Multifunctional molecules that bind to calreticulin and their use

Abstract

Multifunctional molecules are disclosed that include one, two, or all of: i) an antigen-binding domain that binds to a calreticulin protein; (ii) an immune cell engager (e.g., selected from an NK cell engager, a T cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager); (iii) a cytokine molecule; and / or (iv) a stromal-modifying moiety. Also disclosed are nucleic acids encoding the same, methods of producing the molecules, and methods of using the molecules to treat cancer.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 808,779, filed Feb. 21, 2019; U.S. Provisional Application No. 62 / 818,427, filed Mar. 14, 2019; and U.S. Provisional Application No. 62 / 956,866, filed Jan. 3, 2020, the entire contents of each of which are hereby incorporated by reference herein. Sequence Listing This application includes a sequence listing that was electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on Feb. 19, 2020, is named E2070-7021WO_SL.txt and is 1,760,247 bytes in size.

Background Art

[0002] Myeloproliferative neoplasms (MPNs) are a group of conditions in which blood cells grow abnormally in the bone marrow. Common myeloproliferative neoplasms include primary or essential myelofibrosis (MF), essential thrombocythemia (ET), polycythemia vera (PV), and chronic myelogenous leukemia (CML). Primary myelofibrosis is a chronic blood cancer in which excessive scar tissue forms in the bone marrow, impairing the bone marrow's ability to produce normal blood cells. Considering the continuing need for improved treatment of myeloproliferative neoplasms, such as myelofibrosis, new compositions and treatments targeting myeloproliferative neoplasms are highly desirable.

Summary of the Invention

Means for Solving the Problems

[0003] The present disclosure relates, inter alia, to novel multispecific or multifunctional molecules comprising (i) an antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type or mutant calreticulin protein); (ii) an immune cell engager (e.g., selected from an NK cell engager, a T cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager); (iii) a cytokine molecule; and / or (iv) one, two, or all of the stromal modifying moieties. The terms “multispecific” or “multifunctional” are used interchangeably herein.

[0004] Without being bound by theory, the multispecific or multifunctional molecules disclosed herein are expected to target (e.g., localize, bridge, and / or activate) immune cells (e.g., immune effector cells selected from NK cells, T cells, B cells, dendritic cells, or macrophages) to target cells, such as cancer cells that express a calreticulin protein (e.g., a wild-type or mutant calreticulin protein), and / or modify the tumor stroma, e.g., modify the tumor microenvironment near the cancer site. Increasing the proximity and / or activity of immune cells using the multispecific molecules described herein is expected to enhance the immune response against target cells (e.g., cancer cells), thereby achieving a more effective therapy (e.g., a more effective cancer therapy). Without being bound by theory, a targeted and localized immune response against target cells (e.g., cancer cells) is thought to reduce the systemic toxicity effects of the multispecific molecules described herein.

[0005] Accordingly, multispecific molecules (e.g., multispecific or multifunctional antibody molecules) comprising the foregoing moieties, nucleic acids encoding the same, methods of producing the foregoing molecules, and methods of using the foregoing molecules to treat cancer are provided herein, among other things.

[0006] Accordingly, in one aspect, the present disclosure provides (i) a first antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type or mutant calreticulin protein), (ii) a second antigen-binding domain that binds to TCRβV, for example, an anti-TCRβV antigen-binding domain disclosed in any one of Table 1A, Table 2A, Table 3A, Table 10A, Table 11A, Table 12A, or Table 13A, or a second antigen-binding domain that binds to NKp30, for example, an anti-NKp30 antigen-binding domain disclosed in Tables 7 - 10 or 18 characterized by a multifunctional molecule comprising the same.

[0007] In some embodiments, the second antigen-binding domain binds to TCRβV. In some embodiments, the second antigen-binding domain activates T cells or the second antigen-binding domain does not activate T cells.

[0008] In some embodiments, the second antigen-binding domain binds to TCRβV12 or TCRβV6 (e.g., comprising the amino acid sequence of SEQ ID NO: 1044). In some embodiments, the second antigen-binding domain comprises one or more amino acid sequences listed in Table 1A, Table 2A, Table 3A, Table 10A, Table 11A, Table 12A, or Table 13A.

[0009] In some embodiments, the second antigen-binding domain is (a) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein (i) The VH comprises a VHCDR1 having the amino acid sequence of the heavy chain complementarity determining region 1 (VHCDR1) in Table 1A, Table 2A, Table 10A, Table 11A, Table 12A, or Table 13A (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), a VHCDR2 having the amino acid sequence of the VHCDR2 in Table 1A, Table 2A, Table 10A, Table 11A, Table 12A, or Table 13A (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or a VHCDR3 having the amino acid sequence of the VHCDR3 in Table 1A, Table 2A, Table 10A, Table 11A, Table 12A, or Table 13A (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions). (ii) The VL comprises a VLCDR1 having the amino acid sequence of the light chain complementarity determining region 1 (VLCDR1) in Table 1A, Table 2A, Table 10A, Table 11A, Table 12A, or Table 13A (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), a VLCDR2 having the amino acid sequence of the VLCDR2 in Table 1A, Table 2A, Table 10A, Table 11A, Table 12A, or Table 13A (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or a VLCDR3 having the amino acid sequence of the VLCDR3 in Table 1A, Table 2A, Table 10A, Table 11A, Table 12A, or Table 13A (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and a heavy chain variable region (VH) and / or a light chain variable region (VL). (b) A heavy chain variable region (VH) and / or a light chain variable region (VL), wherein (i) The VH comprises the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 3 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 4 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 5 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or (ii) The VL comprises the light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 7 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 8 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), a heavy chain variable region (VH) and / or a light chain variable region (VL); (c) A heavy chain variable region (VH) and / or a light chain variable region (VL), wherein (i) The VH comprises the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 45 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 46 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 47 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or (ii) The variable heavy chain (VH) and / or variable light chain (VL) wherein VL comprises the amino acid sequence of the variable heavy chain complementarity determining region 1 (VHCDR1) of SEQ ID NO: 51 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VHCDR2 of SEQ ID NO: 52 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VHCDR3 of SEQ ID NO: 53 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions); and / or (d) A variable heavy chain (VH) and / or variable light chain (VL) comprising (i) VH comprises the amino acid sequence of the variable heavy chain complementarity determining region 1 (VHCDR1) of SEQ ID NO: 48 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VHCDR2 of SEQ ID NO: 49 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VHCDR3 of SEQ ID NO: 50 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or (ii) VL comprises the amino acid sequence of the variable heavy chain complementarity determining region 1 (VHCDR1) of SEQ ID NO: 54 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VHCDR2 of SEQ ID NO: 55 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VHCDR3 of SEQ ID NO: 56 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), of the variable heavy chain (VH) and / or variable light chain (VL). comprising.

[0010] In some embodiments, the second antigen-binding domain is (a) A variable heavy chain (VH) and / or variable light chain (VL) comprising (i) The VH contains the amino acid sequence of VH in Table 1A, Table 2A, Table 10A, Table 11A, Table 12A, or Table 13A (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and / or (ii) The VL contains the amino acid sequence of VL in Table 1A, Table 2A, Table 10A, Table 11A, Table 12A, or Table 13A (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), (iii) The VH contains the amino acid sequence of SEQ ID NO: 9 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and / or (iv) The VL contains the amino acid sequence of SEQ ID NO: 10 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), a heavy chain variable region (VH) and / or a light chain variable region (VL); (b) A heavy chain variable region (VH) and / or a light chain variable region (VL), wherein (i) The VH contains the amino acid sequence of SEQ ID NO: 9 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and / or (ii) The VL contains the amino acid sequence of SEQ ID NO: 11 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), a heavy chain variable region (VH) and / or a light chain variable region (VL); and / or (c) A heavy chain variable region (VH) and / or a light chain variable region (VL), wherein (i) The VH contains the amino acid sequence of SEQ ID NO: 1312 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and / or (ii) The heavy chain variable region (VH) and / or the light chain variable region (VL), wherein VL comprises the amino acid sequence of SEQ ID NO: 1314 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto). comprises.

[0011] In some embodiments, the second antigen-binding domain is (a) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein (i) VH comprises the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 17 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 18 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 19 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or (ii) VL comprises the light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 20 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 21 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 22 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), a heavy chain variable region (VH) and / or a light chain variable region (VL); (b) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein (i) The VH comprises the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 57 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 58 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 59 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or (ii) The VL comprises the light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 63 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 64 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 65 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), a heavy chain variable region (VH) and / or a light chain variable region (VL); and / or (c) A heavy chain variable region (VH) and / or a light chain variable region (VL), wherein (i) The VH comprises the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 60 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 61 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 62 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or (ii) The heavy chain variable region (VH) and / or the light chain variable region (VL) wherein VL comprises the amino acid sequence of the light chain complementarity determining region 1 (VHCDR1) of SEQ ID NO: 66 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VHCDR2 of SEQ ID NO: 67 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VHCDR3 of SEQ ID NO: 68 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions). comprises.

[0012] In some embodiments, the second antigen-binding domain is (a) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein (i) VH comprises the amino acid sequence of SEQ ID NO: 15 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and / or (ii) VL comprises the amino acid sequence of SEQ ID NO: 16 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), a heavy chain variable region (VH) and / or a light chain variable region (VL); and / or (b) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein (i) VH comprises the amino acid sequence of SEQ ID NO: 23 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), the amino acid sequence of SEQ ID NO: 24 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), or the amino acid sequence of SEQ ID NO: 25 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); and / or (ii) VL The amino acid sequence of SEQ ID NO: 26 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), The amino acid sequence of SEQ ID NO: 27 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), The amino acid sequence of SEQ ID NO: 28 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), The amino acid sequence of SEQ ID NO: 29 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), or A heavy chain variable region (VH) and / or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 30 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto) comprising.

[0013] In some embodiments, the multifunctional molecule is For example, from the N-terminus to the C-terminus, a first polypeptide comprising a first VL and a first CL, For example, from the N-terminus to the C-terminus, a second polypeptide comprising a first VH, a first CH1, a first dimerization domain (e.g., a first Fc), and a first moiety that binds to a TCR (e.g., a first scFv that binds to a TCR (e.g., TCRVβ)), For example, from the N-terminus to the C-terminus, a third polypeptide comprising a second VH, a second CH1, a second dimerization domain (e.g., a second Fc), and optionally a second moiety that binds to a TCR (e.g., a second scFv that binds to a TCR (e.g., TCRVβ)), For example, from the N-terminus to the C-terminus, a fourth polypeptide comprising a second VL and a second CL comprising, The first VL and the first VH form a first antigen-binding domain that binds to a first calreticulin protein, and the second VL and the second VH form a third antigen-binding domain that binds to a second calreticulin protein. Optionally, the first and second calreticulin proteins comprise the amino acid sequence of SEQ ID NO: 6285 or 6286. Optionally, the first and second calreticulin mutant proteins are each independently selected from a molecule comprising the amino acid sequence of SEQ ID NO: 6313 or a molecule comprising the amino acid sequence of SEQ ID NO: 6314. Optionally, the multifunctional molecule comprises the configuration of FIGS. 3A or 3B.

[0014] In some embodiments, the second antigen-binding domain binds to NKp30. In some embodiments, the second antigen-binding domain is selected from an antibody molecule (e.g., an antigen-binding domain) or a ligand that binds to (e.g., activates) NKp30. For example, the second antigen-binding domain is an antibody molecule or ligand that binds to (e.g., activates) NKp30.

[0015] In some embodiments, the second antigen-binding domain is (i) a VHCDR1 having the amino acid sequence of VHCDR1 in Table 7, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having the amino acid sequence of VHCDR2 in Table 7, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 having the amino acid sequence of VHCDR3 in Table 7, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions), and / or (ii) A variable light chain (VL) comprising a VLCDR1 amino acid sequence of Table 8, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of Table 8, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions), and / or a VLCDR3 amino acid sequence of Table 8, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions). comprising.

[0016] In some embodiments, the second antigen-binding domain (i) A variable heavy chain (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 7313 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6001 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 7315 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions); and / or (ii) A variable light chain (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 7326 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 7327 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions), and / or a VLCDR3 amino acid sequence of SEQ ID NO: 7329 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions). comprising.

[0017] In some embodiments, the second antigen-binding domain is (i) a VH comprising the amino acid sequence of SEQ ID NO: 7298 or any of SEQ ID NO: 7300 - 7304 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any of SEQ ID NO: 7298 or SEQ ID NO: 7300 - 7304); and / or (ii) a VL comprising the amino acid sequence of SEQ ID NO: 7299 or any of SEQ ID NO: 7305 - 7309 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to any of SEQ ID NO: 7299 or SEQ ID NO: 7305 - 7309) and comprises.

[0018] In some embodiments, the second antigen-binding domain is (i) a VH comprising the amino acid sequence of SEQ ID NO: 7302 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7302), and a VL comprising the amino acid sequence of SEQ ID NO: 7305 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7305); or (ii) a VH comprising the amino acid sequence of SEQ ID NO: 7302 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7302), and a VL comprising the amino acid sequence of SEQ ID NO: 7309 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7309) and comprises.

[0019] In some embodiments, the second antigen-binding domain is (i) the amino acid sequence of SEQ ID NO: 7310 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7310); or (ii) The amino acid sequence of SEQ ID NO: 7311 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7311) comprises.

[0020] In some embodiments, the second antigen-binding domain (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 6000 heavy chain complementarity determining region 1 (VHCDR1), the amino acid sequence of SEQ ID NO: 6001 VHCDR2, and / or the amino acid sequence of SEQ ID NO: 6002 VHCDR3, and (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 6063 light chain complementarity determining region 1 (VLCDR1), the amino acid sequence of SEQ ID NO: 6064 VLCDR2, and / or the amino acid sequence of SEQ ID NO: 7293 VLCDR3 comprises.

[0021] In some embodiments, the second antigen-binding domain (1) a VHFWR1 having the amino acid sequence of heavy chain framework region 1 (VHFWR1) in Table 7, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, four, five, or six or fewer mutations therefrom, e.g., substitutions, additions, or deletions), a VHFWR2 having the amino acid sequence of VHFWR2 in Table 7, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, four, five, or six or fewer mutations therefrom, e.g., substitutions, additions, or deletions), a VHFWR3 having the amino acid sequence of VHFWR3 in Table 7, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, four, five, or six or fewer mutations therefrom, e.g., substitutions, additions, or deletions), or a VHFWR4 having the amino acid sequence of VHFWR4 in Table 7, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, four, five, or six or fewer mutations therefrom, e.g., substitutions, additions, or deletions) comprising heavy chain variable region (VH), and / or (2) A variable light chain (VL) comprising a VLFWR1 having the amino acid sequence of the light chain framework region 1 (VLFWR1) in Table 8, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, four, five, or six or fewer mutations therefrom, such as substitutions, additions, or deletions), a VLFWR2 having the amino acid sequence of VLFWR2 in Table 8, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, four, five, or six or fewer mutations therefrom, such as substitutions, additions, or deletions), a VLFWR3 having the amino acid sequence of VLFWR3 in Table 8, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, four, five, or six or fewer mutations therefrom, such as substitutions, additions, or deletions), or a VLFWR4 having the amino acid sequence of VLFWR4 in Table 8, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, four, five, or six or fewer mutations therefrom, such as substitutions, additions, or deletions). comprising.

[0022] In some embodiments, the second antigen-binding domain is (1) a variable heavy chain (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6003, the amino acid sequence of VHFWR2 of SEQ ID NO: 6004, the amino acid sequence of VHFWR3 of SEQ ID NO: 6005, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6006, and (3) a variable light chain (VL) comprising the amino acid sequence of the light chain framework region 1 (VLFWR1) of SEQ ID NO: 6066, the amino acid sequence of VLFWR2 of SEQ ID NO: 6067, the amino acid sequence of VLFWR3 of SEQ ID NO: 7292, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6069. comprising.

[0023] In some embodiments, the second antigen-binding domain is (i) a VH comprising the amino acid sequence of the VH in Table 7, Table 9, Table 10, or Table 18 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and / or (ii) a VL comprising the amino acid sequence of Table 8, Table 9, Table 10, or Table 18 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity thereto) comprising

[0024] In some embodiments, the second antigen-binding domain comprises a heavy chain comprising the amino acid sequence of the heavy chain of Table 10 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

[0025] In some embodiments, the second antigen-binding domain comprises a light chain comprising the amino acid sequence of the light chain of Table 10 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

[0026] In some embodiments, the second antigen-binding domain comprises a heavy chain comprising the amino acid sequence of the heavy chain of Table 10 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a light chain comprising the amino acid sequence of the light chain of Table 10 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

[0027] In some embodiments, the multispecific molecule for example, a first polypeptide comprising, from N-terminus to C-terminus, a first VL and a first CL for example, a second polypeptide comprising, from N-terminus to C-terminus, a first VH, a first CH1, a first dimerization domain (e.g., a first Fc), and a first moiety that binds to NKp30 (e.g., a first antibody molecule or ligand that binds to NKp30) for example, a third polypeptide comprising, from N-terminus to C-terminus, a second VH, a second CH1, a second dimerization domain (e.g., a second Fc), and optionally a second moiety that binds to NKp30 (e.g., a second antibody molecule or ligand that binds to NKp30) For example, from the N-terminus to the C-terminus, a fourth polypeptide comprising a second VL and a second CL comprising The first VL and the first VH form a first antigen-binding domain that binds to the first calreticulin protein, and the second VL and the second VH form a third antigen-binding domain that binds to the second calreticulin protein, Optionally, the first and second calreticulin proteins comprise the amino acid sequence of SEQ ID NO: 6285 or 6286, Optionally, the first and second calreticulin mutant proteins are each independently selected from a molecule comprising the amino acid sequence of SEQ ID NO: 6313, or a molecule comprising the amino acid sequence of SEQ ID NO: 6314, Optionally, the multifunctional molecule comprises the configuration of FIGS. 3A or 3B.

[0028] In some embodiments, the calreticulin protein comprises an amino acid sequence selected from SEQ ID NOs: 6285-6312, and optionally, the calreticulin protein comprises an amino acid sequence selected from SEQ ID NOs: 6313-6346.

[0029] In some embodiments, the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285. In some embodiments, the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286.

[0030] In some embodiments, the first antigen-binding domain binds to an epitope located within the C-terminus of the calreticulin protein, and optionally, the first antigen-binding domain binds to an epitope located within the amino acid sequence of SEQ ID NO: 6285 or 6286.

[0031] In some embodiments, the multispecific molecule is A third antigen-binding domain that binds to a second calreticulin protein, wherein, for example, the second calreticulin mutant protein comprises the amino acid sequence of SEQ ID NO: 6285 or 6286, the third antigen-binding domain further comprises, and optionally (i) the third antigen-binding domain is different from the first antigen-binding domain, or (ii) the third antigen-binding domain is the same as the first antigen-binding domain.

[0032] In some embodiments, the second calreticulin molecule is the same as the calreticulin molecule bound by the first antigen-binding domain. In some embodiments, the second calreticulin molecule is different from the calreticulin molecule bound by the first antigen-binding domain.

[0033] In some embodiments, the second calreticulin protein comprises an amino acid sequence selected from SEQ ID NOs: 6285-6312, and optionally, the second calreticulin protein comprises an amino acid sequence selected from SEQ ID NOs: 6313-6346.

[0034] In some embodiments, the calreticulin protein bound by the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 6285, and the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286.

[0035] In some embodiments, the third antigen-binding domain binds to an epitope located within the C-terminus of the second calreticulin protein, and optionally, the third antigen-binding domain binds to an epitope located within the amino acid sequence of SEQ ID NO: 6285 or 6286.

[0036] In some embodiments, the first antigen-binding domain (i) A heavy chain variable region (VH) comprising a VHCDR1 having the amino acid sequence of the heavy chain complementarity determining region 1 (VHCDR1) in Table 4, Table 7A, or Table 17 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), a VHCDR2 having the amino acid sequence of the VHCDR2 in Table 4, Table 7A, or Table 17 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or a VHCDR3 having the amino acid sequence of the VHCDR3 in Table 4, Table 7A, or Table 17 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions); (ii) A light chain variable region (VL) comprising a VHCDR1 having the amino acid sequence of the light chain complementarity determining region 1 (VLCDR1) in Table 5, Table 7A, or Table 18 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), a VHCDR2 having the amino acid sequence of the VLCDR2 in Table 5, Table 7A, or Table 18 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or a VHCDR3 having the amino acid sequence of the VLCDR3 in Table 5, Table 7A, or Table 18 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions); (iii) A VH comprising the amino acid sequence of the VH in Table 7A or Table 16 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); (iv) A VL comprising the amino acid sequence of the VL in Table 7A or Table 16 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity thereto); (v) A VH comprising a VHFWR1 having the amino acid sequence of the heavy chain framework region 1 (VHFWR1) in Table 4 or Table 6 (or a sequence having one, two, three, four, five, six, seven, eight, or nine or fewer mutations, such as substitutions, additions, or deletions), a VHFWR2 having the amino acid sequence of the VHFWR2 in Table 4 or Table 6 (or a sequence having one, two, three, four, five, six, seven, eight, or nine or fewer mutations, such as substitutions, additions, or deletions), a VHFWR3 having the amino acid sequence of the VHFWR3 in Table 4 or Table 6 (or a sequence having one, two, three, four, five, six, seven, eight, or nine or fewer mutations, such as substitutions, additions, or deletions), and / or a VHFWR4 having the amino acid sequence of the VHFWR4 in Table 4 or Table 6 (or a sequence having one, two, three, four, five, six, seven, eight, or nine or fewer mutations, such as substitutions, additions, or deletions), and / or (vi) A VL comprising a VLFWR1 having the amino acid sequence of the light chain framework region 1 (VLFWR1) in Table 5 or Table 6 (or a sequence having one, two, three, four, five, six, seven, eight, or nine or fewer mutations, such as substitutions, additions, or deletions), a VLFWR2 having the amino acid sequence of the VLFWR2 in Table 5 or Table 6 (or a sequence having one, two, three, four, five, six, seven, eight, or nine or fewer mutations, such as substitutions, additions, or deletions), a VLFWR3 having the amino acid sequence of the VLFWR3 in Table 5 or Table 6 (or a sequence having one, two, three, four, five, six, seven, eight, or nine or fewer mutations, such as substitutions, additions, or deletions), and / or a VLFWR4 having the amino acid sequence of the VLFWR4 in Table 5 or Table 6 (or a sequence having one, two, three, four, five, six, seven, eight, or nine or fewer mutations, such as substitutions, additions, or deletions) is included.

[0037] In some embodiments, the multifunctional molecule further comprises a tumor targeting moiety. In some embodiments, the tumor targeting moiety binds to a tumor antigen. In some embodiments, the tumor antigen is selected from G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1.

[0038] In some embodiments, the tumor targeting moiety comprises an antibody molecule that binds to a tumor antigen selected from, for example, G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1.

[0039] In some embodiments, the tumor targeting moiety comprises, for example, VH and / or VL sequences listed in Table A or Table 20. In some embodiments, the multifunctional molecule binds preferentially to myeloproliferative neoplasm cells over non-tumor cells, and optionally, the binding of the multifunctional molecule to myeloproliferative neoplasm cells is at least 10, 20, 30, 40, or 50 times greater than the binding of the multifunctional molecule to non-tumor cells.

[0040] In some embodiments, the myeloproliferative neoplasm cells are selected from myelofibrosis cells, essential thrombocythemia cells, polycythemia vera cells, or chronic myeloid leukemia cells, and optionally, the myeloproliferative neoplasm cells do not contain the JAK2 V617F mutation, or the myeloproliferative neoplasm cells do not contain the MPL mutation.

[0041] In some embodiments, the multispecific molecule further comprises a linker, for example, a linker between the first antigen-binding domain and the second antigen-binding domain. In some embodiments, the linker is selected from a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helix linker, or a non-helix linker.

[0042] In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker contains Gly and Ser. In some embodiments, the peptide linker contains an amino acid sequence selected from SEQ ID NOs: 6214 - 6217, or 6220 - 6221 and 77 - 78.

