Multifunctional molecules for binding to calreticulin and uses thereof

Abstract

To provide multifunctional molecules that bind to calreticulin and uses thereof.SOLUTION: Multifunctional molecules that include i) an antigen binding domain that binds to a calreticulin protein; and one, two or all of: (ii) an immune cell engager (e.g., chosen 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 are disclosed. Additionally disclosed are nucleic acids encoding the same, methods of producing the aforesaid molecules, and methods of treating a cancer using the aforesaid molecules.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Myeloproliferative neoplasms (MPNs) are a group of conditions that cause blood cells to grow abnormally in the bone marrow. Common myeloproliferative neoplasms include primary or idiopathic myelofibrosis (MF), essential thrombocytosis (ET), polycythemia vera (PV), and chronic myeloid 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. Given the ongoing need for improved treatment of myeloproliferative neoplasms, such as myelofibrosis, new compositions and treatments that target myeloproliferative neoplasms are highly desirable. Summary of the Invention [Means for solving the problem]

[0003] The present disclosure relates to novel multispecific or multifunctional molecules that include, inter alia, one, two, or all of: (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) a stromal-modifying moiety. The terms "multispecific" and "multifunctional" are used interchangeably herein.

[0004] Without being bound by theory, it is expected that the multispecific or multifunctional molecules disclosed herein will target (e.g., localize, crosslink, 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, e.g., cancer cells expressing a calreticulin protein (e.g., a wild-type or mutant calreticulin protein), and / or alter the tumor stroma, e.g., alter 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 the target cells (e.g., cancer cells), thereby achieving a more effective therapy (e.g., a more effective cancer therapy). Without being bound by theory, it is believed that a targeted, localized immune response against the target cells (e.g., cancer cells) reduces the systemic toxic effects of the multispecific molecules described herein.

[0005] Thus, provided herein, inter alia, are multispecific molecules (e.g., multispecific or multifunctional antibody molecules) comprising the aforementioned portions, nucleic acids encoding same, methods of producing the aforementioned molecules, and methods of treating cancer using the aforementioned molecules.

[0006] Thus, in one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (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 to 10 or 18; The present invention features a multifunctional molecule comprising:

[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 (eg, 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 comprises: (a) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) the VH comprises a VHCDR1 having the amino acid sequence of heavy chain complementarity determining region 1 (VHCDR1) in Table 1A, Table 2A, Table 10A, Table 11A, Table 12A, or Table 13A (or a sequence with 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 1A, Table 2A, Table 10A, Table 11A, Table 12A, or Table 13A (or a sequence with 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 1A, Table 2A, Table 10A, Table 11A, Table 12A, or Table 13A (or a sequence with one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions), (ii) VL has the amino acid sequence of 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, e.g., substitutions, additions, or deletions), VLCDR1 in Table 1A, Table 2A, Table 10A, Table 11A, Table 12A, or Table 13A (or a sequence having one, two, three, or fewer mutations, e.g., substitutions, additions, or deletions), VLCDR2 in Table 1A, Table 2A, Table 10A, Table 11A, Table 12A, or Table 13A (or a sequence having one, two, three, or fewer mutations, e.g., substitutions, additions, or deletions), a heavy chain variable region (VH) and / or a light chain variable region (VL) comprising a VLCDR2 having no more than four mutations, e.g., a sequence having no more than four mutations, e.g., substitutions, additions, or deletions), and / or a VLCDR3 having the amino acid sequence of a VLCDR3 in Table 1A, Table 2A, Table 10A, Table 11A, Table 12A, or Table 13A (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); (b) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) the VH comprises the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 3 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 4 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 5 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or (ii) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 7 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 8 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); (c) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) the VH comprises the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 45 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 46 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 47 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); and / or (ii) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 51 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 52 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 53 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); and / or (d) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) the VH comprises the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 48 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 49 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 50 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or (ii) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 54 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 55 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 56 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); Includes:

