequence EVS, and SEQ ID NO: 187, respectively; or t) SEQ ID NO: 173, 174, 175, 188, 189, and 190, respectively.

30. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1 -29, wherein the antibody, or the antigen-binding fragment thereof, comprises a variable heavy chain region (VH) comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 63; b) the VH of SEQ ID NO: 95; c) the VH of SEQ ID NO: 127; or d) the VH of SEQ ID NO: 191.

31. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1-30, wherein the antibody, or the antigen-binding fragment thereof, comprises or further comprises a variable light chain region (VL) comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 64; b) the VL of SEQ ID NO: 96; c) the VL of SEQ ID NO: 128; or d) the VL of SEQ ID NO: 192.

32. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1-31, wherein the antibody, or the antigen-binding fragment thereof, comprises: a) the VH of SEQ ID NO: 63 and the VL of SEQ ID NO: 64; b) the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 96; c) the VH of SEQ ID NO: 127 and the VL of SEQ ID NO: 128; or d) the VH of SEQ ID NO: 191 and the VL of SEQ ID NO: 192.

33. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1-32, wherein the antibody, or the antigen-binding fragment thereof, comprises a scFv or a spFV which comprises, from the N- to C-terminus, a VH, a linker and a VL in the format VH-L-VL or a VL, a linker and a VH in the format VL-L-VH.

34. The antibody, or the antigen-binding fragment thereof, according to claim 33, wherein the VH comprises the amino acid sequence of SEQ ID NO: 191, the VL comprises the amino acid sequence of SEQ ID NO: 192, and the linker comprises the amino acid sequence of SEQ ID NO: 221.

35. The antibody, or the antigen-binding fragment thereof, according to claim 33, wherein the linker comprises the amino acid sequence of SEQ ID NO: 221.

36. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1-35, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:194, 196, 198, or 202.

37. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1-36, comprising a light chain comprising the amino acid sequence of SEQ ID NO:195, 197 or 199.

38. The antibody, or the antigen-binding fragment thereof, according to claim 36 or 37, wherein the antibody or the antigen-binding fragment thereof, comprises: a) an HC1 comprising the amino acid sequence of SEQ ID NO: 194 and a first light chain (LC1) comprising the amino acid sequence of SEQ ID NO: 195; b) an HC1 comprising the amino acid sequence of SEQ ID NO: 196 and an LC1 comprising the amino acid sequence of SEQ ID NO: 197; or c) an HC1 comprising the amino acid sequence of SEQ ID NO: 198 and an LC1 comprising the amino acid sequence of SEQ ID NO: 199.

39. An antibody, or an antigen-binding fragment thereof, that binds to the same epitope of EMR2 as the antibody, or the antigen-binding fragment thereof, according to any one of claims 28-38.

40. An antibody, or an antigen-binding fragment thereof, that competes for binding to the same epitope of EMR2 with the antibody, or the antigen-binding fragment thereof, according to any one of claims 28-39.

41. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1 -40, wherein the antibody, or the antigen-binding fragment thereof, is a monospecific antibody.

42. The antibody, or the antigen-binding fragment thereof, according to any one of claims 1 -40, wherein the antibody, or the antigen-binding fragment thereof, is a bispecific antibody which specifically binds to EMR2 and to a second antigen.

43. The antibody, or the antigen-binding fragment thereof, according to claim 42, wherein the second antigen is T-cell Receptor Variable 19 (TRBV19).

44. An isolated polynucleotide encoding the antibody, or the antigen-binding fragment thereof, according to any one of claims 1 -42.

45. The isolated polynucleotide of claim 44, wherein the polynucleotide comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 63; b) the VH of SEQ ID NO: 95 c) the VH of SEQ ID NO: 127; or d) the VH of SEQ ID NO: 191.

46. The isolated polynucleotide of claim 44 or claim 45, wherein the polynucleotide comprises or further comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 64; b) the VL of SEQ ID NO: 96; c) the VL of SEQ ID NO: 128; or d) the VL of SEQ ID NO: 192.