[0043] In another aspect, the disclosure provides a nucleic acid molecule encoding a multifunctional molecule described herein. In another aspect, the disclosure provides a vector, e.g., an expression vector, comprising a nucleic acid molecule described herein.

[0044] In another aspect, the disclosure provides a host cell comprising a nucleic acid molecule or vector described herein. In another aspect, the disclosure provides a method of making, e.g., producing, a multifunctional molecule described herein, the method comprising culturing a host cell described herein under suitable conditions, e.g., conditions suitable for gene expression and / or homo- or hetero-dimerization.

[0045] In another aspect, the disclosure provides a pharmaceutical composition comprising a multifunctional molecule described herein and a pharmaceutically acceptable carrier, excipient, or stabilizer. In another aspect, the disclosure provides a method of treating cancer, the method comprising administering to a subject in need thereof a multifunctional molecule disclosed herein, wherein the multifunctional molecule is administered in an amount effective to treat cancer.

[0046] In another aspect, the present disclosure provides for the use of the multifunctional molecules described herein in treating cancer. In another aspect, the present disclosure provides multifunctional molecules disclosed herein for use in treating cancer.

[0047] In some embodiments, the subject has cancer cells that express the first and / or second calreticulin protein. In some embodiments, the subject has a JAK2 V617F mutation.

[0048] In some embodiments, the subject does not have a JAK2 V617F mutation. In some embodiments, the subject has an MPL mutation. In some embodiments, the subject does not have an MPL mutation.

[0049] In some embodiments, the cancer is a blood cancer, and optionally, the cancer is a myeloproliferative neoplasm, such as primary or idiopathic myelofibrosis (MF), essential thrombocythemia (ET), polycythemia vera (PV), or chronic myelogenous leukemia (CML), and optionally, the cancer is myelofibrosis.

[0050] In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the method or use further comprises the step of administering a second therapeutic treatment. In some embodiments, the second therapeutic treatment includes a therapeutic agent (e.g., a chemotherapeutic agent, a biological agent, hormone therapy), radiation, or surgery.

[0051] In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent or a biological agent. In another aspect, the present disclosure provides (i) a first antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type or mutant calreticulin protein), and (ii)(a) An immune cell engager selected from a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager; (b) A cytokine molecule; (c) A stromal modifying moiety; or (d) A tumor targeting moiety that binds to a tumor antigen selected from, for example, G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1 One, two, or all of characterized by a multifunctional molecule (e.g., a polypeptide or nucleic acid encoding the same).

[0052] In one aspect, the disclosure is directed to (i) A first antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type or mutant calreticulin protein), and (ii) A second antigen-binding domain that includes an immune cell engager (e.g., a T cell engager, e.g., an antigen-binding domain that binds to TCRβV as described herein) and characterized by a multifunctional molecule (e.g., a polypeptide or nucleic acid encoding the same).

[0053] In one aspect, the disclosure is directed to (i) A first antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type or mutant calreticulin protein), and (ii) A second antigen-binding domain that includes a tumor targeting moiety that binds to a tumor antigen selected from, for example, G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1 and Characterized by a multifunctional molecule (e.g., a polypeptide or nucleic acid encoding the same).

[0054] In certain embodiments, the present disclosure (i) a first antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type or mutant calreticulin protein), and (ii) a second antigen-binding domain comprising an immune cell engager (e.g., a T cell engager, e.g., an antigen-binding domain that binds to TCRβV as described herein, e.g., an anti-TCRβV antibody molecule as described herein), and (iii) a third antigen-binding domain comprising a tumor targeting moiety that binds to a tumor antigen selected from, for example, G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1 Characterized by a multifunctional molecule (e.g., a polypeptide or nucleic acid encoding the same).

[0055] In some embodiments, the multifunctional molecule further comprises a cytokine molecule or a modulator of a cytokine molecule, e.g., a TGF-β inhibitor as described herein. In some embodiments, the multifunctional molecule further comprises an NK cell engager, e.g., an antigen-binding domain that binds to Nkp30 as described herein.

[0056] In some embodiments, the calreticulin protein (e.g., a wild-type or mutant calreticulin protein) comprises the amino acid sequence of SEQ ID NO: 6285 or 6286. In some embodiments, the wild-type calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285. In some embodiments, the calreticulin mutant protein comprises the amino acid sequence of SEQ ID NO: 6286.

[0057] In some embodiments, the first antigen-binding domain comprises a heavy-chain variable region (VH) comprising the heavy-chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6224, the VHFWR2 amino acid sequence of SEQ ID NO: 6226, the VHFWR3 amino acid sequence of SEQ ID NO: 6228, or the VHFWR4 amino acid sequence of SEQ ID NO: 6230. In some embodiments, the first antigen-binding domain comprises a heavy-chain variable region (VH) comprising the heavy-chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6232, the VHFWR2 amino acid sequence of SEQ ID NO: 6234, the VHFWR3 amino acid sequence of SEQ ID NO: 6236, or the VHFWR4 amino acid sequence of SEQ ID NO: 6230. In some embodiments, the first antigen-binding domain comprises a light-chain variable region (VL) comprising the light-chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6238, the VLFWR2 amino acid sequence of SEQ ID NO: 6240, the VLFWR3 amino acid sequence of SEQ ID NO: 6242, or the VLFWR4 amino acid sequence of SEQ ID NO: 6244.

[0058] In some embodiments, the calreticulin protein (e.g., wild-type or mutant calreticulin protein) comprises an amino acid sequence selected from SEQ ID NOs: 6285 to 6312. In some embodiments, the calreticulin protein (e.g., wild-type or mutant calreticulin protein) comprises an amino acid sequence selected from SEQ ID NOs: 6313 to 6346. In some embodiments, the calreticulin protein (e.g., wild-type or mutant calreticulin protein) is the calreticulin protein (e.g., wild-type or mutant calreticulin protein) disclosed in Table 2 or 3. In some embodiments, the calreticulin protein (e.g., wild-type or mutant calreticulin protein) comprises the amino acid sequence of SEQ ID NO: 6287. In some embodiments, the calreticulin protein (e.g., wild-type or mutant calreticulin protein) comprises the amino acid sequence of SEQ ID NO: 6313. In some embodiments, the calreticulin protein (e.g., wild-type or mutant calreticulin protein) comprises the amino acid sequence of SEQ ID NO: 6288. In some embodiments, the calreticulin protein (e.g., wild-type or mutant calreticulin protein) comprises the amino acid sequence of SEQ ID NO: 6314.

[0059] In some embodiments, the multifunctional molecule further comprises a second antigen-binding domain that preferentially binds to a second calreticulin protein (e.g., a wild-type or mutant calreticulin protein). In some embodiments, the second calreticulin protein (e.g., a wild-type or mutant calreticulin protein) comprises the amino acid sequence of SEQ ID NO: 6286. In some embodiments, the second antigen-binding domain is different from the first antigen-binding domain. In some embodiments, the second antigen-binding domain is the same as the first antigen-binding domain. In some embodiments, the second calreticulin protein (e.g., a wild-type or mutant calreticulin protein) comprises an amino acid sequence selected from SEQ ID NOs: 6287-6312. In some embodiments, the second calreticulin protein (e.g., a wild-type or mutant calreticulin protein) comprises an amino acid sequence selected from SEQ ID NOs: 6313-6346. In some embodiments, the second calreticulin protein (e.g., a wild-type or mutant calreticulin protein) is the calreticulin protein (e.g., a wild-type or mutant calreticulin protein) disclosed in Table 2 or 3. In some embodiments, the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6287. In some embodiments, the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6313. In some embodiments, the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6288. In some embodiments, the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6314.

[0060] In some embodiments, the first calreticulin protein (e.g., wild-type or mutant calreticulin protein) is a type 1 calreticulin protein (e.g., wild-type or mutant calreticulin protein), and the second calreticulin protein (e.g., wild-type or mutant calreticulin protein) is a type 2 calreticulin protein (e.g., wild-type or mutant calreticulin protein). In some embodiments, the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6287, and the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6288. In some embodiments, the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6313, and the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6314.

[0061] In some embodiments, the wild-type calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285. In some embodiments, the first antigen-binding domain has substantially the same affinity (e.g., equal affinity) for the first calreticulin protein (e.g., mutant calreticulin protein) and the wild-type calreticulin protein.

[0062] In some embodiments, the second antigen-binding domain has substantially the same affinity (e.g., equal affinity) for the second calreticulin protein (e.g., mutant calreticulin protein) and the wild-type calreticulin protein.

[0063] In some embodiments, the first antigen-binding domain has a higher affinity for the first calreticulin mutant protein than for the wild-type calreticulin protein. In some embodiments, the K D for the binding of the first antigen-binding domain to the first calreticulin mutant protein is the K Dis 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01% or less. In some embodiments, the first antigen-binding domain binds to an epitope located within the C-terminus of the first calreticulin mutant protein. In some embodiments, the first antigen-binding domain binds to an epitope located within the amino acid sequence of SEQ ID NO: 6286. In some embodiments, the first antigen-binding domain does not bind to the wild-type calreticulin protein. In some embodiments, the wild-type calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285.

[0064] In some embodiments, the second antigen-binding domain has a higher affinity for the second calreticulin mutant protein than for the wild-type calreticulin protein. In some embodiments, the K D for the binding of the second antigen-binding domain to the second calreticulin mutant protein is D 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01% or less of the K

[0065] In some embodiments, the multifunctional molecule preferentially binds to myeloproliferative neoplasm cells over non-tumor cells. In some embodiments, the binding of the multifunctional molecule to myeloproliferative neoplasm cells is greater than 10, 20, 30, 40, or 50-fold greater than the binding of the multifunctional molecule to non-tumor cells. In some embodiments, the myeloproliferative neoplasm cells are selected from myelofibrosis cells, essential thrombocythemia cells, polycythemia vera cells, or chronic myelogenous leukemia cells. In some embodiments, the myeloproliferative neoplasm cells do not contain the JAK2 V617F mutation. In some embodiments, the myeloproliferative neoplasm cells do not contain the MPL mutation.

[0066] In some embodiments, the first and / or second antigen-binding domain comprises a variable heavy chain (VH) region comprising the amino acid sequence of heavy chain complementarity determining region 1 (VHCDR1) of SEQ ID NO: 6253 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VHCDR2 of SEQ ID NO: 6254 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VHCDR3 of SEQ ID NO: 6255 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions). In some embodiments, the first and / or second antigen-binding domain comprises a variable light chain (VL) region comprising the amino acid sequence of light chain complementarity determining region 1 (VLCDR1) of SEQ ID NO: 6259 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VLCDR2 of SEQ ID NO: 6260 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VLCDR3 of SEQ ID NO: 6261 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions).

[0067] In some embodiments, the first and / or second antigen-binding domain is (i) A heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain complementarity determining region 1 (VHCDR1) of SEQ ID NO: 6253 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VHCDR2 of SEQ ID NO: 6254 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VHCDR3 of SEQ ID NO: 6255 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and (ii) A light chain variable region (VL) comprising the amino acid sequence of the light chain complementarity determining region 1 (VLCDR1) of SEQ ID NO: 6259 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VLCDR2 of SEQ ID NO: 6260 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VLCDR3 of SEQ ID NO: 6261 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions) comprising.

[0068] In some embodiments, the first and / or second antigen-binding domain comprises a VH comprising the amino acid sequence of VHCDR1 of SEQ ID NO: 6253, the amino acid sequence of VHCDR2 of SEQ ID NO: 6254, and the amino acid sequence of VHCDR3 of SEQ ID NO: 6255. In some embodiments, the first and / or second antigen-binding domain comprises a VL comprising the amino acid sequence of VLCDR1 of SEQ ID NO: 6259, the amino acid sequence of VLCDR2 of SEQ ID NO: 6260, and the amino acid sequence of VLCDR3 of SEQ ID NO: 6261.

[0069] In some embodiments, the first and / or second antigen-binding domain is (i) a VH comprising the amino acid sequence of VHCDR1 of SEQ ID NO: 6253, the amino acid sequence of VHCDR2 of SEQ ID NO: 6254, and the amino acid sequence of VHCDR3 of SEQ ID NO: 6255, and (ii) VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 6259, the VLCDR2 amino acid sequence of SEQ ID NO: 6260, and the VLCDR3 amino acid sequence of SEQ ID NO: 6261 comprises.

[0070] In some embodiments, the first and / or second antigen-binding domain comprises a VH comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6224, the VHFWR2 amino acid sequence of SEQ ID NO: 6226, the VHFWR3 amino acid sequence of SEQ ID NO: 6228, and / or the VHFWR4 amino acid sequence of SEQ ID NO: 6230. In some embodiments, the first and / or second antigen-binding domain comprises a VL comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6238, the VLFWR2 amino acid sequence of SEQ ID NO: 6240, the VLFWR3 amino acid sequence of SEQ ID NO: 6242, and / or the VLFWR4 amino acid sequence of SEQ ID NO: 6244.

[0071] In some embodiments, the first and / or second antigen-binding domain is (i) a VH comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6224, the VHFWR2 amino acid sequence of SEQ ID NO: 6226, the VHFWR3 amino acid sequence of SEQ ID NO: 6228, and / or the VHFWR4 amino acid sequence of SEQ ID NO: 6230, and (ii) a VL comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6238, the VLFWR2 amino acid sequence of SEQ ID NO: 6240, the VLFWR3 amino acid sequence of SEQ ID NO: 6242, and / or the VLFWR4 amino acid sequence of SEQ ID NO: 6244 comprises.

[0072] In some embodiments, the first and / or second antigen-binding domain comprises a VH comprising the VHFWR1 amino acid sequence of SEQ ID NO: 6263 (or a sequence having one, two, three, four, five, or six or fewer mutations, e.g., substitutions, additions, or deletions), the VHFWR2 amino acid sequence of SEQ ID NO: 6264 (or a sequence having one, two, three, four, five, or six or fewer mutations, e.g., substitutions, additions, or deletions), the VHFWR3 amino acid sequence of SEQ ID NO: 6265 (or a sequence having one, two, three, four, five, six, seven, eight, nine, ten, or eleven or fewer mutations, e.g., substitutions, additions, or deletions), and / or the VHFWR4 amino acid sequence of SEQ ID NO: 228. In some embodiments, the first and / or second antigen-binding domain comprises a VL comprising the VLFWR1 amino acid sequence of SEQ ID NO: 6277 (or a sequence having one, two, or three or fewer mutations, e.g., substitutions, additions, or deletions), the VLFWR2 amino acid sequence of SEQ ID NO: 6278 (or a sequence having one or fewer mutations, e.g., substitutions, additions, or deletions), the VLFWR3 amino acid sequence of SEQ ID NO: 6279 (or a sequence having one or fewer mutations, e.g., substitutions, additions, or deletions), and / or the VLFWR4 amino acid sequence of SEQ ID NO: 6280.

[0073] In some embodiments, the first and / or second antigen-binding domain is (i) a VH comprising the VHFWR1 amino acid sequence of SEQ ID NO: 6263 (or a sequence having one, two, three, four, five, or six or fewer mutations, e.g., substitutions, additions, or deletions), the VHFWR2 amino acid sequence of SEQ ID NO: 6264 (or a sequence having one, two, three, four, five, or six or fewer mutations, e.g., substitutions, additions, or deletions), the VHFWR3 amino acid sequence of SEQ ID NO: 6265 (or a sequence having one, two, three, four, five, six, seven, eight, nine, ten, or eleven or fewer mutations, e.g., substitutions, additions, or deletions), and / or the VHFWR4 amino acid sequence of SEQ ID NO: 228, and (ii) The VLFWR1 amino acid sequence of SEQ ID NO: 6277 (or a sequence having one, two, or three or fewer mutations, such as substitutions, additions, or deletions), the VLFWR2 amino acid sequence of SEQ ID NO: 6278 (or a sequence having one or fewer mutations, such as substitutions, additions, or deletions), the VLFWR3 amino acid sequence of SEQ ID NO: 6279 (or a sequence having one or fewer mutations, such as substitutions, additions, or deletions), and / or the VLFWR4 amino acid sequence of SEQ ID NO: 6280 comprising.

[0074] In some embodiments, the first and / or second antigen-binding domain comprises a VH comprising the VHFWR1 amino acid sequence of SEQ ID NO: 6263, the VHFWR2 amino acid sequence of SEQ ID NO: 6264, the VHFWR3 amino acid sequence of SEQ ID NO: 6265, and / or the VHFWR4 amino acid sequence of SEQ ID NO: 228. In some embodiments, the first and / or second antigen-binding domain comprises a VL comprising the VLFWR1 amino acid sequence of SEQ ID NO: 6277, the VLFWR2 amino acid sequence of SEQ ID NO: 6278, the VLFWR3 amino acid sequence of SEQ ID NO: 6279, and / or the VLFWR4 amino acid sequence of SEQ ID NO: 6280.

[0075] In some embodiments, the first and / or second antigen-binding domain (i) a VH comprising the VHFWR1 amino acid sequence of SEQ ID NO: 6263, the VHFWR2 amino acid sequence of SEQ ID NO: 6264, the VHFWR3 amino acid sequence of SEQ ID NO: 6265, and / or the VHFWR4 amino acid sequence of SEQ ID NO: 228, and (ii) a VL comprising the VLFWR1 amino acid sequence of SEQ ID NO: 6277, the VLFWR2 amino acid sequence of SEQ ID NO: 6278, the VLFWR3 amino acid sequence of SEQ ID NO: 6279, and / or the VLFWR4 amino acid sequence of SEQ ID NO: 6280 comprising.

[0076] In some embodiments, the first and / or second antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6247 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6247). In some embodiments, the first and / or second antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6249 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6249).

[0077] In some embodiments, the first and / or second antigen-binding domain is (i) a VH comprising the amino acid sequence of SEQ ID NO: 6247 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6247), and (ii) a VL comprising the amino acid sequence of SEQ ID NO: 6249 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6249). comprises.

[0078] In some embodiments, the first and / or second antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6247. In some embodiments, the first and / or second antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6249. In some embodiments, the first and / or second antigen-binding domain comprises (i) a VH comprising the amino acid sequence of SEQ ID NO: 6247, and (ii) a VL comprising the amino acid sequence of SEQ ID NO: 6249.

[0079] In some embodiments, the first and / or second antigen-binding domain comprises a VH comprising an amino acid sequence having at least 70% or 75% sequence identity to SEQ ID NO: 6250. In some embodiments, the first and / or second antigen-binding domain comprises a VL comprising an amino acid sequence having at least 85% or 90% sequence identity to SEQ ID NO: 6252. In some embodiments, the first and / or second antigen-binding domain comprises (i) a VH comprising an amino acid sequence having at least 70% or 75% sequence identity to SEQ ID NO: 6250, and (ii) a VL comprising an amino acid sequence having at least 85% or 90% sequence identity to SEQ ID NO: 6252.

[0080] In some embodiments, the first and / or second antigen-binding domain comprises a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6256 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 6257 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 6258 or 116 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions). In some embodiments, the first and / or second antigen-binding domain comprises a light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6259 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the VLCDR2 amino acid sequence of SEQ ID NO: 6260 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the VLCDR3 amino acid sequence of SEQ ID NO: 6261 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions).

[0081] In some embodiments, the first and / or second antigen-binding domain is (i) A heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain complementarity determining region 1 (VHCDR1) of SEQ ID NO: 6256 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VHCDR2 of SEQ ID NO: 6257 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VHCDR3 of SEQ ID NO: 6258 or 116 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and (ii) A light chain variable region (VL) comprising the amino acid sequence of the light chain complementarity determining region 1 (VLCDR1) of SEQ ID NO: 6259 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VLCDR2 of SEQ ID NO: 6260 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VLCDR3 of SEQ ID NO: 6261 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions) comprising.

[0082] In some embodiments, the first and / or second antigen-binding domain comprises a VH comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6232, the amino acid sequence of VHFWR2 of SEQ ID NO: 6234, the amino acid sequence of VHFWR3 of SEQ ID NO: 6236, and / or the amino acid sequence of VHFWR4 of SEQ ID NO: 6230. In some embodiments, the first and / or second antigen-binding domain comprises a VL comprising the amino acid sequence of the light chain framework region 1 (VLFWR1) of SEQ ID NO: 6238, the amino acid sequence of VLFWR2 of SEQ ID NO: 6240, the amino acid sequence of VLFWR3 of SEQ ID NO: 6242, and / or the amino acid sequence of VLFWR4 of SEQ ID NO: 6244.

[0083] In some embodiments, the first and / or second antigen-binding domain is (i) a VH comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6232, the VHFWR2 amino acid sequence of SEQ ID NO: 6234, the VHFWR3 amino acid sequence of SEQ ID NO: 6236, and / or the VHFWR4 amino acid sequence of SEQ ID NO: 6230, and (ii) a VL comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6238, the VLFWR2 amino acid sequence of SEQ ID NO: 6240, the VLFWR3 amino acid sequence of SEQ ID NO: 6242, and / or the VLFWR4 amino acid sequence of SEQ ID NO: 6244 (iii) comprising.

[0084] In some embodiments, the first and / or second antigen-binding domains comprise a VH comprising the heavy chain framework 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6266 (or a sequence having one, two, three, four, five, six, seven, eight, or nine or fewer mutations, such as substitutions, additions, or deletions), the VHFWR2 amino acid sequence of SEQ ID NO: 6267 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the VHFWR3 amino acid sequence of SEQ ID NO: 6268 (or a sequence having one, two, three, four, five, six, seven, eight, nine, ten, or eleven or fewer mutations, such as substitutions, additions, or deletions), and / or the VHFWR4 amino acid sequence of SEQ ID NO: 6269. In some embodiments, the first and / or second antigen-binding domains comprise a VL comprising the VLFWR1 amino acid sequence of SEQ ID NO: 6277 (or a sequence having one, two, or three or fewer mutations, such as substitutions, additions, or deletions), the VLFWR2 amino acid sequence of SEQ ID NO: 6278 (or a sequence having one or fewer mutations, such as substitutions, additions, or deletions), the VLFWR3 amino acid sequence of SEQ ID NO: 6279 (or a sequence having one or fewer mutations, such as substitutions, additions, or deletions), and / or the VLFWR4 amino acid sequence of SEQ ID NO: 6280.

[0085] In some embodiments, the first and / or second antigen-binding domains are (i) The VH comprising the heavy chain framework 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6266 (or a sequence having one, two, three, four, five, six, seven, eight, or nine or fewer mutations, such as substitutions, additions, or deletions), the VHFWR2 amino acid sequence of SEQ ID NO: 6267 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the VHFWR3 amino acid sequence of SEQ ID NO: 6268 (or a sequence having one, two, three, four, five, six, seven, eight, nine, ten, or eleven or fewer mutations, such as substitutions, additions, or deletions), and / or the VHFWR4 amino acid sequence of SEQ ID NO: 6269, and (ii) The VL comprising the VLFWR1 amino acid sequence of SEQ ID NO: 6277 (or a sequence having one, two, or three or fewer mutations, such as substitutions, additions, or deletions), the VLFWR2 amino acid sequence of SEQ ID NO: 6278 (or a sequence having one or fewer mutations, such as substitutions, additions, or deletions), the VLFWR3 amino acid sequence of SEQ ID NO: 6279 (or a sequence having one or fewer mutations, such as substitutions, additions, or deletions), and / or the VLFWR4 amino acid sequence of SEQ ID NO: 6280 comprising.

[0086] In some embodiments, the first and / or second antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6248 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6248). In some embodiments, the first and / or second antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6249 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6249).

[0087] In some embodiments, the first and / or second antigen-binding domain is (i) A VH comprising the amino acid sequence of SEQ ID NO: 6248 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6248), and (ii) A VL comprising the amino acid sequence of SEQ ID NO: 6249 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6249). It comprises.

[0088] In some embodiments, the first and / or second antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6248. In some embodiments, the first and / or second antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6249. In some embodiments, the first and / or second antigen-binding domain comprises (i) a VH comprising the amino acid sequence of SEQ ID NO: 6248, and (ii) a VL comprising the amino acid sequence of SEQ ID NO: 6249.