47. The isolated polynucleotide of any one of claims 44-46, wherein the polynucleotide comprises a sequence comprising a nucleotide sequence encoding: a) the amino acid sequence of SEQ ID NO: 63 and / or SEQ ID NO: 64; b) the amino acid sequence of SEQ ID NO: 95 and / or SEQ ID NO: 96; c) the amino acid sequence of SEQ ID NO: 127 and / or SEQ ID NO: 128; or d) the amino acid sequence of SEQ ID NO: 191 and / or the SEQ ID NO: 192.

48. The isolated polynucleotide according to claim 46 or claim 47, wherein the polynucleotide comprises a sequence encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 194, 196, 198, or 202.

49. The isolated polynucleotide according to any one of claims 44-48, wherein the polynucleotide comprises a sequence encoding a light chain comprising the amino acid sequence of SEQ ID NO: 195, 197, or 199.

50. The isolated polynucleotide of any one of claims 44-49, comprising a nucleotide sequence encoding: a) the amino acid sequence of SEQ ID NO: 194 and / or SEQ ID NO: 195; b) the amino acid sequence of SEQ ID NO: 196 and / or SEQ ID NO: 197; c) the amino acid sequence of SEQ ID NO: 198 and / or the amino acid sequence of SEQ ID NO: 199; or d) the amino acid sequence of SEQ ID NO: 202.

51. A vector comprising the isolated polynucleotide according to any one of claims 44- 50.

52. The vector according to claim 51, wherein the isolated polynucleotide is operably linked to an expression control sequence.

53. The vector according to claim 51 or claim 52, wherein the vector is a viral vector.

54. The vector according to claim 53, wherein the viral vector is selected from an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpes simplex virus vector, and a poxvirus vector.

55. A pharmaceutical composition comprising (i) the antibody, or the antigen-binding fragment thereof, according to any one of claims 1-43, or the isolated polynucleotide according to any one of claims 44-50, or the vector according to any one of claims 51-54, and (ii) a pharmaceutically acceptable carrier or excipient.

56. A host cell expressing the antibody, or the antigen-binding fragment thereof, according to any one of claims 1-43.

57. The host cell according to claim 56, wherein the cell is a hybridoma.

58. The host cell according to claim 56, wherein the antibody, or the antigen-binding fragment thereof, is recombinantly produced.

59. A host cell comprising the isolated polynucleotide according to any one of claims 44- 50, or the vector according to any one of claims 51-54.

60. A method for treating a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody, or the antigen-binding fragment thereof, according to any one of claims 1-43, or the isolated polynucleotide according to any one of claims 44-50, or the vector according to any one of claims 51-54, or the pharmaceutical composition of claim 55, or the host cell according to any one of claims 56-59.

61. A method for inducing cytotoxicity of a cancer cell or redirecting immune or T cells to cancer cells, the method comprising administering to the cancer cell an effective amount of the antibody or, the antigen-binding fragment thereof, according to any one of claims 1-43, or the isolated polynucleotide according to any one of claims 44-50, or the vector according to any one of claims 51-54, or the pharmaceutical composition of claim 55, or the host cell according to any one of claims 56-59, wherein the effective amount is sufficient to inhibit the growth or proliferation of the cancer cell.

62. The method according to claim 61, wherein the cancer cell is in a subject and the antibody, or the antigen-binding fragment thereof, the polynucleotide, the vector, the pharmaceutical composition, or the host cell is administered to the subject.

63. The method according to claim 61 or claim 62, wherein the administration is conducted ex vivo.

64. The method according to any one of claims 61-63, wherein the cancer is an EMR2- expressing cancer.

65. The method according to claim 64, wherein the EMR2-expressing cancer is a hematological cancer.

66. The method according to claim 65, wherein the hematological cancer is a myeloid malignancy.

67. The method according to claim 66, wherein the hematological cancer is acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), or myelodysplastic neoplasms.

68. The method according to any one of claims 60-67, further comprising administering a second therapeutic agent.

69. The method according to claim 68, wherein the second therapeutic agent is a surgery, a chemotherapy, an androgen deprivation therapy, a radiation, or any combination thereof.