[0089] In some embodiments, the first and / or second antigen-binding domain comprises a VH comprising an amino acid sequence having at least 70% or 74% sequence identity to SEQ ID NO: 6251. In some embodiments, the first and / or second antigen-binding domain comprises a VL comprising an amino acid sequence having at least 85% or 90% sequence identity to SEQ ID NO: 6252. In some embodiments, the first and / or second antigen-binding domain comprises (i) a VH comprising an amino acid sequence having at least 70% or 74% sequence identity to SEQ ID NO: 6251, and / or (ii) a VL comprising an amino acid sequence having at least 85% or 90% sequence identity to SEQ ID NO: 6252.

[0090] In some embodiments, the multifunctional molecule comprises an immune cell engager selected from a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager. In some embodiments, the immune cell engager binds to and activates an immune cell, such as an effector cell. In some embodiments, the immune cell engager binds to, but does not activate, an immune cell, such as an effector cell.

[0091] In some embodiments, the immune cell engager is a T cell engager, such as a T cell engager that mediates binding to and activation of T cells, or a T cell engager that mediates binding to T cells but does not mediate their activation. In some embodiments, the T cell engager binds to CD3, TCRα, TCRβ, TCRγ, TCRζ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. In some embodiments, the T cell engager is an anti-CD3 antibody molecule. In some embodiments, the T cell engager is an anti-TCRβ antibody molecule, such as an anti-TCRβV antibody molecule described herein.

[0092] In some embodiments, the immune cell engager is an NK cell engager, e.g., an NK cell engager that mediates binding to and activation of NK cells, or an NK cell engager that mediates binding to NK cells but does not mediate their activation. In some embodiments, the NK cell engager is selected from an antibody molecule (e.g., an antigen-binding domain) or a ligand that binds to (e.g., activates) NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b, or both), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (also known as SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, or CD160. In some embodiments, the NK cell engager is an antibody molecule or ligand that binds to (e.g., activates) NKp30. In some embodiments, the NK cell engager is an antibody molecule, e.g., an antigen-binding domain. In some embodiments, the NK cell engager is an antibody molecule, e.g., an antigen-binding domain, that binds to NKp30 or NKp46. In some embodiments, the NK cell engager is a ligand, and optionally, the ligand further comprises an immunoglobulin constant region, e.g., an Fc region. In some embodiments, the NK cell engager is a ligand for NKp44 or NKp46, e.g., viral HA. In some embodiments, the NK cell engager is a ligand for DAP10, e.g., a co-receptor for NKG2D. In some embodiments, the NK cell engager is a ligand for CD16, e.g., a CD16a / b ligand, e.g., a CD16a / b ligand that further comprises an antibody Fc region. In some embodiments, the immune cell engager mediates binding to, activation of, or both, one or more of B cells, macrophages, and / or dendritic cells.

[0093] In some embodiments, the immune cell engager comprises a B cell, macrophage, and / or dendritic cell engager selected from CD40 ligand (CD40L) or CD70 ligand; an antibody molecule that binds to CD40 or CD70; an antibody molecule against OX40; OX40 ligand (OX40L); an agonist of a Toll-like receptor (e.g., TLR4, e.g., constitutively active TLR4 (caTLR4), or a TLR9 agonist); 41BB; a CD2 agonist; CD47; or a STING agonist, or one or more combinations thereof. In some embodiments, the immune cell engager is a B cell engager, such as CD40L, OX40L, or CD70 ligand, or an antibody molecule that binds to OX40, CD40, or CD70. In some embodiments, the immune cell engager is a macrophage cell engager, such as a CD2 agonist; CD40L; OX40L; an antibody molecule that binds to OX40, CD40, or CD70; an agonist of a Toll-like receptor (TLR) (e.g., TLR4, e.g., constitutively active TLR4 (caTLR4), or a TLR9 agonist); CD47; or a STING agonist. In some embodiments, the immune cell engager is a dendritic cell engager, such as a CD2 agonist, an OX40 antibody, OX40L, a 41BB agonist, a Toll-like receptor agonist or a fragment thereof (e.g., TLR4, e.g., constitutively active TLR4 (caTLR4)), a CD47 agonist, or a STING agonist. In some embodiments, the STING agonist comprises a cyclic dinucleotide, such as cyclic di-GMP (cdGMP), cyclic di-AMP (cdAMP), or a combination thereof, optionally having a 2′,5′ or 3′,5′ phosphate linkage, e.g., the STING agonist is covalently coupled to a multifunctional molecule.

[0094] In some embodiments, the multifunctional molecule comprises a cytokine molecule or a modulator thereof. In some embodiments, the cytokine molecule is selected from TGF-β, interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), or interferon gamma, or a fragment or variant thereof, or any combination of the above cytokines. In some embodiments, the cytokine molecule is a monomer or a dimer. In some embodiments, the cytokine molecule further comprises a receptor dimerization domain, for example, the IL15R alpha dimerization domain. In some embodiments, the cytokine molecule (e.g., IL-15) and the receptor dimerization domain (e.g., the IL15R alpha dimerization domain) are not covalently linked, for example, they are non-covalently associated.

[0095] In some embodiments, the modulator of the cytokine molecule comprises a TGF-β inhibitor. In some embodiments, the multifunctional molecule comprises a stromal modifying moiety. In some embodiments, the stromal modifying moiety causes one or more of: a decrease in the level or production of stromal or extracellular matrix (ECM) components; a decrease in tumor fibrosis; an increase in stromal tumor transport; an improvement in tumor perfusion; an expansion of tumor microvessels; a decrease in interstitial fluid pressure (IFP) in the tumor; or a decrease or enhancement in the penetration or diffusion of a drug, such as a cancer therapeutic or cell therapy, into the tumor or tumor vasculature. In some embodiments, the stromal or ECM component that is decreased is selected from glycosaminoglycans or extracellular proteins, or combinations thereof. In some embodiments, the glycosaminoglycan is selected from hyaluronan (also known as hyaluronic acid or HA), chondroitin sulfate, chondroitin, dermatan sulfate, heparan sulfate, heparin, entactin, tenascin, aggrecan, or keratan sulfate. In some embodiments, the extracellular protein is selected from collagen, laminin, elastin, fibrinogen, fibronectin, or vitronectin. In some embodiments, the stromal modifying moiety comprises an enzyme molecule that degrades the tumor stroma or extracellular matrix (ECM). In some embodiments, the enzyme molecule is selected from hyaluronidase molecules, collagenase molecules, chondroitinase molecules, matrix metalloproteinase molecules (e.g., macrophage metalloelastase), or variants (e.g., fragments) of any of the foregoing. In some embodiments, the stromal modifying moiety decreases the level or production of hyaluronic acid. In some embodiments, the stromal modifying moiety comprises a hyaluronan-degrading enzyme, an agent that inhibits hyaluronan synthesis, or an antibody molecule against hyaluronic acid. In some embodiments, the hyaluronan-degrading enzyme is a hyaluronidase molecule or a variant thereof (e.g., a fragment thereof). In some embodiments, the hyaluronan-degrading enzyme is active at a neutral or acidic pH, such as a pH of about 4-5. In some embodiments, the hyaluronidase molecule is a mammalian hyaluronidase molecule, such as a recombinant human hyaluronidase molecule or a variant thereof (e.g., a shortened form thereof).In some embodiments, the hyaluronidase molecule is selected from HYAL1, HYAL2, or PH-20 / SPAM1, or a variant thereof (e.g., a truncated form thereof). In some embodiments, the truncated form lacks the C-terminal glycosylphosphatidylinositol (GPI) binding site or a portion of the GPI binding site. In some embodiments, the hyaluronidase molecule is glycosylated, e.g., contains at least one N-linked glycan. In some embodiments, the hyaluronidase molecule has an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO: 6213, or a fragment thereof, or has (e.g., is 95% - 99.9% identical thereto, or has at least one amino acid change to the amino acid sequence of SEQ ID NO: 6213, but 5, 10, or 15 or fewer changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions)). In some embodiments, the hyaluronidase molecule contains amino acid residues 36 - 464 of SEQ ID NO: 6213. In some embodiments, the hyaluronidase molecule contains amino acid residues 36 - 481, 36 - 482, or 36 - 483 of PH20, where PH20 has the amino acid sequence of SEQ ID NO: 6213. In some embodiments, the hyaluronidase molecule contains an amino acid sequence having at least 95% - 100% sequence identity to the polypeptide of the amino acid sequence of SEQ ID NO: 6213 or a truncated form thereof. In some embodiments, the hyaluronidase molecule contains an amino acid sequence having 30, 20, 10, 5, or fewer amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 6213. In some embodiments, the hyaluronidase molecule contains an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, 100%) identical to the amino acid sequence of SEQ ID NO: 6213. In some embodiments, the hyaluronidase molecule is encoded by a nucleotide sequence that is at least 95% (e.g., at least 96%, 97%, 98%, 99%, 100%) identical to the nucleotide sequence of SEQ ID NO: 6213. In some embodiments, the hyaluronidase molecule is PH20, e.g., rHuPH20.In some embodiments, the hyaluronidase molecule is HYAL1 and comprises the amino acid sequence of SEQ ID NO: 6218, or a fragment thereof, or an amino acid sequence that is substantially identical thereto (e.g., 95% - 99.9% identical thereto or having at least one amino acid change to the amino acid sequence of SEQ ID NO: 6218 but 5, 10, or 15 or fewer changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions)). In some embodiments, the hyaluronan-degrading enzyme, e.g., the hyaluronidase molecule, further comprises a polymer, e.g., is conjugated to a polymer, e.g., PEG. In some embodiments, the hyaluronan-degrading enzyme is a PEGylated PH20 enzyme (PEGPH20). In some embodiments, the hyaluronan-degrading enzyme, e.g., the hyaluronidase molecule, further comprises an immunoglobulin heavy chain constant region selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, or IgG4, more specifically, the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4 (e.g., the Fc region). In some embodiments, the immunoglobulin constant region (e.g., the Fc region) is linked, e.g., covalently linked, to the hyaluronan-degrading enzyme, e.g., the hyaluronidase molecule. In some embodiments, the immunoglobulin heavy chain constant region (e.g., the Fc region) is modified, e.g., mutated, to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function. In some embodiments, the hyaluronan-degrading enzyme, e.g., the hyaluronidase molecule, forms a dimer. In some embodiments, the stromal modification moiety comprises an inhibitor of hyaluronan synthesis, e.g., an inhibitor of HA synthase. In some embodiments, the inhibitor comprises a sense or antisense nucleic acid molecule to HA synthase or is a small molecule drug. In some embodiments, the inhibitor is 4-methylumbelliferone (MU) or a derivative thereof (e.g., 6,7-dihydroxy-4-methylcoumarin or 5,7-dihydroxy-4-methylcoumarin), or leflunomide or a derivative thereof.In some embodiments, the stromal modification moiety comprises a collagenase molecule, such as a mammalian collagenase molecule, or a variant thereof (e.g., a fragment). In some embodiments, the collagenase molecule is substantially identical to, for example, the amino acid sequence of SEQ ID NO: 6219, or a fragment thereof, or has at least one amino acid change relative thereto, but 5, 10, or 15 or fewer changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 6219, and is a collagenase IV molecule comprising an amino acid sequence.

[0096] In some embodiments, the multifunctional molecule comprises an immune cell engager (e.g., a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager) and a cytokine molecule. In some embodiments, the multifunctional molecule comprises an immune cell engager (e.g., a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager) and a stromal modification moiety. In some embodiments, the multifunctional molecule comprises a cytokine molecule and a stromal modification moiety. In some embodiments, the multifunctional molecule comprises an immune cell engager (e.g., a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager), a cytokine molecule, and a stromal modification moiety.

[0097] In some embodiments, the multifunctional molecule comprises at least two non - contiguous polypeptide chains. In some embodiments, the multifunctional molecule has the following configuration: A,B - [dimerization module] - C, - D For example, it comprises the configurations shown in FIGS. 1A, 1B, and 1C, where (1) The dimerization module includes a constant domain of an immunoglobulin, such as a heavy chain constant domain (e.g., a homodimeric or heterodimeric heavy chain constant region, such as an Fc region), or a constant domain of an immunoglobulin variable region (e.g., a Fab region); (2) A, B, C, and D are each independently absent or (i) an antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type calreticulin protein or a mutant calreticulin protein), wherein the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286; (ii) an immune cell engager selected from a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager; (iii) a cytokine molecule; or (iv) a stromal modifying moiety, provided that at least one, two, or three of A, B, C, and D is an antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and any of the remaining A, B, C, and D is absent or comprises one of an immune cell engager, a cytokine molecule, or a stromal modifying moiety is a condition.

[0098] In some embodiments, (i) A comprises an antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and B, C, or D comprises an immune cell engager, such as a T cell engager, such as an anti-CD3 antibody molecule; (ii) A is an antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the calreticulin protein contains the amino acid sequence of SEQ ID NO: 6286, and does B, C, or D contain a cytokine molecule? (iii) A is an antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the calreticulin protein contains the amino acid sequence of SEQ ID NO: 6286, and does B, C, or D contain a stromal modification moiety? (iv) A is a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein contains the amino acid sequence of SEQ ID NO: 6286, and B is a second antigen-binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein contains the amino acid sequence of SEQ ID NO: 6286, and does C or D contain an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule? (v) A is a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein contains the amino acid sequence of SEQ ID NO: 6286, and B is a second antigen-binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein contains the amino acid sequence of SEQ ID NO: 6286, and does C or D contain a cytokine molecule? (vi) A is a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, the first antigen-binding domain is included, B is a second antigen-binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, the second antigen-binding domain is included, and does C or D contain a stromal modification moiety? (vii) A is a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, the first antigen-binding domain is included, C is a second antigen-binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, the second antigen-binding domain is included, and does B or D contain an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule? (viii) A is a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, the first antigen-binding domain is included, C is a second antigen-binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, the second antigen-binding domain is included, and does B or D contain a cytokine molecule? (ix) A is a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and includes the first antigen-binding domain; C is a second antigen-binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and includes the second antigen-binding domain; and either B or D includes a stroma-modifying moiety; (x) A is a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and includes the first antigen-binding domain; and either B, C, or D includes (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule and (b) a cytokine molecule; (xi) A is a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and includes the first antigen-binding domain; and either B, C, or D includes (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule and (b) a stroma-modifying moiety; (xii) A is a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and includes the first antigen-binding domain; and either B, C, or D includes (a) a cytokine molecule and (b) a stroma-modifying moiety; (xiii) A is a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and comprises the first antigen-binding domain; B is a second antigen-binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and comprises the second antigen-binding domain; and C or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule, and (b) a cytokine molecule; (xiv) A is a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and comprises the first antigen-binding domain; B is a second antigen-binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and comprises the second antigen-binding domain; and C or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule, and (b) a stromal modifying moiety; (xv) A is a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and includes the first antigen-binding domain; B is a second antigen-binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and includes the second antigen-binding domain; and C or D comprises (a) a cytokine molecule and (b) a stroma-modifying moiety; (xvi) A is a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and includes the first antigen-binding domain; C is a second antigen-binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and includes the second antigen-binding domain; and B or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule, and (b) a cytokine molecule; (xvii) A is a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and includes the first antigen-binding domain, C is a second antigen-binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and includes the second antigen-binding domain, and B or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule, and (b) a stroma-modifying moiety; (xviii) A is a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and includes the first antigen-binding domain, C is a second antigen-binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and includes the second antigen-binding domain, and B or D comprises (a) a cytokine molecule and (b) a stroma-modifying moiety; (xix) A is a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and includes the first antigen-binding domain, and B, C, or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule, (b) a cytokine molecule, and (c) a stroma-modifying moiety; (xx)A is a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and includes the first antigen-binding domain; B is a second antigen-binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and includes the second antigen-binding domain; and C or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule, (b) a cytokine molecule, and (c) a stromal modification moiety; or (xxi)A is a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and includes the first antigen-binding domain; C is a second antigen-binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and includes the second antigen-binding domain; and B or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule, (b) a cytokine molecule, and (c) a stromal modification moiety.

[0099] In some embodiments, the dimerization module comprises one or more immunoglobulin heavy chain constant regions (e.g., Fc region) including one or more of paired holes and protrusions (“knob-in-hole”), electrostatic interactions, or strand exchange. In some embodiments, the one or more immunoglobulin heavy chain constant regions (e.g., Fc region) include amino acid substitutions at one or more positions selected from, for example, positions 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 of the Fc region of human IgG1. In some embodiments, the one or more immunoglobulin heavy chain constant regions (e.g., Fc region) include amino acid substitutions selected from T366S, L368A, or Y407V (e.g., corresponding to a hole or a hole), or T366W (e.g., corresponding to a protrusion or a knob), or combinations thereof.

[0100] In some embodiments, the multifunctional molecule further comprises a linker, for example, between an antigen-binding domain and an immune cell engager, between an antigen-binding domain and a cytokine molecule, between an antigen-binding domain and a stromal modifying moiety, between an immune cell engager and a cytokine molecule, between an immune cell engager and a stromal modifying moiety, between a cytokine molecule and a stromal modifying moiety, between an antigen-binding domain and a dimerization module, between an immune cell engager and a dimerization module, between a cytokine molecule and a dimerization module, or between a stromal modifying moiety and a dimerization module. In some embodiments, the linker is selected from a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker comprises Gly and Ser. In some embodiments, the peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 6214-6217, or 6220-6221 and 77-78.

[0101] In one aspect, the present invention (i) An antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein, for example, the calreticulin mutant protein comprises the amino acid sequence of SEQ ID NO: 6286, the antigen-binding domain, and (ii) a moiety that binds to CD3, such as an antibody molecule that binds to CD3 and A multifunctional molecule comprising is provided.

[0102] In some embodiments, the multifunctional molecule For example, from the N-terminus to the C-terminus, a first polypeptide comprising a first VL and a first CL, For example, from the N-terminus to the C-terminus, a second polypeptide comprising a first VH, a first CH1, a first dimerization domain (e.g., a first Fc), and a first moiety that binds to CD3 (e.g., a first scFv that binds to CD3), For example, from the N-terminus to the C-terminus, a third polypeptide comprising a second VH, a second CH1, a second dimerization domain (e.g., a second Fc), and optionally a second moiety that binds to CD3 (e.g., a second scFv that binds to CD3), For example, from the N-terminus to the C-terminus, a fourth polypeptide comprising a second VL and a second CL Comprising, The first VL and the first VH form a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), the second VL and the second VH form a second antigen-binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), the first and second calreticulin proteins comprise the amino acid sequence of SEQ ID NO: 6286, and optionally, the first and second calreticulin proteins are each independently selected from a molecule comprising the amino acid sequence of SEQ ID NO: 6313, or a molecule comprising the amino acid sequence of SEQ ID NO: 6314.

[0103] In some embodiments, the multifunctional molecule comprises the configuration of FIGS. 2A or 2B. In one aspect, the present invention (i) an antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein, for example, the calreticulin mutant protein comprises the amino acid sequence of SEQ ID NO: 6286, and the antigen-binding domain, (ii) a moiety that binds to a TCR (e.g., TCRβ), for example, an antibody molecule that binds to a TCR (e.g., TCRβ) and provides a multifunctional molecule.

[0104] In some embodiments, the multifunctional molecule for example, from the N-terminus to the C-terminus, a first polypeptide comprising a first VL and a first CL, for example, from the N-terminus to the C-terminus, a second polypeptide comprising a first VH, a first CH1, a first dimerization domain (e.g., a first Fc), and a first moiety that binds to a TCR (e.g., TCRβ) (e.g., a first scFv that binds to a TCR (e.g., TCRβ)), for example, from the N-terminus to the C-terminus, a third polypeptide comprising a second VH, a second CH1, a second dimerization domain (e.g., a second Fc), and optionally a second moiety that binds to a TCR (e.g., TCRβ) (e.g., a second scFv that binds to a TCR (e.g., TCRβ)), for example, from the N-terminus to the C-terminus, a fourth polypeptide comprising a second VL and a second CL and The first VL and the first VH form a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), the second VL and the second VH form a second antigen-binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), the first and second calreticulin proteins comprise the amino acid sequence of SEQ ID NO: 6286, and optionally, the first and second calreticulin proteins are each independently selected from a molecule comprising the amino acid sequence of SEQ ID NO: 6313, or a molecule comprising the amino acid sequence of SEQ ID NO: 6314.

[0105] In some embodiments, the multifunctional molecule comprises the configuration of FIGS. 3A or 3B. In one aspect, the present invention (i) an antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein, for example, the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, the antigen-binding domain, and (ii) a moiety that binds to NKp30, such as an antibody molecule or ligand that binds to (e.g., activates) NKp30 and provides a multifunctional molecule.

[0106] In some embodiments, the multifunctional molecule for example, from the N-terminus to the C-terminus, a first polypeptide comprising a first VL and a first CL, for example, from the N-terminus to the C-terminus, a second polypeptide comprising a first VH, a first CH1, a first dimerization domain (e.g., a first Fc), and a first moiety that binds to NKp30 (e.g., a first antibody molecule or ligand that binds to NKp30) For example, a third polypeptide comprising, from the N-terminus to the C-terminus, a second VH, a second CH1, a second dimerization domain (e.g., a second Fc), and optionally a second moiety that binds to NKp30 (e.g., a second antibody molecule or ligand that binds to NKp30). For example, a fourth polypeptide comprising, from the N-terminus to the C-terminus, a second VL and a second CL comprising The first VL and the first VH form a first antigen-binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), the second VL and the second VH form a second antigen-binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), the first and second calreticulin proteins comprise the amino acid sequence of SEQ ID NO: 6286, and optionally, the first and second calreticulin proteins are each independently selected from a molecule comprising the amino acid sequence of SEQ ID NO: 6313, or a molecule comprising the amino acid sequence of SEQ ID NO: 6314.

[0107] In some embodiments, the multifunctional molecule comprises the configuration of FIGS. 4A or 4B. In another aspect, the disclosure provides an isolated nucleic acid molecule encoding any of the multispecific or multifunctional molecules described herein. In another aspect, the disclosure provides a nucleotide sequence encoding any of the multispecific or multifunctional molecules described herein, or an isolated nucleic acid molecule comprising a nucleotide sequence that is substantially homologous thereto (e.g., at least 80%, 90%, 95%, or 99.9% identical thereto). In another aspect, the disclosure provides a host cell comprising the nucleic acid molecule or vector described herein.

[0108] In another aspect, the present disclosure provides a method of making, e.g., producing, the multispecific or multifunctional molecular polypeptides described herein, the method comprising culturing a host cell described herein under suitable conditions, e.g., conditions suitable for gene expression and / or homoor heterodimerization.

[0109] In another aspect, the present disclosure provides a pharmaceutical composition comprising a multispecific or multifunctional molecular polypeptide described herein and a pharmaceutically acceptable carrier, excipient or stabilizer.

[0110] In another aspect, the present disclosure provides a method of treating cancer, the method comprising administering to a subject in need thereof a multispecific or multifunctional molecular polypeptide described herein, wherein the multispecific antibody is administered in an amount effective to treat cancer. In some embodiments, the subject has cancer cells expressing a first and / or second calreticulin mutant. In some embodiments, the subject has tumor cells expressing a first, second, or third tumor antigen, for example, the subject has tumor cells expressing a tumor antigen selected from G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1. In some embodiments, the subject has a JAK2 V617F mutation. In some embodiments, the subject does not have a JAK2 V617F mutation. In some embodiments, the subject has an MPL mutation. In some embodiments, the subject does not have an MPL mutation. In some embodiments, the cancer is a blood cancer, and optionally, the cancer is a myeloproliferative neoplasm, such as primary or idiopathic myelofibrosis (MF), essential thrombocythemia (ET), polycythemia vera (PV), or chronic myelogenous leukemia (CML). In some embodiments, the cancer is myelofibrosis. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the solid tumor cancer is one or more of pancreatic cancer (e.g., pancreatic adenocarcinoma), breast cancer, colorectal cancer, lung cancer (e.g., small cell or non-small cell lung cancer), skin cancer, ovarian cancer, or liver cancer.