70. The antibody according to any one of claims 1-43, for use in the method according to any one of claims 60-69.

71. The isolated polynucleotide according to any one of claims 44-50, for use in the method according to any one of claims 60-69.

72. The vector according to any one of claims 51-54, for use in the method according to any one of claims 60-69.

73. The pharmaceutical composition according to claim 55, for use in the method according to any one of claims 60-69.

74. The host cell according to any one of claims 56-59, for use in the method according to any one of claims 60-69.

75. A method for generating the antibody, or the antigen-binding fragment thereof, according to any one of claims 1-43, wherein the method comprises culturing the host cell according to any one of claims 56-59, and isolating the antibody, or the antigenbinding fragment thereof.

76. A kit comprising (i) the antibody, or the antigen-binding fragment thereof, according to any one of claims 1-43, and / or the isolated polynucleotide according to any one of claims 44-50, and / or the vector according to any one of claims 51-54, and / or the pharmaceutical composition of claim 55, and / or the host cell according to any one of claims 56-59, and (ii) packaging for the same and / or instructions for use.

77. A bispecific antibody, or a bispecific antigen-binding fragment thereof, that specifically binds (i) TRBV19 with a first antigen-binding site and (ii) the GAIN domain and / or the GPS motif of EMR2 with a second antigen-binding site.

78. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to claim 77, comprising: a) a first heavy chain (HC1); b) optionally a first light chain (LC1);c) a second heavy chain (HC2); and d) optionally a second light chain (LC2), wherein(i) the HC1 and the LC1 form a first antigen-binding site that specifically binds a first antigen,(ii) the HC2 and the LC2 form a second antigen-binding site that specifically binds a second antigen,(iii) the HC1 and HC2 each comprise a Fc domain comprising a CH2-CH3 domain; and wherein the first antigen is TRBV19, and the second antigen is EMR2.

79. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to claim 77 or claim 78, wherein the second antigen-binding site specifically binds the GAIN domain and / or the GPS motif of EMR2.

80. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to claim 79, wherein the GAIN domain comprises amino acid residues D261-Q478 of SEQ ID NO: 205.

81. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to claim 79, wherein the GAIN domain comprises the amino acid sequence of SEQ ID NO: 215.

82. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-81, wherein the GPS motif comprises the amino acid sequence of SEQ ID NO: 216.

83. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-82, wherein the antibody, or the antigen-binding fragment thereof, binds to an epitope comprising the amino acid sequence of SEQ ID NO: 217 or SEQ ID NO: 218.

84. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-83, wherein the first antigen-binding site comprises a Fab or a scFv or a spFv.

85. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-84, wherein the Fc of the first antigen-binding site comprises a CHI domain.

86. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-85, wherein the second antigen-binding site comprises a Fab, a scFv or a spFv.

87. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-86, wherein the Fc of the second antigen-binding site further comprises a CHI domain.

88. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-87, wherein the first antigen-binding site comprises a Fab and the second antigen-binding site comprises a scFv or a spFv.

89. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-88, wherein the first antigen-binding site comprises a scFv or a spFv and the second antigen-binding site comprises a Fab.

90. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-89, wherein the Fc domain of the antibody or the antigenbinding fragment thereof, is an IgA, an IgG, an IgE, or an IgM.

91. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to claim 90, wherein the Fc domain of the antibody, or the antigen-binding fragment thereof, is an IgG.

92. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to claim 91, wherein the IgG is IgGl or IgG4.

93. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-92, wherein the Fc domains of HC1 and HC2 comprise one or more different mutations which promote heterodimerization.

94. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-93, wherein the Fc domain of the HC1 comprises mutations T366S, L368A and Y407V (EU numbering) and the Fc domain of the HC2 comprises mutation T366W (EU numbering).

95. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-94, wherein the Fc domain of the HC2 comprises mutations T366S, L368A and Y407V (EU numbering) and the Fc domain of the HC1 comprises mutation T366W (EU numbering).

96. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-95, wherein the Fc domains of HC1 and / or HC2 further comprise one or more mutations which reduce Fc binding to a Fey receptor.

97. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to claim 96, wherein the Fey receptor is FcyRI, FcyRIIA, Fey RUB, FcyRIIIA, and / or FcyRIIIB.

98. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-97, wherein the Fc domains of HC1 and / or HC2 each comprise one or more mutations selected from L234A, L235A, and D265S (EU numbering).

99. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-98, wherein the Fc domains of HC1 and HC2 each comprise mutations L234A, L235A, and D265S (EU numbering).

100. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-99, wherein the Fc domains of HC1 or HC2 further comprises one or more mutations which reduce Fc binding to protein A.

101. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-100, wherein the Fc domains of HC1 or HC2 comprise mutations H435R and / or Y436F (EU numbering).

102. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-101, wherein the Fc domain of HC1 comprises mutations H435R and Y436F (EU numbering).

103. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-102, wherein the Fc domain of HC2 comprises mutations H435R and Y436F (EU numbering).

104. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-103, wherein the HC1 or HC2 comprise mutation C220S (EU numbering).

105. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-104, wherein the HC1 comprises mutation C220S (EU numbering).

106. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-104, wherein the HC2 comprises mutation C220S (EU numbering).

107. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-106, wherein the bispecific antibody, or the bispecific antigen-binding fragment thereof, comprises a humanized antibody, or an antigen binding fragment thereof, a human antibody, or an antigen binding fragment thereof, a murine antibody, or an antigen binding fragment thereof, a chimeric antibody, or an antigen binding fragment thereof, or a chemically modified derivative thereof.

108. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-107, wherein the first antigen-binding site that specifically binds TRBV19 comprises: a) a HCDR1 , a HCDR2 and a HCDR3 of the VH of SEQ ID NO: 31 and a LCDR1 , a LCDR2 and a LCDR3 of the VL of SEQ ID NO: 32; or b) a HCDR1 , a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 159 and a LCDR1 , LCDR2, and LCDR3 of the VL of SEQ ID NO: 160.

109. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to claim 108, wherein the first antigen-binding site that specifically binds TRBV19 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of: a) SEQ ID NOs: 1, 2, 3, 16, 17, and 18, respectively; b) SEQ ID NOs: 4, 5, 6, 19, 20, and 21, respectively; c) SEQ ID NOs: 7, 8, 9, 22, 23, and 24, respectively; d) SEQ ID NOs: 10, 11, 12, 25, 26, and 27, respectively; e) SEQ ID NOs: 13, 14, 15, 28, 29, and 30, respectively;f) SEQ ID NOs: 129, 130, 131, 144, 145, and 146, respectively; g) SEQ ID NOs: 132, 133, 134, 147, 148, and 149, respectively; h) SEQ ID NOs: 135, 136, 137, 150, 151, and 152, respectively; i) SEQ ID NOs: 138, 139, 140, 153, amino acid sequence KVS, and SEQ ID NO: 155, respectively; or j) SEQ ID NOs: 141, 142, 143, 156, 157, and 158, respectively.

110. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-109, wherein the first antigen-binding site that specifically binds TRBV19 comprises a VH comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 31 ; or b) the VH of SEQ ID NO: 159.

111. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claim 77-110, wherein the first antigen-binding site that specifically binds TRBV19 comprises or further comprises a VL comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 32; or b) the VL of SEQ ID NO: 160.

112. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-111, wherein the first antigen-binding site that specifically binds TRBV19 comprises: a) the VH of SEQ ID NO: 31 and the VL of SEQ ID NO: 32; or b) the VH of SEQ ID NO: 159 and the VL of SEQ ID NO: 160.

113. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-112, wherein the second antigen-binding site that specifically binds EMR2 comprises:a) a HCDR1, a HCDR2 and a HCDR3 of the VH of SEQ ID NO: 63, and a LCDR 1, a LCDR2 and a LCDR3 of the VL of SEQ ID NO: 64; b) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 95, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 96; c) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 127, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 128; or d) a HCDR1, a HCDR2, and a HCDR3 of the VH of SEQ ID NO: 191, and a LCDR1, a LCDR2, and a LCDR3 of the VL of SEQ ID NO: 192.

114. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-113, wherein the second antigen-binding site that specifically binds EMR2 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of: a) SEQ ID NO: 33, 34, 35, 48, 49, and 50, respectively; b) SEQ ID NO: 36, 37, 38, 51, 52, and 53, respectively; c) SEQ ID NO: 39, 40, 41, 54, 55, and 56, respectively; d) SEQ ID NO: 42, 43, 44, 57, 58, and 59, respectively; e) SEQ ID NO: 45, 46, 47, 60, 61, and 62, respectively; f) SEQ ID NO: 65, 66, 67, 80, 81, and 82, respectively; g) SEQ ID NO: 68, 69, 70, 83, 84, and 85, respectively; h) SEQ ID NO: 71, 72, 73, 86, 87, and 88, respectively; i) SEQ ID NO: 74, 75, 76, 89, amino acid sequence AAI, and SEQ ID NO: 91, respectively; j) SEQ ID NO: 77, 78, 79, 92, 93, and 94, respectively; k) SEQ ID NO: 97, 98, 99, 112, 113, and 114, respectively; l) SEQ ID NO: 100, 101, 102, 115, 116, and 117, respectively; m) SEQ ID NO: 103, 104, 105, 118, 119, and 120, respectively; n) SEQ ID NO: 106, 107, 108, 121, ammo acid sequence DNN, and SEQ ID NO: 123, respectively; o) SEQ ID NO: 109, 110, 111, 124, 125, and 126, respectively; p) SEQ ID NO: 161, 162, 163, 176, 177, and 178, respectively; q) SEQ ID NO: 164, 165, 166, 179, 180, and 181, respectively;r) SEQ ID NO: 167, 168, 169, 182, 18, and 184, respectively; s) SEQ ID NO: 170, 171, 172, 185, amino acid sequence EVS, and SEQ ID NO: 187, respectively; or t) SEQ ID NO: 173, 174, 175, 188, 189, and 190, respectively.

115. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-114, wherein the bispecific antibody or the bispecific antigenbinding fragment thereof, comprises a VH comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 63; b) the VH of SEQ ID NO: 95; c) the VH of SEQ ID NO: 127; or d) the VH of SEQ ID NO: 191.

116. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-115, wherein the bispecific antibody or the bispecific antigenbinding fragment thereof, comprises or further comprises a VL comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 64; b) the VL of SEQ ID NO: 96; c) the VL of SEQ ID NO: 128; or d) the VL of SEQ ID NO: 192.

117. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-116, wherein the first antigen-binding site and / or the second antigen-binding site that specifically binds EMR2 comprises: a) the VH of SEQ ID NO: 63 and the VL of SEQ ID NO: 64; b) the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 96; c) the VH of SEQ ID NO: 127 and the VL of SEQ ID NO: 128; or d) the VH of SEQ ID NO: 191 and the VL of SEQ ID NO: 192.

118. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-117, wherein the bispecific antibody or the bispecific antigenbinding fragment thereof, comprises a scFv or a spFV, wherein the scFv or the spFV comprises, from the N- to C-terminus, a VH, a linker and a VL in the format VH-L- VL or a VL, a linker and a VH in the format VL-L-VH.

119. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to claim 118, wherein the VH comprises the amino acid sequence of SEQ ID NO:159, the VL comprises the amino acid sequence of SEQ ID NO: 160, and the linker comprises the amino acid sequence of SEQ ID NO: 221.

120. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to claim 118, wherein the VH comprises the amino acid sequence of SEQ ID NO: 191, the VL comprises the amino acid sequence of SEQ ID NO: 192, and the linker comprises the amino acid sequence of SEQ ID NO: 221.

121. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to claim 118, wherein the linker comprises the amino acid sequence of SEQ ID NO: 221.

122. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-121, wherein the first antigen-binding site specifically binds to TRBV 19 with a KD that is between about 15 nM to about 200 nM.

123. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-122, wherein the first antigen-binding site specifically binds to TRBV19 with an ECso between aboutl nM to about 100 nM.

124. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-123, wherein the second antigen-binding site specifically binds to EMR2 with a KD that is between about 0.01 nM to about 5 nM.

125. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-123, wherein the second antigen-binding site specifically binds to EMR2 with an ECso between about 0.1 nM to about 15 nM.

126. The bispecific antibody, or the bispecific binding fragment thereof, according to any one of claims 77-125, wherein the HC1 comprises the amino acid sequence of SEQ ID NO: 193 or 200.

127. The bispecific antibody, or the bispecific binding fragment thereof, according to any one of claims 77-126, wherein the LC1 comprises the amino acid sequence of SEQ ID NO: 201.

128. The bispecific antibody, or the bispecific binding fragment thereof, according to any one of claims 77-127, wherein a) the HC1 comprises the amino acid sequence of SEQ ID NO: 193; or b) the HC1 comprises the amino acid sequence of SEQ ID NO: 200 and the LC1 comprises the amino acid sequence of SEQ ID NO: 201.

129. The bispecific antibody, or the bispecific binding fragment thereof, according to any one of claims 77-128, wherein the HC2 comprises the amino acid sequence of SEQ ID NO: 194, 196, 198, or 202.

130. The bispecific antibody, or the bispecific binding fragment thereof, according to any one of claims 77-129, wherein the LC2 comprises the amino acid sequence of SEQ ID NO: 195, 197, or 199.

131. The bispecific antibody, or the bispecific binding fragment thereof, according to claim 130, wherein a) the HC2 comprises the amino acid sequence of SEQ ID NO: 194 and the LC2 comprises the amino acid sequence of SEQ ID NO: 195; b) the HC2 comprises the amino acid sequence of SEQ ID NO: 196 and the LC1 comprises the amino acid sequence of SEQ ID NO: 197; c) the HC2 comprises the amino acid sequence of SEQ ID NO: 198 and the LC1 comprises the amino acid sequence of SEQ ID NO: 199; or d) the HC2 comprises the amino acid sequence of SEQ ID NO: 202.

132. The bispecific antibody, or the bispecific binding fragment thereof, according to claim 130 or 131, wherein a) the HC1 comprises the amino acid sequence of SEQ ID NO: 193, the HC2 comprises the amino acid sequence of SEQ ID NO: 194, and the LC2 comprises the amino acid sequence of SEQ ID NO 195;b) the HC1 comprises the amino acid sequence of SEQ ID NO: 193, the HC2 comprises the amino acid sequence of SEQ ID NO: 196, and the LC2 comprises the amino acid sequence of SEQ ID NO: 197; c) the HC1 comprises the amino acid sequence of SEQ ID NO: 193, the HC2 comprises the amino acid sequence of SEQ ID NO: 198, and the LC2 comprises the amino acid sequence of SEQ ID NO: 199; or d) the HC1 comprises the amino acid sequence of SEQ ID NO: 200, the LC1 comprises the amino acid sequence of SEQ ID NO: 201, and the HC2 comprises the amino acid sequence of SEQ ID NO: 202.

133. A bispecific antibody, or a bispecific antigen-binding fragment thereof, that binds to the same epitope as the bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-132.

134. A bispecific antibody, or a bispecific antigen-binding fragment thereof, that competes for binding to the same epitope with the bispecific antibody, or the bispecific antigenbinding fragment thereof, according to any one of claims 77-132.

135. A bispecific antibody, or a bispecific antigen-binding fragment thereof, wherein the bispecific antibody, or the bispecific antigen-binding fragment thereof, comprises an HC1 comprising the amino acid sequence of SEQ ID NO: 193, an HC2 comprising the amino acid sequence of SEQ ID NO: 198, and an LC2 comprising the amino acid sequence of SEQ ID NO: 199.

136. An isolated polynucleotide encoding the bispecific antibody, or the bispecific binding fragment thereof, of any one of claims 77-135.

137. The isolated polynucleotide according to claim 136, wherein the polynucleotide comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 63; b) the VH of SEQ ID NO: 95; c) the VH of SEQ ID NO: 127; or d) the VH of SEQ ID NO: 191.