[0111] In some embodiments, the cancer cells include myeloproliferative neoplasm cells. In embodiments, the myeloproliferative neoplasm cells are selected from myelofibrosis cells, essential thrombocythemia cells, polycythemia vera cells, or chronic myeloid cancer cells. In some embodiments, the myeloproliferative neoplasm cells are myelofibrosis cells. In some embodiments, the myeloproliferative neoplasm cells are essential thrombocythemia cells. In some embodiments, the myeloproliferative neoplasm cells are polycythemia vera cells. In some embodiments, the myeloproliferative neoplasm cells are chronic myeloid cancer cells. In some embodiments, the myeloproliferative neoplasm cells include a JAK2 mutation (e.g., JAK2 V617F mutation). In some embodiments, the myeloproliferative neoplasm cells include a calreticulin mutation. In some embodiments, the myeloproliferative neoplasm cells include an MPL mutation.

[0112] In some embodiments, the method further includes the step of administering a second therapeutic treatment. In some embodiments, the second therapeutic treatment includes a therapeutic agent (e.g., a chemotherapeutic agent, a biological agent, hormone therapy), radiation, or surgery. In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent or a biological agent.

[0113] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0114] Other features and advantages of the present invention will be apparent from the following detailed description and claims. The patent or application file includes at least one drawing created in color. A copy of this patent or patent application publication that includes color drawing(s) will be provided by the Patent Office upon payment of the claims and required fees.

Brief Description of the Drawings

[0115]

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Mode for Carrying Out the Invention

[0116] (i) An antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), wherein, for example, the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285 or 6286, the antigen-binding domain, and (ii) (a) an immunocyte engager selected from a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager, (b) a cytokine molecule, (c) a stromal modification moiety, and (d) a tumor targeting moiety (e.g., one that binds to a tumor antigen selected from G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1), a multifunctional molecule (also referred to herein as a "bispecific molecule") comprising a plurality (e.g., two or more) of functionalities (or binding specificities) including one, two, or all of them are disclosed herein. In some embodiments, the antigen-binding domain binds to a calreticulin protein (e.g., a wild-type calreticulin protein or, for example, a mutant calreticulin protein described herein). In some embodiments, the antigen-binding domain binds to a calreticulin mutant protein disclosed in Table 2 or 3. In some embodiments, the antigen-binding domain binds to a type 1 calreticulin mutant protein disclosed in Table 2 or 3. In some embodiments, the antigen-binding domain binds to a type 2 calreticulin mutant protein disclosed in Table 2 or 3. In some embodiments, the antigen-binding domain binds to type 1 and type 2 calreticulin mutant proteins disclosed in Table 2 or 3. In some embodiments, the T cell engager comprises an additional antigen-binding domain that binds to the variable chain of the beta subunit of the TCR (TCRβV), e.g., TCRβ V6 or TCRβ V12.

[0117] In one embodiment, the multispecific or multifunctional molecule is a bispecific (or bifunctional) molecule, a trispecific (or trifunctional) molecule, or a tetra-specific (or tetra-functional) molecule. In one embodiment, the multispecific or multifunctional molecule is a bispecific molecule.

[0118] Without being bound by theory, the multispecific or multifunctional molecules disclosed herein are predicted to localize (e.g., crosslink) and / or activate calreticulin protein to immune effector cells (e.g., immune cells selected from T cells, NK cells, B cells, dendritic cells or macrophages) in the presence of cells expressing the same on their surface. Increasing the proximity and / or activation of immune cells in the presence of cells expressing calreticulin protein using the multispecific or multifunctional molecules described herein is predicted to enhance the immune response against the target cells, thereby providing a more effective therapy.

[0119] (i) The stromal modification part and (ii) an antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), wherein, for example, the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285 or 6286, and a novel multifunctional, e.g., multispecific molecule is disclosed. Without being bound by theory, the multifunctional molecules disclosed herein are thought to target (e.g., localize to) cancer sites and modify the tumor stroma, e.g., modify the tumor microenvironment near the cancer site. The multifunctional molecule may further comprise one or both of an immune cell engager (e.g., selected from one, two, three, or all of a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager) and / or a cytokine molecule. Accordingly, inter alia, multifunctional, e.g., multispecific molecules comprising the foregoing parts, nucleic acids encoding the same, methods of producing the foregoing molecules, and methods of treating cancer using the foregoing molecules are provided herein.

[0120] Accordingly, inter alia, multispecific or multifunctional molecules (e.g., multispecific or multifunctional antibody molecules) comprising the foregoing parts, nucleic acids encoding the same, methods of producing the foregoing molecules, and methods of treating a disease or disorder, e.g., cancer, using the foregoing molecules are provided herein.

[0121] Definitions In some embodiments, the multifunctional molecule includes an immune cell engager. An "immune cell engager" refers to one or more binding specificities that bind to and / or activate immune cells, e.g., cells involved in an immune response. In embodiments, the immune cells are selected from T cells, NK cells, B cells, dendritic cells, and / or macrophage cells. The immune cell engager can be an antibody molecule, a receptor molecule (e.g., a full-length receptor, a receptor fragment, or a fusion thereof (e.g., a receptor-Fc fusion)), or a ligand molecule that binds to an immune cell antigen (e.g., a T cell, NK cell antigen, B cell antigen, dendritic cell antigen, and / or macrophage cell antigen) (e.g., a full-length ligand, a ligand fragment, or a fusion thereof (e.g., a ligand-Fc fusion)). In embodiments, the immune cell engager specifically binds to a target immune cell, e.g., preferentially binds to the target immune cell. For example, if the immune cell engager is an antibody molecule, it binds to an immune cell antigen (e.g., a T cell antigen, NK cell antigen, B cell antigen, dendritic cell antigen, and / or macrophage cell antigen) with a dissociation constant of less than about 10 nM.

[0122] As used herein, the terms "T cell receptor beta variable chain", "TCRVβ", "TCRVb" and "TCRβV" are used synonymously and refer to the extracellular region of the T cell receptor beta chain that contains the antigen recognition domain of the T cell receptor. The term TCRVβ or TCRβV includes isoforms, mammals, such as human TCRβV, human species homologs and analogs that contain at least one common epitope with TCRβV. Human TCRβV includes, but is not limited to, the TCRβ V6 subfamily, the TCRβ V10 subfamily, the TCRβ V12 subfamily, the TCRβ V5 subfamily, the TCRβ V7 subfamily, the TCRβ V11 subfamily, the TCRβ V14 subfamily, the TCRβ V16 subfamily, the TCRβ V18 subfamily, the TCRβ V9 subfamily, the TCRβ V13 subfamily, the TCRβ V4 subfamily, the TCRβ V3 subfamily, the TCRβ V2 subfamily, the TCRβ V15 subfamily, the TCRβ V30 subfamily, the TCRβ V19 subfamily, the TCRβ V27 subfamily, the TCRβ V28 subfamily, the TCRβ V24 subfamily, the TCRβ V20 subfamily, the TCRβ V25 subfamily, or the TCRβ V29 subfamily, and includes gene families that include subfamilies. In some embodiments, the TCRβ V6 subfamily is TCRβ V6-4 * 01, TCRβ V6-4 * 02, TCRβ V6-9 * 01, TCRβ V6-8 * 01, TCRβ V6-5 * 01, TCRβ V6-6 * 02, TCRβ V6-6 * 01, TCRβ V6-2 * 01, TCRβ V6-3 * 01 or TCRβ V6-1 * 01. In some embodiments, TCRβV includes TCRβ V6-5 * 01. TCRβ V6-5 *01 is also known as TRBV65; TCRBV6S5; TCRBV13S1, or TCRβ V13.1. TCRβ V6-5 * 01, for example, human TCRβ V6-5 * The amino acid sequence of 01 is known in the art and is provided, for example, by IMGT ID L36092. In some embodiments, TCRβ V6-5 * 01 is encoded by the nucleic acid sequence of SEQ ID NO: 1043, or a sequence having 85%, 90%, 95%, 99% or more identity thereto. In some embodiments, TCRβ V6-5 * 01 comprises the amino acid sequence of SEQ ID NO: 1044, or a sequence having 85%, 90%, 95%, 99% or more identity thereto.

[0123] In some embodiments, the multifunctional molecule comprises a cytokine molecule. As used herein, "cytokine molecule" refers to the full length, fragment or variant of a cytokine; further, a cytokine comprising a receptor domain, such as a cytokine receptor dimerization domain; or an agonist of a cytokine receptor that induces activation of at least one of the naturally occurring cytokines, such as an antibody molecule (e.g., agonist antibody) against a cytokine receptor. In some embodiments, the cytokine molecule is selected from interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), or interferon γ, or fragments or variants thereof, or any combination of the aforementioned cytokines. The cytokine molecule can be a monomer or a dimer. In embodiments, the cytokine molecule can further comprise a cytokine receptor dimerization domain. In other embodiments, the cytokine molecule is an agonist of a cytokine receptor, such as an antibody molecule (e.g., agonist antibody) against a cytokine receptor selected from IL-15Ra or IL-21R.

[0124] As used herein, the term "molecule" as used, for example, in an antibody molecule, a cytokine molecule, or a receptor molecule includes a full-length naturally occurring molecule, as well as variants, such as functional variants (e.g., truncations, fragments, mutations (e.g., substantially similar sequences), or derivatized forms thereof), as long as at least one function and / or activity of the unmodified (e.g., naturally occurring) molecule remains.

[0125] In some embodiments, the multifunctional molecule includes a stromal modifying moiety. As used herein, the term "stromal modifying moiety" refers to an agent, such as a protein (e.g., an enzyme), that can modify, e.g., degrade, a component of the stroma. In embodiments, the component of the stroma is selected from, for example, ECM components such as glycosaminoglycans such as hyaluronan (also known as hyaluronic acid or HA), chondroitin sulfate, chondroitin, dermatan sulfate, heparan sulfate, heparin, entactin, tenascin, aggrecan, and keratin sulfate; or extracellular proteins such as collagen, laminin, elastin, fibrinogen, fibronectin, and vitronectin. Certain terms are defined below.

[0126] As used herein, the articles "a" and "an" refer to one or more, e.g., at least one, of the grammatical objects of the article. The use of the term "a" or "an" may mean "one" when used in conjunction with the term "comprising" herein, but is also consistent with the meaning of "one or more," "at least one," and "more than one."

[0127] As used herein, "about" and "substantially" generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within twenty percent (20%) of a given range of values, typically within ten percent (10%), and more typically within five percent (5%).

[0128] As used herein, the term "antibody molecule" refers to a protein, such as an immunoglobulin chain or a fragment thereof, that includes at least one immunoglobulin variable domain sequence. Antibody molecules include antibodies (e.g., full-length antibodies) and antibody fragments. In certain embodiments, an antibody molecule includes an antigen-binding or functional fragment of a full-length antibody, or a full-length immunoglobulin chain. For example, a full-length antibody is an immunoglobulin (Ig) molecule (e.g., an IgG antibody) that occurs naturally or is formed by a recombinant process of normal immunoglobulin gene segments. In embodiments, an antibody molecule refers to an immunologically active antigen-binding portion of an immunoglobulin molecule, such as an antibody fragment. Antibody fragments, such as functional fragments, are portions of an antibody, such as Fab, Fab’, F(ab’)2, F(ab)2, variable fragment (Fv), domain antibody (dAb), or single-chain variable fragment (scFv). Functional antibody fragments bind to the same antigen as that recognized by an intact (e.g., full-length) antibody. The terms "antibody fragment" or "functional fragment" also include isolated fragments consisting of variable regions, such as an "Fv" fragment consisting of the variable regions of the heavy and light chains, or a recombinant single-chain polypeptide molecule in which the variable regions of the light and heavy chains are linked by a peptide linker ("scFv protein"). In some embodiments, an antibody fragment includes a portion of an antibody that does not have antigen-binding activity, such as an Fc fragment or does not include a single amino acid residue. Exemplary antibody molecules include full-length antibodies and antibody fragments, such as dAb (domain antibody), single-chain, Fab, Fab’, and F(ab’)2 fragments, and single-chain variable fragments (scFv).

[0129] As used herein, the term "immunoglobulin variable domain sequence" refers to an amino acid sequence capable of forming the structure of an immunoglobulin variable domain. For example, the sequence may include all or a portion of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two, or more N-terminal or C-terminal amino acids, or may include other modifications that are compatible with the formation of a protein structure.

[0130] In embodiments, the antibody molecule is monospecific and includes, for example, binding specificity for a single epitope. In some embodiments, the antibody molecule is multispecific; for example, it includes a plurality of immunoglobulin variable domain sequences, where the first immunoglobulin variable domain sequence has binding specificity for a first epitope and the second immunoglobulin variable domain sequence has binding specificity for a second epitope. In some embodiments, the antibody molecule is a bispecific antibody molecule. As used herein, a "bispecific antibody molecule" refers to an antibody molecule having specificity for more than one (e.g., 2, 3, 4, or more) epitopes and / or antigens.

[0131] As used herein, "antigen" (Ag) refers to a molecule capable of eliciting an immune response, such as one that is associated with activation of certain immune cells and / or antibody production. Virtually any macromolecule, including most proteins or peptides, can be an antigen. Antigens can also be derived from genomic recombinants or DNA. For example, any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein capable of inducing an immune response encodes an "antigen." In embodiments, an antigen need not be encoded only by the full-length nucleotide sequence of a gene, nor need an antigen be encoded at all by a gene. In embodiments, an antigen can be synthesized or derived from a biological sample, such as a tissue sample, tumor sample, cell, or fluid having other biological components. As used herein, "tumor antigen" or, synonymously, "cancer antigen" includes any molecule present on or associated with a tumor, such as a cancer cell, or the tumor microenvironment that is capable of eliciting an immune response. As used herein, "immune cell antigen" includes any molecule present on or associated with an immune cell that is capable of eliciting an immune response.

[0132] The "antigen-binding site" or "binding site" of an antibody molecule refers to a part of the antibody molecule involved in antigen binding, such as a part of an immunoglobulin (Ig) molecule. In embodiments, the antigen-binding site is formed by amino acid residues in the variable regions (V) of the heavy chain (H) and light chain (L). Three highly divergent stretches within the variable regions of the heavy and light chains, called hypervariable regions, are positioned between more conserved adjacent regions called "framework regions" (FR). The FR is the amino acid sequence naturally found between and adjacent to the hypervariable regions in an immunoglobulin. In embodiments, in an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface complementary to the three-dimensional surface of the binding antigen. Each of the three hypervariable regions of the heavy and light chains is called a "complementary determining region" or "CDR". The framework regions and CDRs are defined and described, for example, in Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917. Each variable chain (e.g., variable heavy chain and variable light chain) typically consists of three CDRs and four FRs, arranged in the order of amino acids: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxy terminus.

[0133] As used herein, "cancer" can include all types of carcinogenic processes and / or cancerous growth. In embodiments, cancer can include primary tumors, as well as metastatic tissues, or cells, tissues, or organs that have been malignantly transformed. In embodiments, cancer includes all histopathologies and stages of cancer, such as invasive / severity stages. In embodiments, cancer includes recurrent cancer and / or resistant cancer. The terms "cancer" and "tumor" can be used interchangeably. For example, both terms can include solid tumors and liquid tumors. As used herein, the term "cancer" or "tumor" includes pre-cancer, as well as malignant cancers and tumors.

[0134] As used herein, "immune cell" refers to any of a variety of cells that function in the immune system, e.g., defend against infectious and foreign agents. In embodiments, the term includes white blood cells, such as neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Innate white blood cells include phagocytes (e.g., macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer cells. Innate white blood cells identify and eliminate pathogens by attacking larger pathogens through contact or by engulfing and killing microorganisms, and are mediators of the activation of the adaptive immune response. Cells of the adaptive immune system are a special type of white blood cell called lymphocytes. B cells and T cells are important types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow. B cells are involved in the humoral immune response, and T cells are involved in the cellular immune response. The term "immune cell" includes immune effector cells.

[0135] The term "immune effector cell" as used herein refers to a cell involved in promoting an immune response, e.g., an immune effector response. Examples of immune effector cells include, but are not limited to, T cells, such as alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T cells, and mast cells.

[0136] The term "effector function" or "effector response" refers to the specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity including the secretion of cytokines.

[0137] The compositions and methods of the present invention include polypeptides and nucleic acids having a specific sequence or a sequence that is substantially identical or similar thereto, for example, a sequence that is at least 80%, 85%, 90%, 95% identical or more identical to a specific sequence. In the context of amino acid sequences, the term "substantially identical" is used herein such that the first and second amino acid sequences can have a common structural domain and / or a common functional activity, i) being identical to the second amino acid sequence, or ii) containing a sufficient or minimal number of amino acid residues that are conservative substitutions of the aligned amino acid residues in the second amino acid sequence. For example, an amino acid sequence containing a common structural domain has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, such as a sequence provided herein.

[0138] In the context of nucleotide sequences, the term "substantially identical" is used herein such that the first and second nucleotide sequences contain a sufficient or minimal number of nucleotides that are identical to the aligned nucleotides in the second nucleic acid sequence so as to encode a polypeptide having a common functional activity or to encode a common polypeptide structural domain or a common polypeptide functional activity. For example, a nucleotide sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, such as a sequence provided herein.

[0139] The term "variant" refers to a polypeptide having an amino acid sequence that is substantially identical to a reference amino acid sequence or encoded by a substantially identical nucleotide sequence. In some embodiments, the variant is a functional variant.

[0140] The term "functional variant" refers to a polypeptide having an amino acid sequence that is substantially identical to a reference amino acid sequence or encoded by a substantially identical nucleotide sequence and having one or more activities of the reference amino acid sequence.

[0141] The calculation of homology or sequence identity between sequences (these terms are used interchangeably herein) is performed as follows. To determine the percent identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences may be disregarded for comparison purposes). In preferred embodiments, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, still more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. Next, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. If the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equal to amino acid or nucleic acid "homology").

[0142] The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

[0143] The comparison of arrays and the determination of the percent identity between two arrays can be accomplished using mathematical algorithms. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Blossum62 matrix or the PAM250 matrix, and either a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6, using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com). In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program of the GCG software package (available at http: / / www.gcg.com), using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and those to be used unless otherwise specified) is the Blossum62 score matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0144] The percent identity between two amino acid sequences or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) incorporated into the ALIGN program (version 2.0), using the PAM120 residue weight table, a gap length penalty of 12, and a gap penalty of 4.

[0145] The nucleic acid and protein sequences described herein can be used, for example, as "query sequences" for performing searches against public databases to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed using the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed using the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing the BLAST and gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov.

[0146] It is understood that the molecules of the present invention may have additional conservative or non-essential amino acid substitutions that do not substantially affect their function. The term "amino acid" is intended to include any molecule, whether natural or synthetic, that contains both amino functionality and acid functionality and can be included in a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and homologs thereof; amino acid analogs having variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes both D or L optical isomers and peptidomimetics.

[0147] "Conservative amino acid substitution" refers to an amino acid residue being replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0148] The terms "polypeptide", "peptide" and "protein" (in the case of single chains) are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. These terms also encompass modified amino acid polymers; for example, any other operation such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component. A polypeptide can be isolated from a natural source, produced by recombinant techniques from a eukaryotic or prokaryotic host, or be the product of synthetic procedures.

[0149] The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence", or "polynucleotide sequence", and "polynucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide can be either single-stranded or double-stranded, and if single-stranded, can be either the coding strand or the non-coding (antisense) strand. A polynucleotide can include modified nucleotides such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, for example, by conjugation with a labeling component. A nucleic acid can be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin that is ligated to another polynucleotide with a non-natural or unnatural sequence.

[0150] The term "isolated", as used herein, refers to a material that has been removed from its original environment or natural environment (e.g., the natural environment if it occurs naturally). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide that has been separated by human intervention from some or all of the coexisting materials in the natural system is isolated. Such a polynucleotide can be part of a vector, and / or such a polynucleotide or polypeptide can be part of a composition, and such a vector or composition can still be isolated in that it is not part of the environment in which it is found naturally.

[0151] As used herein, the term "transforming growth factor beta-1 (TGF-beta1)" refers to the protein encoded by the gene TGFB1, or its ortholog, in humans. Swiss-Prot accession number P01137 provides an exemplary human TGF-beta1 amino acid sequence. The exemplary immature human TGF-beta1 amino acid sequence is provided by SEQ ID NO: 6378. The exemplary mature human TGF-beta1 amino acid sequence is provided by SEQ ID NO: 6395.

[0152] As used herein, the term "transforming growth factor beta-2 (TGF-beta2)" refers to the protein encoded by the gene TGFB2, or its ortholog, in humans. Swiss-Prot accession number P61812 provides an exemplary human TGF-beta2 amino acid sequence. The exemplary immature human TGF-beta2 amino acid sequence is provided by SEQ ID NO: 6379. The exemplary mature human TGF-beta2 amino acid sequence is provided by SEQ ID NO: 6396.

[0153] As used herein, the term "transforming growth factor beta-3 (TGF-beta3)" refers to the protein encoded by the gene TGFB3, or its ortholog, in humans. Swiss-Prot accession number P10600 provides an exemplary human TGF-beta3 amino acid sequence. The exemplary immature human TGF-beta3 amino acid sequence is provided by SEQ ID NO: 6380. The exemplary mature human TGF-beta3 amino acid sequence is provided by SEQ ID NO: 6397.

[0154] As used herein, "TGF-beta receptor polypeptide" refers to a TGF-beta receptor (e.g., TGFBR1, TGFBR2 or TGFBR3), or a fragment thereof, or a variant thereof.

[0155] As used herein, the term "transforming growth factor beta receptor type I (TGFBR1)" (also known as ALK-5 or SKR4) refers to the protein encoded by the gene TGFBR1, or its ortholog, in humans. Swiss-Prot accession number P36897 provides an exemplary human TGFBR1 amino acid sequence. Exemplary immature human TGFBR1 amino acid sequences are provided by SEQ ID NOs: 6381, 6382, and 6383. Exemplary mature human TGFBR1 amino acid sequences are provided by SEQ ID NOs: 6398, 6399, and 6400. As used herein, "TGFBR1 polypeptide" refers to TGFBR1 or a fragment thereof, or a variant thereof.

[0156] As used herein, the term "transforming growth factor beta receptor type II (TGFBR2)" refers to the protein encoded by the gene TGFBR2, or its ortholog, in humans. Swiss-Prot accession number P37173 provides an exemplary human TGFBR2 amino acid sequence. Exemplary immature human TGFBR2 amino acid sequences are provided by SEQ ID NOs: 6384 and 6385. Exemplary mature human TGFBR2 amino acid sequences are provided by SEQ ID NOs: 6401 and 6402. As used herein, "TGFBR2 polypeptide" refers to TGFBR2 or a fragment thereof, or a variant thereof.

[0157] As used herein, the term "transforming growth factor beta receptor type III (TGFBR3)" refers to the protein encoded by the gene TGFBR3, or its ortholog, in humans. Swiss-Prot accession number Q03167 provides an exemplary human TGFBR3 amino acid sequence. Exemplary immature human TGFBR3 amino acid sequences are provided by SEQ ID NOs: 6392 and 6393. Exemplary mature human TGFBR3 amino acid sequences are provided by SEQ ID NOs: 6403 and 6404. As used herein, "TGFBR3 polypeptide" refers to TGFBR3 or a fragment thereof or a variant thereof.

[0158] Various aspects of the present invention are described in further detail below. Additional definitions are set forth throughout the specification.

[0159] Antibody molecule In some embodiments, the multifunctional molecules, multispecific molecules, and / or antigen-binding domains described herein include antibody molecules. In one embodiment, the antibody molecule binds to a cancer antigen, such as a tumor antigen or a stromal antigen. In some embodiments, the cancer antigen is, for example, a mammalian, such as a human, cancer antigen. In other embodiments, the antibody molecule binds to an immune cell antigen, such as a mammalian, such as a human, immune cell antigen. For example, the antibody molecule specifically binds to an epitope on a cancer antigen or an immune cell antigen, such as a linear epitope or a conformational epitope.

[0160] In one embodiment, the antibody molecule is a monospecific antibody molecule that binds to a single epitope. For example, a monospecific antibody molecule has a plurality of immunoglobulin variable domain sequences, each of which binds to the same epitope.

[0161] In one embodiment, the antibody molecule is a multispecific or multifunctional antibody molecule. For example, it includes a plurality of immunoglobulin variable domain sequences, wherein a first one of the plurality of immunoglobulin variable domain sequences has binding specificity for a first epitope and a second one of the plurality of immunoglobulin variable domain sequences has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, such as the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, such as different proteins (or different subunits of a multimeric protein). In one embodiment, the multispecific antibody molecule includes a third, fourth, or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or a tetravalent antibody molecule.

[0162] In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. The bispecific antibody has specificity for only two antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, for example, the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, for example, different proteins (or different subunits of a multimeric protein). In one embodiment, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a half antibody, or a fragment thereof, having binding specificity for a first epitope and a half antibody, or a fragment thereof, having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a scFv or Fab, or a fragment thereof, having binding specificity for a first epitope and a scFv or Fab, or a fragment thereof, having binding specificity for a second epitope.

[0163] In one embodiment, antibody molecules include antigen-binding fragments of antibodies (e.g., Fab, F(ab’)2, and Fv), in addition to diabodies and single-chain molecules. For example, an antibody molecule can include a heavy (H) chain variable domain sequence (abbreviated herein as VH) and a light (L) chain variable domain sequence (abbreviated herein as VL). In one embodiment, an antibody molecule comprises or consists of a heavy chain and a light chain (referred to herein as a half antibody). In another example, an antibody molecule includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen-binding sites, e.g., Fab, Fab’, F(ab’)2, Fc, Fd, Fd’, Fv, single-chain antibodies (e.g., scFv), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by modification of a full-length antibody or synthesized de novo using recombinant DNA technology. These functional antibody fragments retain their ability to selectively bind to their respective antigens or receptors. Antibodies and antibody fragments can be from any class of antibodies, including but not limited to IgG, IgA, IgM, IgD, and IgE, and from any subclass of antibodies (e.g., IgG1, IgG2, IgG3, and IgG4). Preparations of antibody molecules can be monoclonal or polyclonal. Antibody molecules can also be human, humanized, CDR-grafted, or in vitro-generated antibodies. An antibody can have a heavy chain constant region selected, for example, from IgG1, IgG2, IgG3, or IgG4. An antibody can also have a light chain selected, for example, from kappa or lambda. The term “immunoglobulin” (Ig) is used interchangeably herein with the term “antibody”.

[0164] Examples of antigen-binding fragments of antibody molecules include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab’)2 fragments, which are bivalent fragments containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragments, which consist of VH and CH1 domains; (iv) Fv fragments, which consist of the VL and VH domains of a single arm of an antibody; (v) dAb (single domain antibody) fragments, which consist of a VH domain; (vi) camel or camelized variable domains; (vii) single-chain Fv (scFv), see, for example, Bird et al., (1988) Science 242: 423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883; and (viii) single domain antibodies. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0165] Antibody molecules include, in addition to intact molecules, functional fragments thereof. The constant regions of antibody molecules can be modified, for example, mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function).

[0166] The antibody molecule can also be a single-domain antibody. A single-domain antibody can include an antibody in which its complementary determining region is part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, antibodies that are naturally lacking in light chains, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. The single-domain antibody can be any one in the art or any future single-domain antibody. The single-domain antibody can be derived from any species including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit, and cow. According to another aspect of the present invention, the single-domain antibody is a naturally occurring single-domain antibody known as a heavy-chain antibody lacking a light chain. Such single-domain antibodies are disclosed, for example, in WO9404678. For reasons of clarity, this variable domain derived from a heavy-chain antibody that is naturally lacking in a light chain is known herein as VHH or nanobody to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be derived from antibodies produced in camelid species such as camel, llama, alpaca, and guanaco. Other species other than camelids may produce heavy-chain antibodies that are naturally lacking in light chains, and such VHHs are within the scope of the present invention.

[0167] The VH and VL regions can be further divided into hypervariable regions called "complementary determining regions" (CDRs) flanked by more conserved regions called "framework regions" (FR or FW).

[0168] The framework regions and the scope of CDRs are precisely defined in a number of ways (see Kabat, E. A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al., (1987) J. Mol. Biol. 196:901-917; and the definition of AbM used by Oxford Molecular's AbM antibody modeling software. Generally, see, for example, "Protein Sequence and Structure Analysis of Antibody Variable Domains", Antibody Engineering Lab Manual (Duebel, S. and Kontermann, R., eds., Springer-Verlag, Heidelberg)).

[0169] The terms "complementary determining region" and "CDR", as used herein, refer to the amino acid sequences within the antibody variable regions that confer antigen specificity and binding affinity. Generally, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.

[0170] The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described in Kabat et al., (1991), "Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, pages 927-948 ("Chothia" numbering scheme). As used herein, a CDR defined according to the "Chothia" numbering scheme may also be referred to as a "hypervariable loop".

[0171] For example, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).

[0172] Each VH and VL typically contains three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0173] The antibody molecule can be a polyclonal or monoclonal antibody. As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies can be produced by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods).

[0174] Antibodies can be produced recombinantly, for example, by phage display or combinatorial methods. Phage display and combinatorial methods for generating antibodies are known in the art (e.g., Ladner et al., U.S. Patent No. 5,223,409; Kang et al., International Publication WO92 / 18619; Dower et al., International Publication WO91 / 17271; Winter et al., International Publication WO92 / 20791; Markland et al., International Publication WO92 / 15679; Breitling et al., International Publication WO93 / 01288; McCafferty et al., International Publication WO92 / 01047; Garrard et al., International Publication WO92 / 09690; Ladner et al., International Publication WO90 / 02809; Fuchs et al., (1991) Bio / Technology 9:1370-1372; Hay et al., (1992) Hum Antibod Hybridomas 3:81-85; Huse et al., (1989) Science 246:1275-1281; Griffiths et al., (1993) EMBO J 12:725-734; Hawkins et al., (1992) J Mol Biol 226:889-896; Clackson et al., (1991) Nature 352:624-628; Gram et al., (1992) PNAS 89:3576-3580; Garrad et al., (1991) Bio / Technology 9:1373-1377; Hoogenboom et al., (1991) Nuc Acid Res 19:4133-4137; and Barbas et al., (1991) PNAS 88:7978-7982 (the entire contents of all of these are hereby incorporated by reference herein)).

[0175] In one embodiment, the antibody is a fully human antibody (e.g., an antibody made in a mouse genetically engineered to produce an antibody from human immunoglobulin sequences), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Methods for producing rodent antibodies are known in the art.

[0176] Human monoclonal antibodies can be generated using transgenic mice having human immunoglobulin genes rather than mouse strains. Using spleen cells from these transgenic mice immunized with the antigen of interest, hybridomas are produced that secrete human mAbs having specific affinity for epitopes from human proteins (see, e.g., Wood et al., International Application WO91 / 00906; Kucherlapati et al., PCT International Publication WO91 / 10741; Lonberg et al., International Application WO92 / 03918; Kay et al., International Application 92 / 03917; Lonberg, N. et al., 1994 Nature 368:856-859; Green, L.L. et al., 1994 Nature Genet. 7:13-21; Morrison, S.L. et al., 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al., 1993 Year Immunol 7:33-40; Tuaillon et al., 1993 PNAS 90:3720-3724; Bruggeman et al., 1991 Eur J Immunol 21:1323-1326).

[0177] The antibody molecule can be one in which the variable region, or portion thereof, e.g., the CDR, is generated in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the scope of the invention. Antibody molecules generated in a non-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to reduce antigenicity in humans are within the scope of the invention.

[0178] The "effective human" protein is a protein that does not substantially induce a neutralizing antibody response, such as a human anti-mouse antibody (HAMA) response. HAMA can be a problem in a number of situations, for example, in the treatment of chronic or recurrent disease states where antibody molecules are repeatedly administered. The HAMA response can render repeated antibody administration potentially ineffective due to increased antibody clearance from the serum (see, for example, Saleh et al., Cancer Immunol. Immunother., 32:180-190 (1990)) and also due to potential allergic reactions (see, for example, LoBuglio et al., Hybridoma, 5:5117-5123 (1986)).

[0179] Chimeric antibodies can be produced by recombinant DNA techniques known in the art (see Robinson et al., International Publication PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, M., European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., International Application WO86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application No. 125,023; Better et al., (1988 Science 240:1041-1043); Liu et al., (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al., (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[0180] Humanized or CDR-grafted antibodies have at least one or two (of the heavy and / or light immunoglobulin chains), and generally all three, recipient CDRs replaced with donor CDRs. The antibody may be replaced with at least a portion of the non-human CDRs, or only a portion of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding to the antigen. Preferably, the donor is a rodent antibody, such as a rat or mouse antibody, and the recipient is a human framework or human consensus framework. Typically, the immunoglobulin providing the CDRs is called the "donor", and the immunoglobulin providing the framework is called the "acceptor". In one embodiment, the donor immunoglobulin is non-human (e.g., rodent). The acceptor framework is a naturally occurring (e.g., human) framework or consensus framework, or a sequence that is about 85% or higher, preferably 90%, 95%, 99% or more identical thereto.

[0181] As used herein, the term "consensus sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of related sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family of proteins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. When two amino acids occur equally frequently, either may be included in the consensus sequence. "Consensus framework" refers to the framework region in a consensus immunoglobulin sequence.

[0182] Antibody molecules can be humanized by methods known in the art (see, e.g., Morrison, S. L., 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; and Queen et al., U.S. Patent Nos. 5,585,089, 5,693,761, and 5,693,762, the entire contents of all of which are hereby incorporated by reference).

[0183] Humanized or CDR-grafted antibody molecules can be produced by CDR grafting or CDR substitution, in which one, two, or all of the CDRs of an immunoglobulin chain can be replaced. See, e.g., U.S. Patent No. 5,225,539; Jones et al., 1986 Nature 321:552-525; Verhoeyan et al., 1988 Science 239:1534; Beidler et al., 1988 J. Immunol. 141:4053-4060; Winter, U.S. Patent No. 5,225,539 (the entire contents of all of which are hereby expressly incorporated by reference). Winter describes a CDR grafting method that can be used to prepare the humanized antibodies of the present invention (British Patent Application No. 2188638A, filed Mar. 26, 1987; Winter, U.S. Patent No. 5,225,539), the contents of which are hereby incorporated by reference).

[0184] Humanized antibody molecules with specific amino acid substitutions, deletions, or additions are also within the scope of the present invention. The criteria for selecting amino acids from the donor are described in U.S. Patent No. 5,585,089, e.g., columns 12-16 of U.S. Patent No. 5,585,089 (the contents of which are hereby incorporated by reference). Other techniques for humanizing antibodies are described in Padlan et al., EP519596A1, published Dec. 23, 1992.

[0185] The antibody molecule can be a single-chain antibody. The single-chain antibody (scFv) may be engineered (see, e.g., Colcher, D. et al., (1999) Ann N Y Acad Sci 880:263-280; and Reiter, Y., (1996) Clin Cancer Res 2:245-252). The single-chain antibody can dimerize or multimerize to produce a multivalent antibody having specificity for different epitopes of the same target protein.

[0186] In yet other embodiments, the antibody molecule has a heavy chain constant region selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly, for example, the (e.g., human) heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4. In another embodiment, the antibody molecule has a light chain constant region selected from, for example, the (e.g., human) kappa or lambda light chain constant regions. The constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, and / or complement function). In one embodiment, the antibody has effector function and can fix complement. In other embodiments, the antibody does not recruit effector cells and does not fix complement. In another embodiment, the antibody has a reduced or no ability to bind to Fc receptors. For example, it is an isotype or subtype, fragment or other mutant that does not support binding to Fc receptors, e.g., it has a mutagenized Fc receptor-binding region or lacks it.

[0187] Methods for altering the constant region of an antibody are known in the art. Antibodies having altered functions, such as altered affinity for an effector ligand, such as an FcR on a cell, or the C1 component of complement, can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see, e.g., EP388,151A1, U.S. Patent No. 5,624,821, and U.S. Patent No. 5,648,260, the entire contents of all of which are hereby incorporated by reference). Similar types of alterations that reduce or eliminate these functions when applied to mouse, or other species, immunoglobulins can be described.

[0188] Antibody molecules can be derivatized or conjugated to another functional molecule (e.g., another peptide or protein). As used herein, a “derivatized” antibody molecule is a modified antibody molecule. Methods of derivatization include, but are not limited to, the addition of a fluorescent moiety, a radioactive nucleotide, a toxin, an enzyme, or an affinity ligand, such as biotin. Thus, the antibody molecules of the invention are intended to include derivatives and other modified forms of the antibodies described herein, including immunoadhesion molecules. For example, an antibody molecule can be functionally linked (by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association with another molecule of the antibody or antibody portion (e.g., a streptavidin core region or polyhistidine tag).

[0189] One type of derivatized antibody molecule is produced by cross-linking two or more antibodies (e.g., of the same or different types, such as to create a bispecific antibody). Suitable cross-linkers include heterobifunctional ones having two separately reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.

[0190] Multispecific or multifunctional antibody molecules Exemplary structures of the multispecific and multifunctional molecules defined herein are described throughout. Exemplary structures are further described in Weidle U et al., (2013) The Intriguing Options of Multispecific Antibody Formats for Treatment of Cancer. Cancer Genomics & Proteomics 10: 1~18 (2013); and Spiess C et al., (2015) Alternative molecular formats and therapeutic applications for bispecific antibodies. Molecular Immunology 67: 95~106 (the entire contents of each of which are hereby incorporated by reference herein).

[0191] In embodiments, the multispecific antibody molecule can include more than one antigen-binding site where different sites are specific for different antigens. In embodiments, the multispecific antibody molecule can bind to more than one (e.g., two or more) epitopes on the same antigen. In embodiments, the multispecific antibody molecule includes an antigen-binding site specific for a target cell (e.g., a cancer cell) and a different antigen-binding site specific for an immune effector cell. In embodiments, the multispecific antibody molecule is a bispecific, trispecific, or tetra-specific antibody molecule. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibody molecules can be classified into five different structural groups: (i) bispecific immunoglobulin G (BsIgG), (ii) IgG with an additional antigen-binding moiety added, (iii) bispecific antibody fragments, (iv) bispecific fusion proteins, and (v) bispecific antibody conjugates.

[0192] BsIgG is a monovalent format for each antigen. Exemplary BsIgG formats include, but are not limited to, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pair, Fab arm exchange, SEED body, triomab, LUZ-Y, Fcab, κλ body, orthogonal Fab. See Spiess et al., Mol. Immunol. 67 (2015): 95-106. Exemplary BsIgGs include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm) containing an anti-CD3 arm and an anti-EpCAM arm, and ertumaxomab (Neovii Biotech, Fresenius Biotech) targeting CD3 and HER2. In some embodiments, BsIgG comprises heavy chains engineered for heterodimerization. For example, the heavy chains can be engineered for heterodimerization using the "knobs-into-holes" strategy, the SEED platform, common heavy chains (e.g., in a κλ body), and the use of a heterodimeric Fc region. See Spiess et al., Mol. Immunol. 67 (2015): 95-106. Strategies that have been used to avoid homodimeric heavy chain pairing in BsIgG include knobs-in-holes, duobody, azymetric, charge pair, HA-TF, SEED body, and differential protein A affinity. See the same reference. BsIgG can be produced by separate expression of the component antibodies in different host cells and subsequent purification / assembly into BsIgG. BsIgG can also be produced by expression of the component antibodies in a single host cell. BsIgG can be purified, for example, using affinity chromatography using protein A and sequential pH elution.

[0193] IgG with an added additional antigen-binding portion is another format of bispecific antibody molecule. For example, a monospecific IgG can be engineered to have bispecificity by adding an additional antigen-binding unit to the monospecific IgG, for example, at the N- or C-terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single domain antibodies (e.g., variable heavy or variable light chains), engineered protein scaffolds, and paired antibody variable domains (e.g., single chain variable fragments or variable fragments). See the same reference. Examples of added IgG formats include dual variable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (4-in-1). See Spiess et al., Mol. Immunol. 67 (2015): 95-106. An example of IgG-scFv is MM-141 (Merrimack Pharmaceuticals) that binds to IGF-1R and HER3. Examples of DVD-Ig include ABT-981 (AbbVie) that binds to IL-1α and IL-1β, and ABT-122 (AbbVie) that binds to TNF and IL-17A.

[0194] Bispecific antibody fragments (BsAbs) are formats of bispecific antibody molecules that lack some or all of the antibody constant domains. For example, some BsAbs lack the Fc region. In embodiments, the bispecific antibody fragment comprises heavy and light chain regions connected by a peptide linker that permits efficient expression of the BsAb in a single host cell. Exemplary bispecific antibody fragments include, but are not limited to, nanobodies, nanobody-HAS, BiTE, diabodies, DART, TandAb, scDiabody, scDiabody-CH3, diabody-CH3, tribodies, miniantibodies, minibodies, TriBi minibodies, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab’)2, F(ab’)2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, and intrabodies. See the same reference. For example, the BiTE format comprises a tandem scFv, and the component scFvs bind to CD3 on T cells and a surface antigen on cancer cells.

[0195] Examples of bispecific fusion proteins include, for example, antibody fragments linked to other proteins to add additional specificity and / or functionality. An example of a bispecific fusion protein is immTAC, which comprises an anti-CD3 scFv linked to an affinity matured T cell receptor that recognizes an HLA-presented peptide. In embodiments, the dock-and-lock (DNL) method can be used to generate bispecific antibody molecules with higher valency. Also, fusion to an albumin-binding protein or human serum albumin can extend the serum half-life of the antibody fragment. See the same reference.

[0196] In embodiments, chemical conjugation, for example, chemical conjugation of an antibody and / or an antibody fragment, can be used to create the BsAb molecule. See the same reference. Exemplary bispecific antibody conjugates include the CovX body format, in which a low molecular weight drug is site-specifically conjugated to each Fab arm or a single reactive lysine in the antibody or fragment thereof. In embodiments, the conjugation improves the serum half-life of the low molecular weight drug. An exemplary CovX body is CVX-241 (NCT01004822), which comprises an antibody conjugated to two short peptides that inhibit either VEGF or Ang2. See the same reference.

[0197] Antibody molecules can be produced, for example, by recombinant expression of at least one or more components in a host system. Exemplary host systems include eukaryotic cells (e.g., mammalian cells, e.g., CHO cells, or insect cells, e.g., SF9 or S2 cells) and prokaryotic cells (e.g., E. coli). Bispecific antibody molecules can be produced by separate expression of the components in different host cells and subsequent purification / assembly. Alternatively, antibody molecules can be produced by expression of the components in a single host cell. Purification of bispecific antibody molecules can be performed by various methods, such as affinity chromatography, for example, using protein A and sequential pH elution. In other embodiments, affinity tags, such as histidine-containing tags, myc tags, or streptavidin tags, can be used for purification.

[0198] CDR grafted scaffold In an embodiment, the antibody molecule is a CDR-grafted scaffold domain. In an embodiment, the scaffold domain is based on a fibronectin domain, such as a fibronectin type III domain. The overall fold of the fibronectin type III (Fn3) domain is closely related to that of the smallest functional antibody fragment, the variable domain of the antibody heavy chain. There are three loops at the termini of Fn3, and the positions of the BC, DE, and FG loops roughly correspond to those of CDR1, 2, and 3 of the VH domain of an antibody. Fn3 has no disulfide bonds and thus, unlike antibodies and their fragments, is stable under reducing conditions (see, e.g., WO98 / 56915, WO01 / 64942, WO00 / 34784). The Fn3 domain can be modified or altered (e.g., using the CDRs or hypervariable loops described herein) to select a domain that binds to an antigen / marker / cell, for example, as described herein.

[0199] In an embodiment, the scaffold domain, e.g., the folded domain, is based on a "minibody" scaffold created by deleting three beta strands from the heavy chain variable domain of an antibody, e.g., a monoclonal antibody (see, e.g., Tramontano et al., 1994, J Mol. Recognit. 7:9, and Martin et al., 1994, EMBO J. 13:5303-5309). The "minibody" can be used to present two hypervariable loops. In an embodiment, the scaffold domain is a V-like domain (see, e.g., Coia et al., WO99 / 45110), or a domain derived from tendamistatin, a 74-residue six-stranded beta sheet sandwich held together by two disulfide bonds (see, e.g., McConnell and Hoess, 1995, J Mol. Biol. 250:460). For example, the loops of tendamistatin can be modified or altered (e.g., using CDRs or hypervariable loops) to select a domain that binds to, e.g., the markers / antigens / cells described herein. Another exemplary scaffold domain is a beta-sandwich structure derived from the extracellular domain of CTLA-4 (see, e.g., WO00 / 60070).

[0200] Other exemplary scaffold domains include, but are not limited to, T cell receptors, MHC proteins, extracellular domains (e.g., fibronectin type III repeats, EGF repeats), protease inhibitors (e.g., Kunitz domains, echistatin, and BPTI, etc.), TPR repeats, the trifoil structure, zinc finger domains, DNA-binding proteins, particularly monomeric DNA-binding proteins, RNA-binding proteins, enzymes, e.g., proteases (particularly inactivated proteases), RNases, chaperones, e.g., thioredoxin, and heat shock proteins, as well as intracellular signaling domains (e.g., SH2 and SH3 domains). See, for example, U.S. Patent Application Publication No. 20040009530 and U.S. Patent No. 7,501,121, which are incorporated herein by reference.

[0201] In embodiments, the scaffold domain is evaluated and selected by, for example, one or more of the following criteria: (1) amino acid sequence, (2) sequences of several homologous domains, (3) three-dimensional structure, and / or (4) stability data over a range of pH, temperature, salt, organic solvent, and oxidant concentration. In embodiments, the scaffold domain is a small, stable protein domain, e.g., a protein of less than 100, 70, 50, 40, or 30 amino acids. The domain may contain one or more disulfide bonds or may chelate a metal, e.g., zinc.

[0202] Antibody-based fusions A variety of formats can be generated that contain additional binding entities attached to the N or C terminus of an antibody. These fusions having single-chain or disulfide-stabilized Fv or Fab result in the production of tetravalent molecules having bivalent binding specificity for each antigen. Combinations of scFv and scFab with IgG allow the production of molecules capable of recognizing three or more different antigens.

[0203] Antibody-Fab fusions An antibody-Fab fusion is a bispecific antibody that includes a traditional antibody against a first target and a Fab against a second target fused to the C-terminus of the antibody heavy chain. Generally, the antibody and the Fab have a common light chain. The antibody fusion can be produced by (1) manipulating the DNA sequence of the target fusion and (2) transfecting the target DNA into a suitable host cell to express the fusion protein. As described by Coloma, J et al., (1997) Nature Biotech 15:159, the antibody-scFv fusion may be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv.

[0204] antibody-scFv fusion An antibody-scFv fusion is a bispecific antibody that includes a traditional antibody and an scFv of unique specificity fused to the C-terminus of the antibody heavy chain. The scFv can be fused to the C-terminus directly or through the heavy chain of the scFv through a linker peptide. The antibody fusion can be produced by (1) manipulating the DNA sequence of the target fusion and (2) transfecting the target DNA into a suitable host cell to express the fusion protein. As described by Coloma, J et al., (1997) Nature Biotech 15:159, the antibody-scFv fusion may be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv.

[0205] Variable domain immunoglobulin DVD A related format is the dual variable domain immunoglobulin (DVD) composed of VH and VL domains of a second specificity placed at the N-terminus of the V domain by a shorter linker sequence.