138. The isolated polynucleotide according to claim 136 or 137, wherein the polynucleotide comprises or further comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 64; b) the VL of SEQ ID NO: 96; c) the VL of SEQ ID NO: 128; or d) the VL of SEQ ID NO: 192.

139. The isolated polynucleotide according to any one of claims 136-138, wherein the polynucleotide comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VH of SEQ ID NO: 31 ; or b) the VH of SEQ ID NO: 159.

140. The isolated polynucleotide according to any one of claims 136-139, wherein the polynucleotide comprises or further comprises a sequence comprising a nucleotide sequence coding for an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to: a) the VL of SEQ ID NO: 32; or b) the VL of SEQ ID NO: 160.

141. The isolated polynucleotide according to any one of claims 136-140, wherein the polynucleotide comprises a sequence encoding a first antigen-binding site that specifically binds TRBV19, the sequence comprising a nucleotide sequence encoding: a) the amino acid sequence of SEQ ID NO: 31 and / or the amino acid sequence of SEQ ID NO: 32; or b) the amino acid sequence of SEQ ID NO: 159 and / or the amino acid sequence of SEQ ID NO: 160.

142. The isolated polynucleotide according to any one of claims 136-141, wherein the polynucleotide comprises a sequence encoding a second antigen-binding site that specifically binds EMR2, the sequence comprising a nucleotide sequence encoding: a) the amino acid sequence of SEQ ID NO: 63 and / or the amino acid sequence of SEQ ID NO: 64; b) the amino acid sequence of SEQ ID NO: 95 and / or the amino acid sequence of SEQ ID NO: 96; c) the amino acid sequence of SEQ ID NO: 127 and / or the amino acid sequence of SEQ ID NO: 128; or d) the amino acid sequence of SEQ ID NO: 191 and / or the amino acid sequence of SEQ ID NO: 192.

143. The isolated polynucleotide according to any one of claims 136-142, wherein the polynucleotide comprises a sequence encoding an HC1 comprising the amino acid sequence of SEQ ID NO: 193 or 200.

144. The isolated polynucleotide according to any one of claims 136-143, wherein the polynucleotide comprises a sequence encoding a LC1 comprising the amino acid sequence of SEQ ID NO: 201.

145. The isolated polynucleotide according to any one of claims 136-144, comprising a nucleotide sequence encoding: a) the amino acid sequence of SEQ ID NO: 193; or b) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 201.

146. The isolated polynucleotide according to any one of claims 136-145, wherein the polynucleotide comprises a sequence encoding an HC2 comprising the nucleotide sequence of SEQ ID NO: 194, 196, 198, or 202.

147. The isolated polynucleotide according to any one of claims 136-146, wherein the polynucleotide comprises a sequence encoding a LC2 comprising the nucleotide sequence of SEQ ID NO: 195, 197, or 199.

148. The isolated polynucleotide according to any one of claims 136-147, wherein the polynucleotide comprises a sequence encoding:a) the amino acid sequence of SEQ ID NO: 194 and / or the amino acid sequence of SEQ ID NO: 195; b) the amino acid sequence of SEQ ID NO: 196 and / or the amino acid sequence of SEQ ID NO: 197; c) the amino acid sequence of SEQ ID NO: 198 and / or the amino acid sequence of SEQ ID NO: 199; or d) the amino acid sequence of SEQ ID NO: 202.

149. The polynucleotide according to claim any one of claims 136-148, wherein the polynucleotide comprises a sequence encoding: a) the amino acid sequence of SEQ ID NO: 193, the amino acid sequence of SEQ ID NO: 194 and the amino acid of SEQ ID NO: 195; b) the amino acid sequence of SEQ ID NO: 193, the amino acid sequence of SEQ ID NO: 196 and the amino acid sequence of SEQ ID NO: 197; c) the amino acid sequence of SEQ ID NO: 193, the amino acid sequence of SEQ ID NO: 198 and the amino acid sequence of SEQ ID NO: 199; or d) the amino acid sequence of SEQ ID NO: 200, the amino acid sequence of SEQ ID NO: 201, and the amino acid sequence of SEQ ID NO: 202.