[0206] Other exemplary bispecific antibody formats include, for example, the following: U.S. Patent Application Publication No. 20160114057A1, U.S. Patent Application Publication No. 20130243775A1, U.S. Patent Application Publication No. 20140051833, U.S. Patent Application Publication No. 20130022601, U.S. Patent Application Publication No. 20150017187A1, U.S. Patent Application Publication No. 20120201746A1, U.S. Patent Application Publication No. 20150133638A1, U.S. Patent Application Publication No. 20130266568A1, U.S. Patent Application Publication No. 20160145340A1, WO2015127158A1, U.S. Patent Application Publication No. 20150203591A1, U.S. Patent Application Publication No. 20140322221A1, U.S. Patent Application Publication No. 20130303396A1, U.S. Patent Application Publication No. 20110293613, U.S. Patent Application Publication No. 20130017200A1, U.S. Patent Application Publication No. 20160102135A1, WO2015197598A2, WO2015197582A1, U.S. Patent No. 9359437, U.S. Patent Application Publication No. 20150018529, WO2016115274A1, WO2016087416A1, U.S. Patent Application Publication No. 20080069820A1, U.S. Patent No. 9145588B, U.S. Patent No. 7919257, and U.S. Patent Application Publication No. 20150232560A1. Exemplary bispecific molecules utilizing the full-length antibody-Fab / scFab format include the following: U.S. Patent No. 9382323B2, U.S. Patent Application Publication No. 20140072581A1, U.S. Patent Application Publication No. 20140308285A1, U.S. Patent Application Publication No. 20130165638A1, U.S. Patent Application Publication No. 20130267686A1, U.S. Patent Application Publication No. 20140377269A1, U.S. Patent No. 7741446B2, and WO1995009917A1.Exemplary multispecific molecules that utilize a domain exchange format include, among others: those described in U.S. Patent Application Publication No. 20150315296A1, WO2016087650A1, U.S. Patent Application Publication No. 20160075785A1, WO2016016299A1, U.S. Patent Application Publication No. 20160130347A1, U.S. Patent Application Publication No. 20150166670, U.S. Patent No. 8703132B2, U.S. Patent Application Publication No. 20100316645, U.S. Patent No. 8227577B2, and U.S. Patent Application Publication No. 20130078249.

[0207] Fc-containing entity (minibody) Fc-containing entities, also known as minibodies, can be generated by fusing an scFv to the C-terminus of the constant heavy chain domain 3 (CH3-scFv) and / or the hinge region of an antibody having a different specificity (scFv-hinge-Fc). A trivalent entity having a disulfide-stabilized variable domain fused to the C-terminus of the CH3 domain of IgG (without a peptide linker) can also be produced.

[0208] Fc-containing multispecific molecule In some embodiments, the multispecific molecules disclosed herein include an immunoglobulin constant region (e.g., an Fc region). Exemplary Fc regions can be selected from the heavy chain constant regions of IgG1, IgG2, IgG3, or IgG4, more particularly, the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4.

[0209] In some embodiments, the immunoglobulin chain constant region (e.g., an Fc region) is modified, e.g., mutated, to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function.

[0210] In other embodiments, the boundary between the first and second immunoglobulin chain constant regions (e.g., the first and second Fc regions) is altered, e.g., mutated, to increase or decrease dimerization as compared to, e.g., an unmanipulated boundary, e.g., a naturally occurring boundary. For example, dimerization of the immunoglobulin chain constant regions (e.g., Fc regions) can be enhanced by providing one or more of paired holes and protrusions (“knob-in-hole”), electrostatic interactions, or chain exchange at the Fc boundary of the first and second Fc regions, thereby forming a higher ratio of heteromultimer:homomultimer as compared to, e.g., an unmanipulated boundary.

[0211] In some embodiments, the multispecific molecule comprises paired amino acid substitutions at one or more positions selected from 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 of the Fc region of, e.g., human IgG1. For example, the immunoglobulin chain constant region (e.g., Fc region) can comprise paired amino acid substitutions selected from T366S, L368A, or Y407V (e.g., corresponding to a hole or a cavity), and T366W (e.g., corresponding to a protrusion or a knob).

[0212] In other embodiments, the multifunctional molecule comprises a half-life extender, e.g., human serum albumin or an antibody molecule against human serum albumin.

[0213] Heterodimeric antibody molecules and methods of making A variety of methods for producing bispecific antibodies have been disclosed to address the problem of incorrect heavy chain pairing. Exemplary methods are described below. Exemplary bispecific antibody formats and methods of making said bispecific antibodies are also disclosed, for example, in Speiss et al., Molecular Immunology 67 (2015) pages 95 - 106, and Klein et al., mAb 4:6, pages 653 - 663, November / December 2012 (the respective contents of which are hereby incorporated by reference herein).

[0214] Heterodimerizing bispecific antibodies are based on the native IgG structure, with two binding arms recognizing different antigens. IgG-derived formats that allow defined monovalent (and simultaneous) antigen binding are generated by forced heavy chain heterodimerization combined with techniques that minimize light chain mispairing (e.g., common light chain). Forced heavy chain heterodimerization can be obtained, for example, using the knob-in-hole or strand exchange engineering domain (SEED).

[0215] Knob-in-hole The knob-in-hole described in U.S. Patent No. 5,731,116, U.S. Patent No. 7,476,724 and Ridgway, J et al., (1996) Prot. Engineering 9(7):617 - 621 generally involves (1) mutating the CH3 domain of one or both antibodies to promote heterodimerization, and (2) combining the mutated antibodies under conditions that promote heterodimerization. The "knob" or "protrusion" is typically created by replacing a small amino acid in the parental antibody with a larger amino acid (e.g., T366Y or T366W), and the "hole" or "pore" is created by replacing a larger residue in the parental antibody with a smaller amino acid (e.g., Y407T, T366S, L368A and / or Y407V).

[0216] For bispecific antibodies containing an Fc domain, the introduction of specific mutations into the constant region of the heavy chain can be utilized to promote the correct heterodimerization of the Fc portion. Some such techniques are reviewed in Klein et al. (mAb (2012) 4:6, pages 1-11) (the content of which is hereby incorporated by reference in its entirety). These techniques include the "knob-into-hole" (KiH) approach involving the introduction of a bulky residue into one of the CH3 domains of one of the antibody heavy chains. This bulky residue fits into a complementary "hole" in the other CH3 domain of the paired heavy chain, thereby promoting the correct pairing of the heavy chains (see, for example, U.S. Patent No. 7,642,228).

[0217] Exemplary KiH mutations include S354C, T366W in the "knob" heavy chain and Y349C, T366S, L368A, Y407V in the "hole" heavy chain. Other exemplary KiH mutations are provided in Table 1, along with stabilizing Fc cysteine mutations as needed.

[0218]

Table 1

[0219] Other Fc mutations have been provided by Igawa and Tsunoda, who identified three negatively charged residues in the CH3 domain of one chain that pair with three positively charged residues in the CH3 domain of the other chain. These specific charged residue pairs are E356-K439, E357-K370, D399-K409 and their reverse. In addition to the following three mutations in chain A: E356K, E357K and D399K, alone or in combination with newly identified disulfide bridges, by introducing at least two of K370E, K409D, K439E in chain B, they were able to act favorably on very efficient heterodimerization while simultaneously suppressing homodimerization (Martens T et al., "A novel one-armed antic-Met antibody inhibits glioblastoma growth in vivo.", Clin Cancer Res 2006;12:6144-52; PMID:17062691). Xencor defined 41 variant pairs based on a combination of structural calculations and sequence information, which were then screened for maximum heterodimerization to define the combination of S364H, F405A (HA) on chain A and Y349T, T394F (TF) on chain B (Moore GL et al., "A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens.", MAbs 2011;3:546-57; PMID:22123055).

[0220] Other exemplary Fc mutations for promoting heterodimerization of bispecific antibodies include the following references (the content of each of which is hereby incorporated by reference into this specification): WO2016071377A1, US Patent Application Publication No. 20140079689A1, US Patent Application Publication No. 20160194389A1, US Patent Application Publication No. 20160257763, WO2016071376A2, WO2015107026A1, WO2015107025A1, WO2015107015A1, US Patent Application Publication No. 20150353636A1, US Patent Application Publication No. 20140199294A1, US Patent No. 7750128B2, US Patent Application Publication No. 20160229915A1, US Patent Application Publication No. 20150344570A1, US Patent No. 8003774A1, US Patent Application Publication No. 20150337049A1, US Patent Application Publication No. 20150175707A1, US Patent Application Publication No. 20140242075A1, US Patent Application Publication No. 20130195849A1, US Patent Application Publication No. 20120149876A1, US Patent Application Publication No. 20140200331A1, US Patent No. 9309311B2, US Patent No. 8586713, US Patent Application Publication No. 20140037621A1, US Patent Application Publication No. 20130178605A1, US Patent Application Publication No. 20140363426A1, US Patent Application Publication No. 20140051835A1, and US Patent Application Publication No. 20110054151A1.

[0221] Stabilizing cysteine mutations have also been used in combination with KiH and other Fc heterodimerization promoting variants (see, for example, US Patent No. 7183076). Other exemplary cysteine modifications include, for example, those disclosed in US Patent Application Publication No. 20140348839A1, US Patent No. 7855275B2, and US Patent No. 9000130B2.

[0222] Chain Exchange Operation Domain (SEED) There is known a heterodimeric Fc platform that supports the design of bispecific and asymmetric fusion proteins by engineering a lock-exchange operation domain (SEED) C(H)3 heterodimer. These derivatives of the human IgG and IgA C(H)3 domains create complementary human SEED C(H)3 heterodimers composed of alternating segments of the human IgA and IgG C(H)3 sequences. The resulting pairs of SEED C(H)3 domains preferentially associate to form heterodimers when expressed in mammalian cells. SEED body (Sb) fusion proteins may be genetically linked to one or more fusion partners and consist of [IgG1 hinge]-C(H)2-[SEED C(H)3] (see, for example, Davis JH et al., “SEEDbodies: fusion proteins based on strand exchange engineered domain (SEED) CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies.”, Protein Eng Des Sel 2010;23:195-202; PMID:20299542 and U.S. Patent No. 8,871,912, the contents of each of which are hereby incorporated by reference herein).

[0223] Duobody The "duobody" technology for producing bispecific antibodies with correct heavy chain pairing is known. The duobody technology involves three basic steps for generating stable bispecific human IgG1 antibodies in a post-production exchange reaction. In the first step, two IgG1s each containing a single matched mutation in the third constant (CH3) domain are produced separately using a standard mammalian recombinant cell line. These IgG1 antibodies are then purified according to standard methods for recovery and purification. After production and purification (post-production), the two antibodies are recombined under controlled laboratory conditions to yield a bispecific antibody product with a very high yield (typically >95%) (see, for example, Labrijn et al., PNAS 2013;110(13):5145-5150 and Labrijn et al., Nature Protocols 2014;9(10):2450-63, the contents of each of which are hereby incorporated by reference herein).

[0224] Electrostatic interaction Methods for making multispecific antibodies using CH3 amino acid changes with charged amino acids such that homodimer formation is electrostatically disfavored are disclosed. EP1870459 and WO2009089004 describe other strategies that work favorably for heterodimer formation upon co-expression of different antibody domains in host cells. In these methods, the heavy chain constant domain 3 (CH3), one or more residues that make up the CH3-CH3 boundary in both CH3 domains, are replaced with charged amino acids such that homodimer formation is electrostatically disfavored and heterodimerization is electrostatically favored. Additional methods for making multispecific molecules using electrostatic interactions are described in references including US Patent Application Publication No. 20100015133, US Patent No. 8592562B2, US Patent No. 9200060B2, US Patent Application Publication No. 20140154254A1, and US Patent No. 9358286A1, the contents of each of which are hereby incorporated by reference herein).

[0225] Common light chain Light chain mispairing needs to be avoided to generate a homogeneous preparation of bispecific IgG. One way to achieve this is through the use of the common light chain principle, i.e., by combining two binders that share one light chain but still have separate specificities. An exemplary method for enhancing the formation of the desired bispecific antibody from a mixture of monomers is by providing a common variable light chain for interaction with each of the heteromeric variable heavy chain regions of the bispecific antibody. Compositions of bispecific antibodies having a common light chain and methods for their production are disclosed, for example, in U.S. Patent No. 7,183,076 B2, U.S. Patent Application Publication No. 2011 / 0177073 A1, EP 2847231 A1, WO 2016 / 079081 A1, and EP 3055329 A1 (the contents of each of which are hereby incorporated by reference herein).

[0226] CrossMab Another option for reducing light-chain mispairing is CrossMab technology, which avoids non-specific L-chain mispairing by exchanging the CH1 and CL domains in the Fab of one half of a bispecific antibody. Such crossover variants retain binding specificity and affinity, but the two arms are made different so that L-chain mispairing is prevented. (As reviewed by Klein et al., supra) CrossMab technology involves domain swapping between the heavy and light chains to facilitate the formation of correct pairings. Briefly, a two-step modification process is applied to construct a bispecific IgG-like CrossMab antibody that can bind to two antigens by using two separate light-chain-heavy-chain pairs. First, the dimerization interface is engineered into the C-terminus of each heavy chain using a heterodimerization approach, such as the knob-into-hole (KiH) technology, to ensure that only heterodimers of two separate heavy chains from one antibody (e.g., antibody A) and a second antibody (e.g., antibody B) are efficiently formed. Next, the constant heavy chain 1 (CH1) and constant light chain (CL) domains of one antibody (antibody A) are exchanged while keeping the variable heavy chain (VH) and variable light chain (VL) domains consistent. The exchange of the CH1 and CL domains ensures that only the desired bispecific CrossMab is efficiently formed because the modified antibody (antibody A) light chain dimerizes efficiently only with the modified antibody (antibody A) heavy chain and the unmodified antibody (antibody B) light chain dimerizes efficiently only with the unmodified antibody (antibody B) heavy chain (see, for example, Cain, C., SciBX 4(28); doi:10.1038 / scibx.2011.783, the content of which is hereby incorporated by reference herein).

[0227] Common heavy chain Exemplary methods for enhancing the formation of a desired bispecific antibody from a mixture of monomers include providing a common variable heavy chain for interacting with each of the heteromeric variable light chain regions of the bispecific antibody. Compositions of bispecific antibodies having a common heavy chain and methods for their production are disclosed, for example, in U.S. Patent Application Publication No. 20120184716, U.S. Patent Application Publication No. 20130317200, and U.S. Patent Application Publication No. 20160264685A1, the contents of each of which are hereby incorporated by reference herein.

[0228] Amino acid modifications Alternative compositions of multispecific antibodies having correct light chain pairing and methods for their production include various amino acid modifications. For example, Zymeworks describes heterodimers having one or more amino acid modifications in the CH1 and / or CL domains, one or more amino acid modifications in the VH and / or VL domains, or combinations thereof, which are part of the interface between the light and heavy chains and create preferential pairing of each heavy chain with a desired light chain, such that when two heavy chains and two light chains of a heterodimer pair are co-expressed in a cell, the heavy chain of the first heterodimer preferentially pairs with one of the light chains over the others (see, for example, WO2015181805). Other exemplary methods are described in WO2016026943 (Argen-X), U.S. Patent Application Publication No. 20150211001, U.S. Patent Application Publication No. 20140072581A1, U.S. Patent Application Publication No. 20160039947A1, and U.S. Patent Application Publication No. 20150368352.

[0229] Lambda / kappa format Multispecific molecules (e.g., multispecific antibody molecules) comprising lambda light chain polypeptides and kappa light chain polypeptides can be used to enable heterodimerization. Methods for generating bispecific antibody molecules comprising lambda light chain polypeptides and kappa light chain polypeptides are disclosed in PCT Publication No. WO2018057955 (corresponding to PCT / US17 / 53053, filed September 22, 2017), which is hereby incorporated by reference in its entirety.

[0230] In embodiments, the multispecific molecule comprises a multispecific antibody molecule, e.g., an antibody molecule comprising two binding specificities, e.g., a bispecific antibody molecule. The multispecific antibody molecule lambda light chain polypeptide 1 (LLCP1) specific for a first epitope, heavy chain polypeptide 1 (HCP1) specific for the first epitope, kappa light chain polypeptide 2 (KLCP2) specific for a second epitope, and heavy chain polypeptide 2 (HCP2) specific for the second epitope is included.

[0231] "Lambda light chain polypeptide 1 (LLCP1)", as the term is used herein, refers to a polypeptide comprising a light chain (LC) sequence sufficient to mediate specific binding to its epitope and complex with HCP1 when combined with its cognate heavy chain variable region. In one embodiment, it includes all or a fragment of the CH1 region. In one embodiment, LLCP1 comprises LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3, FR4, and CH1, or sequences therefrom sufficient to mediate specific binding to its epitope and complex with HCP1. LLCP1, together with its HCP1, provides specificity for the first epitope (KLCP2, together with its HCP2, provides specificity for the second epitope). As described elsewhere herein, LLCP1 has a higher affinity for HCP1 than for HCP2.

[0232] "Kappa light chain polypeptide 2 (KLCP2)", as the term is used herein, refers to a polypeptide that, when combined with its cognate heavy chain variable region, contains a light chain (LC) sequence sufficient to mediate specific binding to its epitope and to complex with HCP2. In one embodiment, it includes all or a fragment of the CH1 region. In one embodiment, KLCP2 includes LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3, FR4, and CH1, or sequences therefrom sufficient to mediate specific binding to its epitope and to complex with HCP2. KLCP2, together with its HCP2, provides specificity for a second epitope (LLCP1, together with its HCP1, provides specificity for a first epitope).

[0233] "Heavy chain polypeptide 1 (HCP1)", as the term is used herein, refers to a polypeptide that, when combined with its cognate LLCP1, contains a heavy chain (HC) sequence sufficient to mediate specific binding to its epitope and to complex with HCP1, e.g., an HC variable region sequence. In one embodiment, it includes all or a fragment of the CH1 region. In one embodiment, it includes all or a fragment of the CH2 and / or CH3 regions. In one embodiment, HCP1 includes HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or (i) sequences therefrom sufficient to mediate specific binding to its epitope and to complex with LLCP1, (ii) to preferentially complex with LLCP1 as contrasted with KLCP2 as described herein, and (iii) to preferentially complex with HCP2 as contrasted with another molecule of HCP1 as described herein. HCP1, together with its LLCP1, provides specificity for a first epitope (KLCP2, together with its HCP2, provides specificity for a second epitope).

[0234] "Heavy chain polypeptide 2 (HCP2)", as the term is used herein, refers to a polypeptide comprising a heavy chain (HC) sequence, e.g., an HC variable region sequence, that is sufficient to mediate specific binding to its epitope and complex with HCP1 when combined with cognate LLCP1. In one embodiment, it comprises all or a fragment of the CH1 region. In one embodiment, it comprises all or a fragment of the CH2 and / or CH3 regions. In one embodiment, HCP1 comprises HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or (i) sequences sufficient to mediate specific binding to its epitope, complex with KLCP2, (ii) preferentially complex with KLCP2 as contrasted with LLCP1 as described herein, and (iii) preferentially complex with HCP1 as contrasted with another molecule of HCP2 as described herein. HCP2, together with its KLCP2, provides specificity for a second epitope (LLCP1, together with its HCP1, provides specificity for a first epitope).

[0235] In some embodiments of the multispecific antibody molecules disclosed herein, LLCP1 has a higher affinity for HCP1 than for HCP2, and / or KLCP2 has a higher affinity for HCP2 than for HCP1.

[0236] In embodiments, the affinity of LLCP1 for HCP1 is sufficiently higher than its affinity for HCP2 such that, under preselected conditions, e.g., in an aqueous buffer at pH 7, e.g., in physiological saline at pH 7, or under physiological conditions, at least 75, 80, 90, 95, 98, 99, 99.5, or 99.9% of the multispecific antibody molecules have LLCP1 complexed with or bound to HCP1.

[0237] In some embodiments of the multispecific antibody molecules disclosed herein, HCP1 has a higher affinity for HCP2 than for a second molecule of HCP1, and / or HCP2 has a higher affinity for HCP1 than for a second molecule of HCP2.

[0238] In embodiments, the affinity of HCP1 for HCP2 is sufficiently higher than its affinity for a second molecule of HCP1 such that, under preselected conditions, e.g., in an aqueous buffer at pH 7, e.g., in physiological saline at pH 7, or under physiological conditions, at least 75%, 80, 90, 95, 98, 99, 99.5 or 99.9% of the multispecific antibody molecules have HCP1 complexed with or bound to HCP2.

[0239] In another aspect, methods of making or producing multispecific antibody molecules are disclosed herein. The methods comprise providing (i)-(iv) under conditions such that (i) a first heavy chain polypeptide (e.g., a heavy chain polypeptide comprising one, two, three or all of a first heavy chain variable region (first VH), first CH1, first heavy chain constant region (e.g., first CH2, first CH3, or both)), (ii) a second heavy chain polypeptide (e.g., a heavy chain polypeptide comprising one, two, three or all of a second heavy chain variable region (second VH), second CH1, second heavy chain constant region (e.g., second CH2, second CH3, or both)), (iii) a lambda chain polypeptide (e.g., a lambda light chain variable region (VLλ), lambda light chain constant chain (VLλ), or both) that preferentially associates with the first heavy chain polypeptide (e.g., first VH), and (iv) a kappa chain polypeptide (e.g., a lambda light chain variable region (VLκ), lambda light chain constant chain (VLκ), or both) that preferentially associates with the second heavy chain polypeptide (e.g., second VH). are included.

[0240] In an embodiment, the first and second heavy chain polypeptides form an Fc boundary that enhances heterodimerization. In an embodiment, (i)-(iv) (e.g., nucleic acids encoding (i)-(iv)) are introduced into a single cell, e.g., a single mammalian cell, e.g., a CHO cell. In an embodiment, (i)-(iv) are expressed in the cell.

[0241] In an embodiment, (i)-(iv) (e.g., nucleic acids encoding (i)-(iv)) are introduced into different cells, e.g., different mammalian cells, e.g., two or more CHO cells. In an embodiment, (i)-(iv) are expressed in the cells.

[0242] In one embodiment, the method further comprises purifying the cell-expressed antibody molecule, e.g., using lambda and / or kappa specific purification, e.g., affinity chromatography.

[0243] In an embodiment, the method further comprises evaluating the cell-expressed multispecific antibody molecule. For example, the purified cell-expressed multispecific antibody molecule can be analyzed by techniques known in the art, including mass spectrometry. In one embodiment, the purified cell-expressed antibody molecule is digested with a cleavage, e.g., papain, to yield Fab portions and evaluated using mass spectrometry.

[0244] In an embodiment, the method produces correctly paired kappa / lambda multispecific, e.g., bispecific, antibody molecules in high yields, e.g., at least 75%, 80, 90, 95, 98, 99, 99.5 or 99.9%.

[0245] In other embodiments, the multispecific, e.g., bispecific, antibody molecules are (i) A first heavy chain polypeptide (HCP1) (e.g., a heavy chain polypeptide comprising one, two, three, or all of a first heavy chain variable region (first VH), a first CH1, a first heavy chain constant region (e.g., a first CH2, a first CH3, or both)), which binds, for example, to a first epitope, HCP1, (ii) A second heavy chain polypeptide (HCP2) (e.g., a heavy chain polypeptide comprising one, two, three, or all of a second heavy chain variable region (second VH), a second CH1, a second heavy chain constant region (e.g., a second CH2, a second CH3, or both)), which binds, for example, to a second epitope, HCP2, (iii) A lambda light chain polypeptide (LLCP1) (e.g., a lambda light chain variable region (VLl), a lambda light chain constant chain (VLl), or both) that preferentially associates with a first heavy chain polypeptide (e.g., a first VH), which binds, for example, to a first epitope, LLCP1, and (iv) A kappa light chain polypeptide (KLCP2) (e.g., a lambda light chain variable region (VLk), a lambda light chain constant chain (VLk), or both) that preferentially associates with a second heavy chain polypeptide (e.g., a second VH), which binds, for example, to a second epitope, KLCP2 comprising.