150. A vector comprising the isolated polynucleotide according to any one of claims 136- 149.

151. The vector according to claim 150, wherein the polynucleotide is operably linked to an expression control sequence.

152. The vector according to claim 150 or claim 151, wherein the vector is a viral vector.

153. The vector according to claim 152, wherein the viral vector is selected from an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpes simplex virus vector, and a poxvirus vector.

154. A pharmaceutical composition comprising (i) the bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-135, or the isolated polynucleotide according to any one of claims 136-149, or the vectoraccording to any one of claims 150-153, and (ii) a pharmaceutically acceptable carrier or excipient.

155. A host cell expressing the bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-135.

156. The host cell according to claim 155, wherein the cell is a hybridoma.

157. The host cell according to claim 155, wherein the bispecific antibody, or the bispecific antigen-binding fragment thereof, is recombinantly produced.

158. A host cell comprising the isolated polynucleotide according to any one of claims 136-149 or the vector according to any one of claims 150-153.

159. A method for treating a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-135, or the polynucleotide according to any one of claims 136-149, or the vector according to any one of claims 150-153, or the pharmaceutical composition of claim 154.

160. A method for inducing cytotoxicity of a cancer cell or redirecting immune or T cells against cancer cells, the method comprising administering to the cancer cells an effective amount of the bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-135, or the isolated polynucleotide according to any one of claims 136-149, or the vector according to any one of claims 150-153, or the pharmaceutical composition of claim 154, or the host cell according to any one of claims 155-158, wherein the effective amount is sufficient to inhibit the growth or proliferation of the cancer cells.

161. The method according to claim 160, wherein the cancer cells are in a subject and the bispecific antibody, or the bispecific antigen-binding fragment thereof, the polynucleotide, the vector, the pharmaceutical composition, or the host cell is administered to the subject.

162. The method according to claim 160 or 161, wherein the administration is conducted ex vivo.

163. A method of redirecting a T cell to EMR2-expressing cancer cells in a subject in need thereof, the method comprising administering to the subject a therapeuticallyeffective amount of the bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-135, or the polynucleotide according to any one of claims 136-149, or the vector according to any one of claims 150-153, or the pharmaceutical composition of claim 154, or the host cell according to any one of claims 155-158.

164. The method, according to claim 163, wherein the therapeutically effective amount administered is sufficient to redirect the T cell response to the cancer cells.

165. The method, according to any one of claims 159-164, wherein the cancer is an EMR2-expressing cancer.

166. The method, according to claim 165, wherein the EMR2-expressing cancer is a hematological cancer.

167. The method, according to claim 166, wherein the hematological cancer is a myeloid malignancy.

168. The method, according to claim 167, wherein the cancer is acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), or myelodysplastic neoplasms.

169. The method, according to any one of claims 159-168, further comprising administering a second therapeutic agent.

170. The method, according to claim 169, wherein the second therapeutic agent is a surgery, a chemotherapy, an androgen deprivation therapy, or a radiation, or any combination thereof.

171. The bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-135, for use in the method according to any one of claims 159-170.

172. The polynucleotide according to any one of claims 136-149, for use in the method according to any one of claims 159-170.

173. The vector according to any one of claims 150-153 for use in the method according to any one of claims 159-170.

174. The pharmaceutical composition according to claim 154 for use in the method according to any one of claims 159-170.

175. The host cell according to any one of claims 155-158 for use in the method according to any one of claims 159-170.

176. A method for generating the bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-135, wherein the method comprises culturing the host cell according to any one of claims 155-158, and isolating the bispecific antibody, or the bispecific antigen-binding fragment thereof.

177. A kit comprising (i) the bispecific antibody, or the bispecific antigen-binding fragment thereof, according to any one of claims 77-135, or the polynucleotide according to any one of claims 136-149, or the vector according to any one of claims 150-153, or the pharmaceutical composition of claim 154, or the host cell according to any one of claims 155-157, and (ii) packaging for the same and / or instructions for use.

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