[0246] In embodiments, the first and second heavy chain polypeptides form an Fc boundary that enhances heterodimerization. In embodiments, the multispecific antibody molecule comprises a first binding specificity comprising a hybrid VLl-CLl heterodimerized to a first heavy chain variable region connected to an Fc constant, CH2-CH3 domain (having a knob modification), and a second binding specificity comprising a hybrid VLk-CLk heterodimerized to a second heavy chain variable region connected to an Fc constant, CH2-CH3 domain (having a hole modification). Calreticulin-targeting antigen-binding domain The present disclosure provides, inter alia, multispecific (e.g., bispecific, trispecific, tetraspecific) or multifunctional molecules that include, for example, are engineered to contain, one or more antigen-binding domains that bind to calreticulin, such as a wild-type calreticulin protein or a calreticulin mutant protein. In some embodiments, the multifunctional molecule binds to a wild-type calreticulin protein and a calreticulin mutant protein having a similar affinity. In some embodiments, the multifunctional molecule preferentially binds to a calreticulin mutant protein over a wild-type calreticulin protein.

[0247] An exemplary wild-type human calreticulin is shown as SEQ ID NO: 6285. EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGLQTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNSLDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKDDEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASKPEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEPPVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGVLGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQDEEQRLKEEEEDKKRKEEEEAEDKEDDEDKDEDEEDEEDKEEDEEEDVPGQAKDEL (SEQ ID NO: 6285)

[0248] Calreticulin mutant proteins have been identified and found to be associated with myeloid cancer, see, for example, Nangalia et al., N Engl J Med. December 19, 2013; 369(25):2391-2405, Klampfl et al., N Engl J Med. December 19, 2013; 369(25):2379-90 and US Patent International Publication No. 20170269092 (the entire contents of which are hereby incorporated by reference herein). The mutant calreticulin has a frameshift in exon 9 of the coding sequence of wild-type calreticulin, resulting in substitution of negatively charged amino acids at the C-terminus of wild-type calreticulin mainly by positively charged polypeptides. Table 2 discloses the full-length amino acid sequences of 36 calreticulin mutant proteins. Table 3 discloses the C-terminal amino acid sequences of 36 calreticulin mutant proteins. All 36 calreticulin mutant proteins contain the amino acid sequence of RRKMSPARPRTSCREACLQGWTEA (SEQ ID NO: 6286).

[0249] The main mutations of calreticulin are type 1 and type 2 mutations (see Tables 2 and 3). The type 1 mutation is a 52bp deletion (c.1092_1143del), while the type 2 mutation is a 5bp insertion (c.1154_1155insTTGTC).

[0250]

Table 2-1

[0251]

Table 2-2

[0252]

Table 2-3

[0253]

Table 2-4

[0254]

Table 2-5

[0255]

Table 2-6

[0256]

Table 2-7

[0257]

Table 3-1

[0258]

Table 3-2

[0259] In some embodiments, the carretcurin-targeted antigen-binding domain comprises any CDR amino acid sequence, framework region (FWR) amino acid sequence, or variable region amino acid sequence disclosed in Tables 4-7.

[0260] In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising, for example, one, two, or three CDRs derived from the mouse 16B11.1 antibody described in Table 4. For example, in some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising the heavy chain complementarity-determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6358 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 6360 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 227 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions). In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising the heavy chain complementarity-determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6358 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 6360 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 227 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions). In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising the heavy chain complementarity-determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6358 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 6360 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 227 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions).In some embodiments, the anti-calretinin targeting antigen-binding domain comprises a VH comprising the VH complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6358 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 6360 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 227 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions).

[0261] Alternatively, or in combination with a calreticulin-targeted antigen-binding domain comprising a VH comprising one, two, or three CDRs derived from the mouse 16B11.1 antibody, the calreticulin-targeted antigen-binding domain comprises, for example, a VL comprising one, two, or three CDRs derived from the mouse 16B11.1 antibody described in Table 4. For example, in some embodiments, the calreticulin-targeted antigen-binding domain comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 251 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 246 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or a VLCDR3 amino acid sequence of SEQ ID NO: 248 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 251, the VLCDR2 amino acid sequence of SEQ ID NO: 253, and the VLCDR3 amino acid sequence of SEQ ID NO: 255. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 258, the VLCDR2 amino acid sequence of SEQ ID NO: 260, and the VLCDR3 amino acid sequence of SEQ ID NO: 262. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 265, the VLCDR2 amino acid sequence of SEQ ID NO: 267, and the VLCDR3 amino acid sequence of SEQ ID NO: 269. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 272, the VLCDR2 amino acid sequence of SEQ ID NO: 274, and the VLCDR3 amino acid sequence of SEQ ID NO: 276. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 279, the VLCDR2 amino acid sequence of SEQ ID NO: 281, and the VLCDR3 amino acid sequence of SEQ ID NO: 283.

[0262] In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6253 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 6254 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 6255 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions). In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising the VHCDR1 amino acid sequence of SEQ ID NO: 6253, the VHCDR2 amino acid sequence of SEQ ID NO: 6254, and / or the VHCDR3 amino acid sequence of SEQ ID NO: 6255.

[0263] In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VL comprising the light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6259 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), the VLCDR2 amino acid sequence of SEQ ID NO: 6260 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions), and / or the VLCDR3 amino acid sequence of SEQ ID NO: 6261 (or a sequence having one, two, three, or four or fewer mutations, such as substitutions, additions, or deletions). In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 6259, the VLCDR2 amino acid sequence of SEQ ID NO: 6260, and the VLCDR3 amino acid sequence of SEQ ID NO: 6261.

[0264] In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising one, two, three, or four framework regions derived from, for example, the humanized 16B11.1 antibody described in Table 4. For example, in some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6357, the VHFWR2 amino acid sequence of SEQ ID NO: 6359, the VHFWR3 amino acid sequence of SEQ ID NO: 6361, and / or the VHFWR4 amino acid sequence of SEQ ID NO: 6273. In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6362, the VHFWR2 amino acid sequence of SEQ ID NO: 6363, the VHFWR3 amino acid sequence of SEQ ID NO: 226, and / or the VHFWR4 amino acid sequence of SEQ ID NO: 228. In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 229, the VHFWR2 amino acid sequence of SEQ ID NO: 6369, the VHFWR3 amino acid sequence of SEQ ID NO: 6371, and / or the VHFWR4 amino acid sequence of SEQ ID NO: 228. In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6373, the VHFWR2 amino acid sequence of SEQ ID NO: 6369, the VHFWR3 amino acid sequence of SEQ ID NO: 6371, and / or the VHFWR4 amino acid sequence of SEQ ID NO: 228.

[0265] In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6374, the VLFWR2 amino acid sequence of SEQ ID NO: 6375, the VLFWR3 amino acid sequence of SEQ ID NO: 247, and / or the VLFWR4 amino acid sequence of SEQ ID NO: 249. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 250, the VLFWR2 amino acid sequence of SEQ ID NO: 252, the VLFWR3 amino acid sequence of SEQ ID NO: 254, and / or the VLFWR4 amino acid sequence of SEQ ID NO: 256. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 257, the VLFWR2 amino acid sequence of SEQ ID NO: 259, the VLFWR3 amino acid sequence of SEQ ID NO: 261, and / or the VLFWR4 amino acid sequence of SEQ ID NO: 263. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 264, the VLFWR2 amino acid sequence of SEQ ID NO: 266, the VLFWR3 amino acid sequence of SEQ ID NO: 268, and / or the VLFWR4 amino acid sequence of SEQ ID NO: 270. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 271, the VLFWR2 amino acid sequence of SEQ ID NO: 273, the VLFWR3 amino acid sequence of SEQ ID NO: 275, and / or the VLFWR4 amino acid sequence of SEQ ID NO: 277. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 278, the VLFWR2 amino acid sequence of SEQ ID NO: 280, the VLFWR3 amino acid sequence of SEQ ID NO: 282, and / or the VLFWR4 amino acid sequence of SEQ ID NO: 284.

[0266] In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the variable heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6224, the VHFWR2 amino acid sequence of SEQ ID NO: 6226, the VHFWR3 amino acid sequence of SEQ ID NO: 6228, and / or the VHFWR4 amino acid sequence of SEQ ID NO: 6230. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the variable light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6238, the VLFWR2 amino acid sequence of SEQ ID NO: 6240, the VLFWR3 amino acid sequence of SEQ ID NO: 6242, and / or the VLFWR4 amino acid sequence of SEQ ID NO: 6244. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the VHFWR1 amino acid sequence of SEQ ID NO: 6263 (or a sequence having one, two, three, four, five, or six or fewer mutations, e.g., substitutions, additions, or deletions), the VHFWR2 amino acid sequence of SEQ ID NO: 6264 (or a sequence having one, two, three, four, five, or six or fewer mutations, e.g., substitutions, additions, or deletions), the VHFWR3 amino acid sequence of SEQ ID NO: 6265 (or a sequence having one, two, three, four, five, six, seven, eight, nine, ten, or eleven or fewer mutations, e.g., substitutions, additions, or deletions), and / or the VHFWR4 amino acid sequence of SEQ ID NO: 228. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the VLFWR1 amino acid sequence of SEQ ID NO: 6277 (or a sequence having one, two, or three or fewer mutations, e.g., substitutions, additions, or deletions), the VLFWR2 amino acid sequence of SEQ ID NO: 6278 (or a sequence having one or fewer mutations, e.g., substitutions, additions, or deletions), the VLFWR3 amino acid sequence of SEQ ID NO: 6279 (or a sequence having one or fewer mutations, e.g., substitutions, additions, or deletions), and / or the VLFWR4 amino acid sequence of SEQ ID NO: 6280.In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the VHFWR1 amino acid sequence of SEQ ID NO: 6263, the VHFWR2 amino acid sequence of SEQ ID NO: 6264, the VHFWR3 amino acid sequence of SEQ ID NO: 6265, and / or the VHFWR4 amino acid sequence of SEQ ID NO: 228. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the VLFWR1 amino acid sequence of SEQ ID NO: 6277, the VLFWR2 amino acid sequence of SEQ ID NO: 6278, the VLFWR3 amino acid sequence of SEQ ID NO: 6279, and / or the VLFWR4 amino acid sequence of SEQ ID NO: 6280.

[0267] In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6347 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6347). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6348 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6348). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6349 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6349). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6350 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6350). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6351 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6351). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6352 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6352). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6353 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6353). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6354 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6354).In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6355 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6355). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6356 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6356).

[0268] In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6247 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6247). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6249 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6249). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6247. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6249. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6247 and a VL comprising the amino acid sequence of SEQ ID NO: 6249.

[0269] [Table 4]

[0270] [Table 5]

[0271] [Table 6-1]

[0272]

Table 6-2

[0273]

Table 7-1

[0274]

Table 7-2

[0275] Additional calreticulin-targeting antigen-binding domain In some embodiments, the calreticulin-targeted antigen-binding domain comprises any of the CDR amino acid sequences or variable region amino acid sequences disclosed in Tables 16-19. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6253 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 243 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 6255 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the VHCDR1 amino acid sequence of SEQ ID NO: 6253, the VHCDR2 amino acid sequence of SEQ ID NO: 243, and / or the VHCDR3 amino acid sequence of SEQ ID NO: 6255. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6259 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions), the VLCDR2 amino acid sequence of SEQ ID NO: 6260 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions), and / or the VLCDR3 amino acid sequence of SEQ ID NO: 6261 (or a sequence having one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 6259, the VLCDR2 amino acid sequence of SEQ ID NO: 6260, and the VLCDR3 amino acid sequence of SEQ ID NO: 6261. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 244 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 245 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 244 and / or a VL comprising the amino acid sequence of SEQ ID NO: 245. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372, 234, 235, 236, or 237, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 238, 239, 240, 241, or 242, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 238 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372 and a VL comprising the amino acid sequence of SEQ ID NO: 238. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 234 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 238 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 234 and a VL comprising the amino acid sequence of SEQ ID NO: 238.In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 238 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 and a VL comprising the amino acid sequence of SEQ ID NO: 238. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 238 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 and a VL comprising the amino acid sequence of SEQ ID NO: 238. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 238 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 and a VL comprising the amino acid sequence of SEQ ID NO: 238. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 239 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto).In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372 and a VL comprising the amino acid sequence of SEQ ID NO: 239. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 234 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 239 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 234 and a VL comprising the amino acid sequence of SEQ ID NO: 239. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 239 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 and a VL comprising the amino acid sequence of SEQ ID NO: 239. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 239 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 and a VL comprising the amino acid sequence of SEQ ID NO: 239.In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 239 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 and a VL comprising the amino acid sequence of SEQ ID NO: 239. In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 240 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372 and a VL comprising the amino acid sequence of SEQ ID NO: 240. In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 234 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 240 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 234 and a VL comprising the amino acid sequence of SEQ ID NO: 240. In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 240 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto).In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 and a VL comprising the amino acid sequence of SEQ ID NO: 240. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 240 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain comprises SEQ ID NO: 236. It includes a VH containing an amino acid sequence and a VL containing the amino acid sequence of SEQ ID NO: 240. In some embodiments, the calreticulin-targeted antigen-binding domain includes a VH containing the amino acid sequence of SEQ ID NO: 237 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL containing the amino acid sequence of SEQ ID NO: 240 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain includes a VH containing the amino acid sequence of SEQ ID NO: 237 and a VL containing the amino acid sequence of SEQ ID NO: 240. In some embodiments, the calreticulin-targeted antigen-binding domain includes a VH containing the amino acid sequence of SEQ ID NO: 6372 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL containing the amino acid sequence of SEQ ID NO: 241 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain includes a VH containing the amino acid sequence of SEQ ID NO: 6372 and a VL containing the amino acid sequence of SEQ ID NO: 241. In some embodiments, the calreticulin-targeted antigen-binding domain includes a VH containing the amino acid sequence of SEQ ID NO: 234 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL containing the amino acid sequence of SEQ ID NO: 241 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain includes a VH containing the amino acid sequence of SEQ ID NO: 234 and a VL containing the amino acid sequence of SEQ ID NO: 241.In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 241 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 and a VL comprising the amino acid sequence of SEQ ID NO: 241. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 241 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 and a VL comprising the amino acid sequence of SEQ ID NO: 241. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 241 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 and a VL comprising the amino acid sequence of SEQ ID NO: 241. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 242 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto).In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372 and a VL comprising the amino acid sequence of SEQ ID NO: 242. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 234 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 242 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 234 and a VL comprising the amino acid sequence of SEQ ID NO: 242. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 242 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 and a VL comprising the amino acid sequence of SEQ ID NO: 242. In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 242 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 and a VL comprising the amino acid sequence of SEQ ID NO: 242.In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 242 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 and a VL comprising the amino acid sequence of SEQ ID NO: 242.

[0276] In some embodiments, the calreticulin-targeted antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 and a VL comprising the amino acid sequence of SEQ ID NO: 238.

[0277] [Table 8-1]

[0278] [Table 8-2]

[0279] [Table 9]

[0280] [Table 10]

[0281] [Table 11]

[0282] Immune cell engager The immune cell engagers of the multispecific or multifunctional molecules disclosed herein can mediate binding to immune cells, such as immune effector cells, and / or their activation. In some embodiments, the immune cells are selected from T cells, NK cells, B cells, dendritic cells, or macrophage cells, or combinations thereof. In some embodiments, the immune cell engager is selected from one, two, three, or all of a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager, or combinations thereof. The immune cell engager can be an agonist of the immune system. In some embodiments, the immune cell engager can be an antibody molecule, a ligand molecule (e.g., a ligand further comprising an immunoglobulin constant region, such as an Fc region), a small molecule, a nucleotide molecule. T cell engager The present disclosure provides, inter alia, multispecific (e.g., bispecific, trispecific, tetra-specific) or multifunctional molecules engineered to contain one or more T cell engagers that mediate binding to and / or activation of T cells. Thus, in some embodiments, the T cell engager is selected from antigen-binding domains or ligands that bind to (e.g., in some embodiments, activate) one or more of the variable chain of the beta subunit of the TCR (e.g., TCRβV), CD3, TCRα, TCRβ, TCRγ, TCRζ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2 or CD226. In other embodiments, the T cell engager is selected from antigen-binding domains or ligands that bind to one or more of TCRβV, CD3, TCRα, TCRβ, TCRγ, TCRζ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2 or CD226 and do not activate it. In some embodiments, the T cell engager binds to TCRβV. Human T cell receptor (TCR) complex The T cell receptor (TCR) can be found on the surface of T cells. The TCR recognizes an antigen, such as a peptide, that is presented on the surface of a cell, for example, an antigen-presenting cell, and that binds to, for example, a major histocompatibility complex (MHC) molecule. The TCR is a heterodimeric molecule and can include an alpha chain, a beta chain, a gamma chain, or a delta chain. A TCR that includes an alpha chain and a beta chain is also called TCRαβ. The TCRβ chain consists of the following regions (also known as segments): variable (V), diverse (D), joining (J), and constant (C) (see Mayer G. and Nyland J. (2010) Chapter 10: Major Histocompatibility Complex and T-cell Receptors - Role in Immune Responses. In: Microbiology and Immunology on-line, University of South Carolina School of Medicine). The TCRα chain consists of V, J, and C regions. Somatic recombination of the T cell receptor (TCR) by V (variable), D (diverse), J (joining), and C (constant) regions is a decisive event in T cell development and maturation. TCR gene rearrangement occurs in the thymus.

[0283] The TCR can include a TCR heterodimer that includes an alpha chain and a beta chain, and a receptor complex known as a TCR complex that includes a dimeric signaling molecule, such as a CD3 coreceptor, such as CD3δ / ε and / or CD3γ / ε.

[0284] TCR beta V (TCRβV) The diversity of the immune system enables defense against a vast number of pathogens. Since the size of the germline genome is limited, diversity is achieved not only through the process of V(D)J recombination but also through deletions at the junctions of nucleotides (junctions between V-D segments and D-J segments) and the addition of nucleotides that are pseudorandom and not templated. The TCR beta gene undergoes genetic rearrangement to generate diversity.

[0285] The TCR V beta repertoire varies between individuals and populations due to, for example, seven frequently occurring inactivating polymorphisms in the functional gene segments, and large insertion / deletion related polymorphisms encompassing two V beta gene segments. The present disclosure provides, for example, antibody molecules and fragments thereof that bind, such as specifically bind, to, for example, human TCR beta V chains (TCRβV), such as TCRβV gene families (also called groups), such as TCRβV subfamilies (also called subgroups), as described herein. TCR beta V families and subfamilies are known in the art and are described, for example, in Yassai et al. (2009) Immunogenetics 61(7):493-502; Wei S. and Concannon P. (1994) Human Immunology 41(3):201-206. The antibodies described herein can be recombinant antibodies, such as recombinant non-mouse antibodies, such as recombinant human or humanized antibodies.

[0286] In one aspect, the present disclosure provides anti-TCRβV antibody molecules that bind to human TCRβV, such as a TCRβV family, such as a gene family or variants thereof. In some embodiments, the TCRBV gene family includes one or more subfamilies, such as described herein, such as described in FIG. 3, Table 8A or 8B. In some embodiments, the TCRβV gene family includes the TCRβ V6 subfamily, the TCRβ V10 subfamily, the TCRβ V12 subfamily, the TCRβ V5 subfamily, the TCRβ V7 subfamily, the TCRβ V11 subfamily, the TCRβ V14 subfamily, the TCRβ V16 subfamily, the TCRβ V18 subfamily, the TCRβ V9 subfamily, the TCRβ V13 subfamily, the TCRβ V4 subfamily, the TCRβ V3 subfamily, the TCRβ V2 subfamily, the TCRβ V15 subfamily, the TCRβ V30 subfamily, the TCRβ V19 subfamily, the TCRβ V27 subfamily, the TCRβ V28 subfamily, the TCRβ V24 subfamily, the TCRβ V20 subfamily, the TCRβ V25 subfamily, the TCRβ V29 subfamily, the TCRβ V1 subfamily, the TCRβ V17 subfamily, the TCRβ V21 subfamily, the TCRβ V23 subfamily, or the TCRβ V26 subfamily.

[0287] In some embodiments, the TCRβ V6 subfamily is also known as TCRβ V13.1. In some embodiments, the TCRβ V6 subfamily is TCRβ V6-4 * 01, TCRβ V6-4 * 02, TCRβ V6-9 * 01, TCRβ V6-8 * 01, TCRβ V6-5 * 01, TCRβ V6-6 * 02, TCRβ V6-6 * 01, TCRβ V6-2 * 01, TCRβ V6-3 * 01 or TCRβ V6-1 *01, or variants thereof. In some embodiments, TCRβ V6 is TCRβ V6-4 * 01, or a variant thereof. In some embodiments, TCRβ V6 is TCRβ V6-4 * 02, or a variant thereof. In some embodiments, TCRβ V6 is TCRβ V6-9 * 01, or a variant thereof. In some embodiments, TCRβ V6 is TCRβ V6-8 * 01, or a variant thereof. In some embodiments, TCRβ V6 is TCRβ V6-5 * 01, or a variant thereof. In some embodiments, TCRβ V6 is TCRβ V6-6 * 02, or a variant thereof. In some embodiments, TCRβ V6 is TCRβ V6-6 * 01, or a variant thereof. In some embodiments, TCRβ V6 is TCRβ V6-2 * 01, or a variant thereof. In some embodiments, TCRβ V6 is TCRβ V6-3 * 01, or a variant thereof. In some embodiments, TCRβ V6 is TCRβ V6-1 * 01, or a variant thereof.

[0288] In some embodiments, TCRβ V6 is TCRβ V6-5 * 01, or a variant thereof. In some embodiments, TCRβ V6, e.g., TCRβ V6-5 * 01 is recognized, e.g., bound, by SEQ ID NO: 1 and / or SEQ ID NO: 2. In some embodiments, TCRβ V6, e.g., TCRβ V6-5 * 01 is recognized, e.g., bound, by SEQ ID NO: 9 and / or SEQ ID NO: 10. In some embodiments, TCRβ V6 is recognized, e.g., bound, by SEQ ID NO: 9 and / or SEQ ID NO: 11.

[0289] In some embodiments, the TCRβ V10 subfamily is also known as TCRβ V12. In some embodiments, the TCRβ V10 subfamily is also known as TCRβ V10-1 * 01. TCRβ V10-1 * 02. TCRβ V10-3 * 01 or TCRβ V10-2 * 01, or any variant thereof.

[0290] In some embodiments, the TCRβ V12 subfamily is also known as TCRβ V8.1. In some embodiments, the TCRβ V12 subfamily is also known as TCRβ V12-4. * 01. TCRβ V12-3 * 01, or TCRβ V12-5 * 01, or variants thereof. In some embodiments, TCR β V12 is recognized, e.g., bound, by SEQ ID NO: 15 and / or SEQ ID NO: 16. In some embodiments, TCR β V12 is recognized, e.g., bound, by any one of SEQ ID NOs: 23-25, and / or any one of SEQ ID NOs: 26-30.

[0291] In some embodiments, the TCRβ V5 subfamily is TCRβ V5-5 * 01. TCRβ V5-6 * 01. TCRβ V5-4 * 01. TCRβ V5-8 * 01. TCRβ V5-1 * 01, or a variant thereof.

[0292] In some embodiments, the TCRβ V7 subfamily is TCRβ V7-7 * 01. TCRβ V7-6 * 01. TCRβ V7-8 * 02. TCRβ V7-4 * 01. TCRβ V7-2 * 02. TCRβ V7-2 * 03. TCRβ V7-2 *01, TCRβ V7-3 * 01, TCRβ V7-9 * 03, or TCRβ V7-9 * 01, or includes their variants.

[0293] In some embodiments, the TCRβ V11 subfamily is TCRβ V11-1 * 01, TCRβ V11-2 * 01 or TCRβ V11-3 * 01, or includes their variants.

[0294] In some embodiments, the TCRβ V14 subfamily is TCRβ V14 * 01, or includes its variant. In some embodiments, the TCRβ V16 subfamily is TCRβ V16 * 01, or includes its variant.

[0295] In some embodiments, the TCRβ V18 subfamily is TCRβ V18 * 01, or includes its variant. In some embodiments, the TCRβ V9 subfamily is TCRβ V9 * 01 or TCRβ V9 * 02, or includes its variant.

[0296] In some embodiments, the TCRβ V13 subfamily is TCRβV13 * 01, or includes its variant. In some embodiments, the TCRβ V4 subfamily is TCRβ V4-2 * 01, TCRβ V4-3 * 01, or TCRβ V4-1 * 01, or includes their variants.

[0297] In some embodiments, the TCRβ V3 subfamily is TCRβ V3-1 * 01, or includes its variant. In some embodiments, the TCRβ V2 subfamily comprises TCRβ V2 * 01, or a variant thereof.

[0298] In some embodiments, the TCRβ V15 subfamily comprises TCRβ V15 * 01, or a variant thereof. In some embodiments, the TCRβ V30 subfamily comprises TCRβ V30 * 01, or TCRβ V30 * 02, or a variant thereof.

[0299] In some embodiments, the TCRβ V19 subfamily comprises TCRβ V19 * 01, or TCRβ V19 * 02, or a variant thereof. In some embodiments, the TCRβ V27 subfamily comprises TCRβ V27 * 01, or a variant thereof.

[0300] In some embodiments, the TCRβ V28 subfamily comprises TCRβ V28 * 01, or a variant thereof. In some embodiments, the TCRβ V24 subfamily comprises TCRβ V24-1 * 01, or a variant thereof.

[0301] In some embodiments, the TCRβ V20 subfamily comprises TCRβ V20-1 * 01, or TCRβ V20-1 * 02, or a variant thereof. In some embodiments, the TCRβ V25 subfamily comprises TCRβ V25-1 * 01, or a variant thereof.

[0302] In some embodiments, the TCRβ V29 subfamily comprises TCRβ V29-1 * 01, or a variant thereof.

[0303]

Table 12

[0304]

Table 13

[0305] Anti-TCRβV antibody Despite having low sequence similarity (e.g., low sequence identity among different antibody molecules that recognize different TCRβV subfamilies), a novel class of antibodies that recognize structurally conserved regions, such as domains, on the TCRβV protein and have similar functions (e.g., similar cytokine profiles) is disclosed herein, namely, the anti-TCRβV antibody molecules disclosed herein. Thus, the anti-TCRβV antibody molecules disclosed herein share structure-function relationships.

[0306] In some embodiments, the anti-TCRβV antibody molecules disclosed herein do not recognize, e.g., do not bind to, the interface of the TCRβV:TCR alpha complex. In some embodiments, the anti-TCRβV antibody molecules disclosed herein do not recognize, e.g., do not bind to, the constant region of the TCRβV protein. An exemplary antibody that binds to the constant region of the TCRBV region is JOVI.1 described by Viney et al. (Hybridoma. 1992 Dec;11(6):701-13).

[0307] In some embodiments, the anti-TCRβV antibody molecules disclosed herein do not recognize, e.g., do not bind to, one or more (e.g., all) of the complementarity determining regions (e.g., CDR1, CDR2, and / or CDR3) of the TCRβV protein.

[0308] In some embodiments, the anti-TCRβV antibody molecules disclosed herein bind to (e.g., specifically bind to) the TCRβV region. In some embodiments, the binding of the anti-TCRβV antibody molecules disclosed herein results in a cytokine profile that is different from the cytokine profile of a T cell engager (a "non-TCRβV-binding T cell engager") that binds to a receptor or molecule other than the TCRβV region. In some embodiments, the non-TCRβV-binding T cell engager includes an antibody that binds to a CD3 molecule (e.g., a CD3 epsilon (CD3e) molecule), or a TCR alpha (TCRα) molecule. In some embodiments, the non-TCRβV-binding T cell engager is an OKT3 antibody or an SP34-2 antibody.

[0309] In one aspect, the disclosure provides anti-TCRβV antibody molecules that bind to one or more of human TCRβV, e.g., TCRβV gene families, e.g., TCRβV subfamilies, as described herein, e.g., in FIG. 3, Table 8A, or Table 8B. In some embodiments, the anti-TCRβV antibody molecule binds to one or more TCRβV subfamilies selected from the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily, TCRβ V29 subfamily, TCRβ V1 subfamily, TCRβ V17 subfamily, TCRβ V21 subfamily, TCRβ V23 subfamily, or TCRβ V26 subfamily, or variants thereof.

[0310] In some embodiments, the anti-TCRβV antibody molecule binds to the TCRβ V6 subfamily that includes TCRβ V6-4 * 01, TCRβ V6-4 * 02, TCRβ V6-9 * 01, TCRβ V6-8 * 01, TCRβ V6-5 * 01, TCRβ V6-6 * 02, TCRβ V6-6 * 01, TCRβ V6-2 * 01, TCRβ V6-3 * 01 or TCRβ V6-1 * 01, or binds to the TCRβ V6 subfamily that includes TCRβ V6-5 or a variant thereof. In some embodiments, the TCRβ V6 subfamily includes TCRβ V6-5 * 01, or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-4 * 01, or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-4 * 02, or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-9 * 01, or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-8 * 01, or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-5 * 01, or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-6 * 02, or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-6 * 01, or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-2 * 01, or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-3 * 01, or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-1 * 01, or a variant thereof.

[0311] In some embodiments, the anti-TCRβV antibody molecule binds to the TCRβ V10 subfamily comprising TCRβ V10-1 * 01, TCRβ V10-1 * 02, TCRβ V10-3 * 01 or TCRβ V10-2 * 01, or a variant thereof.

[0312] In some embodiments, the anti-TCRβV antibody molecule binds to the TCRβ V12 subfamily comprising TCRβ V12-4 * 01, TCRβ V12-3 * 01 or TCRβ V12-5 * 01, or a variant thereof.

[0313] In some embodiments, the anti-TCRβV antibody molecule binds to the TCRβ V5 subfamily comprising TCRβ V5-5 * 01, TCRβ V5-6 * 01, TCRβ V5-4 * 01, TCRβ V5-8 * 01, TCRβ V5-1 * 01, or a variant thereof.

[0314] In some embodiments, the anti-TCRβV antibody molecule does not bind to TCRβ V12 or binds to TCRβ V12 with an affinity and / or binding specificity lower (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2, 5, or 10 times lower) than the affinity and / or binding specificity of the 16G8 mouse antibody or its humanized version described in U.S. Patent No. 5,861,155.

[0315] In some embodiments, the anti-TCRβV antibody molecule binds to TCRβ V12 with an affinity and / or binding specificity that is higher (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2, 5, or 10 times higher) than the affinity and / or binding specificity of the 16G8 mouse antibody or its humanized version described in U.S. Patent No. 5,861,155.

[0316] In some embodiments, the anti-TCRβV antibody molecule binds to a TCRβV region other than TCRβ V12 (e.g., a TCRβV region described herein, e.g., the TCRβ V6 subfamily (e.g., TCRβ V6-5 * 01) with an affinity and / or binding specificity that is higher (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2, 5, or 10 times higher) than the affinity and / or binding specificity of the 16G8 mouse antibody or its humanized version described in U.S. Patent No. 5,861,155.

[0317] In some embodiments, the anti-TCRβV antibody molecule does not bind to TCRβ V5-5 * 01 nor to TCRβ V5-1 * 01, or binds to TCRβ V5-5 * 01 or TCRβ V5-1 * 01 with an affinity and / or binding specificity that is lower (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2, 5, or 10 times lower) than the affinity and / or binding specificity of mouse antibody C or its humanized version described in U.S. Patent No. 5,861,155.

[0318] In some embodiments, the anti-TCRβV antibody molecule binds to TCRβ V5-5 with an affinity and / or binding specificity that is higher (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2, 5, or 10 times higher) than the affinity and / or binding specificity of mouse antibody C or its humanized version described in U.S. Patent No. 5,861,155.* 01 or TCRβ V5-1 * binds to 01.

[0319] In some embodiments, the anti-TCRβV antibody molecule has a higher affinity and / or binding specificity (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2, 5, or 10 times higher) for TCRβ V5-5 than the affinity and / or binding specificity of the murine antibody C or its humanized version described in U.S. Patent No. 5,861,155 * 01 or TCRβ V5-1 * TCRβV regions other than 01 (e.g., TCRβV regions described herein, e.g., TCRβ V6 subfamily (e.g., TCRβ V6-5 * 01).

[0320] Anti-TCRβ V6 antibody Thus, in one aspect, the disclosure provides an anti-TCRβV antibody molecule that binds to a TCRβ V6 subfamily comprising, e.g., human TCRβ V6, e.g., TCRβ V6-4 * 01, TCRβ V6-4 * 02, TCRβ V6-9 * 01, TCRβ V6-8 * 01, TCRβ V6-5 * 01, TCRβ V6-6 * 02, TCRβ V6-6 * 01, TCRβ V6-2 * 01, TCRβ V6-3 * 01 or TCRβ V6-1 * 01. Provided are anti-TCRβV antibody molecules that bind to a TCRβ V6 subfamily comprising 01. In some embodiments, the TCRβ V6 subfamily comprises TCRβ V6-5 * 01 or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-4 * 01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-4 * 02, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-9 *01, or a variant thereof. In some embodiments, TCRβ V6 is TCRβ V6-8 * 01, or a variant thereof. In some embodiments, TCRβ V6 is TCRβ V6-5 * 01, or a variant thereof. In some embodiments, TCRβ V6 is TCRβ V6-6 * 02, or a variant thereof. In some embodiments, TCRβ V6 is TCRβ V6-6 * 01, or a variant thereof. In some embodiments, TCRβ V6 is TCRβ V6-2 * 01, or a variant thereof. In some embodiments, TCRβ V6 is TCRβ V6-3 * 01, or a variant thereof. In some embodiments, TCRβ V6 is TCRβ V6-1 * 01, or a variant thereof.

[0321] In some embodiments, TCRβ V6-5 * 01 is encoded by the nucleic acid sequence of SEQ ID NO: 43, or a sequence having 85%, 90%, 95%, 99% or higher identity thereto. SEQ ID NO: 43 ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACTC

[0322] In some embodiments, TCRβ V6-5* 01 comprises the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having 85%, 90%, 95%, 99% or higher identity thereto. SEQ ID NO: 44 MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSY In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule is a non-mouse antibody molecule, e.g., a human or humanized antibody molecule. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule is a human antibody molecule. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule is a humanized antibody molecule.

[0323] In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule is isolated or recombinant. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule comprises at least one antigen-binding region, e.g., a variable region or an antigen-binding fragment thereof, from an antibody selected from any one of the antibodies described herein, e.g., any one of A-H.1 to A-H.68, e.g., A-H.1, A-H.2 or A-H.68, or described in Table 1A, or encoded by a nucleotide sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to the nucleotide sequence in Table 1A or any of the foregoing sequences.

[0324] In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule comprises at least one, two, three or four variable regions from an antibody selected from any one of the antibodies described herein, e.g., any one of A-H.1 to A-H.68, e.g., A-H.1, A-H.2 or A-H.68, or described in Table 1A, or a nucleotide sequence in Table 1A, or an antibody encoded by a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0325] In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule comprises at least one or two heavy chain variable regions from an antibody molecule selected from any one of the antibodies described herein, e.g., any one of A-H.1 to A-H.68, e.g., A-H.1, A-H.2 or A-H.68, or described in Table 1A, or a nucleotide sequence in Table 1A, or an antibody molecule encoded by a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0326] In some embodiments, the anti-TCRβV antibody molecule comprises a heavy chain variable region (VH) having the consensus sequence of SEQ ID NO: 231 or 3290. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) The antibody molecule comprises at least one or two light chain variable regions from an antibody selected from any one of the antibodies described herein, such as any one of A-H.1 to A-H.68, such as A-H.1, A-H.2 or A-H.68, or as described in Table 1A, or the nucleotide sequence in Table 1A, or an antibody encoded by a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0327] In some embodiments, the anti-TCRβV antibody molecule comprises a light chain variable region (VL) having the consensus sequence of SEQ ID NO: 230 or 3289. In some embodiments, the anti-TCRβV antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises a heavy chain constant region of IgG4, such as human IgG4. In yet another embodiment, the anti-TCRβV antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises a heavy chain constant region of IgG1, such as human IgG1. In one embodiment, the heavy chain constant region comprises the amino acid sequence shown in Table 3A, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) thereto.

[0328] In some embodiments, the anti-TCRβV antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises a kappa light chain constant region, such as human kappa light chain constant region. In one embodiment, the light chain constant region comprises the amino acid sequence shown in Table 3A, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) thereto.

[0329] In some embodiments, the anti-TCRβV antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) The antibody molecule comprises at least one, two, or three complementarity-determining regions (CDRs) from the variable heavy chain region (VH) of an antibody encoded by a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any one of the antibodies described herein, such as an antibody selected from any one of A-H.1 to A-H.68, such as A-H.1, A-H.2 or A-H.68, or as described in Table 1A, or the nucleotide sequence in Table 1A, or any of the foregoing sequences.

[0330] In some embodiments, the anti-TCRβV antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises at least one, two, or three CDRs (or all of the CDRs together) from the variable heavy chain region comprising the amino acid sequence shown in Table 1A or encoded by the nucleotide sequence shown in Table 1A. In one embodiment, one or more of the CDRs (or all of the CDRs together) have one, two, three, four, five, six or more changes, such as amino acid substitutions or deletions, compared to the amino acid sequence shown in Table 1A or encoded by the nucleotide sequence shown in Table 1A.

[0331] In some embodiments, the anti-TCRβV antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises at least one, two, or three complementarity-determining regions (CDRs) from the variable light chain region of an antibody encoded by a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any one of the antibodies described herein, such as an antibody selected from any one of A-H.1 to A-H.68, such as A-H.1, A-H.2 or A-H.68, or as described in Table 1A, or the nucleotide sequence in Table 1A, or any of the foregoing sequences.

[0332] In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule comprises at least one, two, or three CDRs (or all of the CDRs together) from the light chain variable region comprising the amino acid sequence shown in Table 1A or encoded by the nucleotide sequence shown in Table 1A. In one embodiment, one or more (or all of the CDRs together) of the CDRs have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, compared to the amino acid sequence shown in Table 1A or encoded by the nucleotide sequence shown in Table 1A.

[0333] In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule comprises at least one, two, three, four, five or six CDRs (or all of the CDRs together) from the heavy and light chain variable regions comprising the amino acid sequence shown in Table 1A or encoded by the nucleotide sequence shown in Table 1A. In one embodiment, one or more (or all of the CDRs together) of the CDRs have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, compared to the amino acid sequence shown in Table 1A or encoded by the nucleotide sequence shown in Table 1A.

[0334] In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) The antibody molecule comprises an antibody described herein, for example, a...

Claims

**Claim 1** (i) a first antigen-binding domain that binds to a calreticulin protein; and (ii) a second antigen-binding domain that binds to a T cell receptor beta variable chain (TCRβV) and activates T cells, wherein the bispecific antibody comprises: the bispecific antibody comprises a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide are discontinuous, the first polypeptide comprises a first member of a dimerization module, and the second polypeptide comprises a second member of the dimerization module; the first antigen-binding domain that binds to the calreticulin protein is linked to the first member; and the second antigen-binding domain that binds to TCRβV is linked to the second member. **Claim 2** wherein the first antigen-binding domain is: a heavy chain variable region (VH) comprising the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO: 6358, the amino acid sequence of VHCDR2 of SEQ ID NO: 6360, and the amino acid sequence of VHCDR3 of SEQ ID NO: 227, and a light chain variable region (VL) comprising the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO: 251, the amino acid sequence of VLCDR2 of SEQ ID NO: 246, and the amino acid sequence of VLCDR3 of SEQ ID NO: 248; a VH comprising the amino acid sequence of VHCDR1 of SEQ ID NO: 6253, the amino acid sequence of VHCDR2 of SEQ ID NO: 6254, and the amino acid sequence of VHCDR3 of SEQ ID NO: 6255, and a VL comprising the amino acid sequence of VLCDR1 of SEQ ID NO: 6259, the amino acid sequence of VLCDR2 of SEQ ID NO: 6260, and the amino acid sequence of VLCDR3 of SEQ ID NO: 6261; a VH comprising the amino acid sequence of VHCDR1 of SEQ ID NO: 6253, the amino acid sequence of VHCDR2 of SEQ ID NO: 243, and the amino acid sequence of VHCDR3 of SEQ ID NO: 6255, and a VL comprising the amino acid sequence of VLCDR1 of SEQ ID NO: 6259, the amino acid sequence of VLCDR2 of SEQ ID NO: 6260, and the amino acid sequence of VLCDR3 of SEQ ID NO: 6261; VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 6256, the VH CDR2 amino acid sequence of SEQ ID NO: 6257, and the VH CDR3 amino acid sequence of SEQ ID NO: 6258, and VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 6259, the VL CDR2 amino acid sequence of SEQ ID NO: 6260, and the VL CDR3 amino acid sequence of SEQ ID NO: 6261; or VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 6256, the VH CDR2 amino acid sequence of SEQ ID NO: 6257, and the VH CDR3 amino acid sequence of SEQ ID NO: 6262, and VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 6259, the VL CDR2 amino acid sequence of SEQ ID NO: 6260, and the VL CDR3 amino acid sequence of SEQ ID NO: 6261 The multispecific antibody according to claim 1, comprising

3. The first antigen-binding domain that binds to the calreticulin protein is (i) (a) SEQ ID NO: 6232, SEQ ID NO: 6234, SEQ ID NO: 6236, and SEQ ID NO: 6230, respectively; (b) SEQ ID NO: 6362, SEQ ID NO: 6363, SEQ ID NO: 226, and SEQ ID NO: 228, respectively; (c) SEQ ID NO: 229, SEQ ID NO: 6369, SEQ ID NO: 6371, and SEQ ID NO: 228, respectively; (d) SEQ ID NO: 6373, SEQ ID NO: 6369, SEQ ID NO: 6371, and SEQ ID NO: 228, respectively; (e) SEQ ID NO: 6263, SEQ ID NO: 6264, SEQ ID NO: 6265, and SEQ ID NO: 228, respectively; (f) SEQ ID NO: 6357, SEQ ID NO: 6359, SEQ ID NO: 6361, and SEQ ID NO: 6273, respectively; (g) SEQ ID NO: 6266, SEQ ID NO: 6267, SEQ ID NO: 6268, and SEQ ID NO: 6269, respectively; (h) SEQ ID NO: 6270, SEQ ID NO: 6271, SEQ ID NO: 6272, and SEQ ID NO: 6273, respectively; (i) SEQ ID NO: 6274, SEQ ID NO: 6275, SEQ ID NO: 6276, and SEQ ID NO: 6273, respectively; or (j) SEQ ID NO: 6362, SEQ ID NO: 6363, SEQ ID NO: 226, and SEQ ID NO: 228, respectively of VH comprising heavy chain framework region 1 (VHFR1), heavy chain framework region 2 (VHFR2), heavy chain framework region 3 (VHFR3), and heavy chain framework region 4 (VHFR4) and (ii) (a) SEQ ID NO: 6238, SEQ ID NO: 6240, SEQ ID NO: 6242, and SEQ ID NO: 6244, respectively; (b) SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, and SEQ ID NO: 256, respectively; (c) SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, and SEQ ID NO: 263, respectively; (d) SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 268, and SEQ ID NO: 270, respectively; (e) SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, and SEQ ID NO: 277, respectively; (f) SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, and SEQ ID NO: 284, respectively; (g) SEQ ID NO: 6277, SEQ ID NO: 6278, SEQ ID NO: 6279, and SEQ ID NO: 6280, respectively; (h) SEQ ID NO: 6374, SEQ ID NO: 6375, SEQ ID NO: 247, and SEQ ID NO: 249, respectively; or (i) SEQ ID NO: 6281, SEQ ID NO: 6282, SEQ ID NO: 6283, and SEQ ID NO: 6284, respectively of a light chain variable region (VL) comprising a light chain framework region 1 (VLFR1), a light chain framework region 2 (VLFR2), a light chain framework region 3 (VLFR3), and a light chain framework region 4 (VLFR4) The multispecific antibody according to claim 2, comprising [

4. ] The multispecific antibody according to claim 2 or 3, wherein the first antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) having a sequence with at least 90% sequence identity to the sequences of SEQ ID NOs: 6247 and 6249, respectively. [

5. ] The multispecific antibody according to claim 2 or 3, wherein the first antigen-binding domain comprises a VH and a VL having a sequence with at least 90% sequence identity to the sequences of SEQ ID NOs: 6248 and 6249, respectively. [

6. ] The multispecific antibody according to claim 2 or 3, wherein the first antigen-binding domain comprises a VH having a sequence with at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 6250, SEQ ID NO: 6372, 234-237, and 244, and a VL having a sequence with at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 6252, 238-242, and 245. [

7. ] The multispecific antibody according to claim 2 or 3, wherein the first antigen-binding domain comprises a VH and a VL having a sequence with at least 90% sequence identity to the sequences of SEQ ID NOs: 6251 and 6252, respectively. [

8. ] The multispecific antibody according to claim 2 or 3, wherein the first antigen-binding domain comprises a VH comprising a sequence having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6347 and 6349-6351, and a VL comprising a sequence having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6348 and 6352-6356.

9. The multispecific antibody according to any one of claims 1-8, wherein the second antigen-binding domain binds to TCRβV12, TCRβV6, or TCRβV5.

10. The multispecific antibody according to any one of claims 1-9, wherein the second antigen-binding domain comprises a VH comprising VHCDR1 of SEQ ID NO: 17, VHCDR2 of SEQ ID NO: 18, and VHCDR3 of SEQ ID NO: 19, and a VL comprising VLCDR1 of SEQ ID NO: 20, VLCDR2 of SEQ ID NO: 21, and VLCDR3 of SEQ ID NO:

22.

11. The multispecific antibody according to any one of claims 1-9, wherein the second antigen-binding domain comprises a VH comprising VHCDR1 of SEQ ID NO: 3, VHCDR2 of SEQ ID NO: 4, and VHCDR3 of SEQ ID NO: 5, and a VL comprising VLCDR1 of SEQ ID NO: 6, VLCDR2 of SEQ ID NO: 7, and VLCDR3 of SEQ ID NO:

8.

12. The multispecific antibody according to any one of claims 1-9, wherein the second antigen-binding domain comprises a VH comprising VHCDR1 of SEQ ID NO: 1315, VHCDR2 of SEQ ID NO: 1316, and VHCDR3 of SEQ ID NO: 1317, and a VL comprising VLCDR1 of SEQ ID NO: 1321, VLCDR2 of SEQ ID NO: 1322, and VLCDR3 of SEQ ID NO: 1323.

13. The multispecific antibody according to any one of claims 1-9, wherein the second antigen-binding domain comprises a VH comprising VHCDR1 of SEQ ID NO: 1298, VHCDR2 of SEQ ID NO: 1299, and VHCDR3 of SEQ ID NO: 1300, and a VL comprising VLCDR1 of SEQ ID NO: 1305, VLCDR2 of SEQ ID NO: 1306, and VLCDR3 of SEQ ID NO: 1307.

14. The multispecific antibody according to any one of claims 1-13, further comprising a cytokine molecule.

15. The multispecific antibody according to any one of claims 1 to 13, further comprising a stromal modification moiety selected from a hyaluronidase molecule, a collagenase molecule, a chondroitinase molecule, a matrix metalloproteinase molecule, a hyaluronan-degrading enzyme, and an antibody molecule against hyaluronic acid.

16. A polynucleotide comprising a sequence encoding the multispecific antibody according to any one of claims 1 to 15.

17. A pharmaceutical composition comprising the multispecific antibody according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier, excipient or stabilizer.

18. Use of a pharmaceutical composition comprising the multispecific antibody according to any one of claims 1 to 15 in the manufacture of a medicament for use in treating cancer in a subject in need of treatment.

19. The use according to claim 18, wherein the cancer is a hematological cancer or a solid tumor cancer.

20. Use of the pharmaceutical composition according to claim 18, wherein the cancer is a myeloproliferative neoplasm.

21. The use of the pharmaceutical composition according to claim 20, wherein the myeloproliferative neoplasm is primary or idiopathic myelofibrosis (MF), essential thrombocythemia (ET), polycythemia vera (PV), or chronic myelogenous leukemia (CML).

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