[0010] In some embodiments, the second antigen-binding domain comprises: (a) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) the VH comprises an amino acid sequence of a 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 comprises an amino acid sequence of a 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) VH has 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) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VL comprises 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); (b) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) the VH comprises 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) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VL comprises 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); and / or (c) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) the VH comprises 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) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the 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); Includes:

[0011] In some embodiments, the second antigen-binding domain comprises: (a) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) the VH comprises the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 17 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 18 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 19 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or (ii) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 20 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 21 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 22 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); (b) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) the VH comprises the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 57 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 58 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 59 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or (ii) VL has a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 63 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 64 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 65 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions). a heavy chain variable region (VH) and / or a light chain variable region (VL) containing up to four mutations (e.g., sequences with substitutions, additions, or deletions); and / or (c) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) the VH comprises the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 60 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 61 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 62 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or (ii) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 66 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 67 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 68 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); Includes:

[0012] In some embodiments, the second antigen-binding domain comprises: (a) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) the 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) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the 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); and / or (b) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) VH is 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 comprising 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 is 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). Includes:

[0013] In some embodiments, the multifunctional molecule is 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 portion that binds to a TCR (e.g., a TCR Vβ) (e.g., a first scFv that binds to a TCR (e.g., a TCR Vβ)); 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 portion that binds to a TCR (e.g., a TCR Vβ) (e.g., a second scFv that binds to a TCR (e.g., a TCR Vβ)); For example, a fourth polypeptide comprising, from N-terminus to C-terminus, a second VL and a second CL, 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 structure of Figure 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, e.g., the second antigen-binding domain is an antibody molecule or a ligand that binds to (e.g., activates) NKp30.

[0015] In some embodiments, the second antigen-binding domain comprises: (i) a heavy chain variable region (VH) comprising a VHCDR1 having the amino acid sequence of heavy chain complementarity determining region 1 (VHCDR1) of Table 7, Table 9, Table 10, or Table 18 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having the amino acid sequence of VHCDR2 of Table 7, Table 9, Table 10, or Table 18 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 having the amino acid sequence of VHCDR3 of Table 7, Table 9, Table 10, or Table 18 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and or (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) having the 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 having the amino acid sequence of VLCDR2 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 having the amino acid sequence of VLCDR3 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). Includes:

[0016] In some embodiments, the second antigen-binding domain comprises: (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 7313 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6001 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 7315 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); and / or (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 7326 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 7327 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VLCDR3 amino acid sequence of SEQ ID NO: 7329 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions). Includes:

[0017] In some embodiments, the second antigen-binding domain comprises: (i) a VH comprising the amino acid sequence of any one of SEQ ID NOs: 7298 or 7300 to 7304 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 7298 or 7300 to 7304); and / or (ii) a VL comprising the amino acid sequence of any one of SEQ ID NOs: 7299 or 7305 to 7309 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 7299 or 7305 to 7309); Includes:

[0018] In some embodiments, the second antigen-binding domain comprises: (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). Includes:

[0019] In some embodiments, the second antigen-binding domain comprises: (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). Includes:

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

[0021] In some embodiments, the second antigen-binding domain comprises: (1) VHFWR1 having the amino acid sequence of heavy chain framework region 1 (VHFWR1) of Table 7, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, four, five, or six or fewer mutations, e.g., substitutions, additions, or deletions therefrom); VHFWR2 having the amino acid sequence of VHFWR2 of Table 7, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, four, five, or six or fewer mutations, e.g., substitutions, additions, or deletions therefrom); a heavy chain variable region (VH) comprising a VHFWR3 having the amino acid sequence of VHFWR3 in Table 9, Table 10, or Table 18 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom); or a VHFWR4 having the amino acid sequence of VHFWR4 in Table 7, Table 9, Table 10, or Table 18 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom); and / or (2) VLFWR1 having the amino acid sequence of light chain framework region 1 (VLFWR1) of Table 8, 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 therefrom); VLFWR2 having the amino acid sequence of VLFWR2 of Table 8, 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 therefrom). a light chain variable region (VL) comprising 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, e.g., substitutions, additions, or deletions therefrom); 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, e.g., substitutions, additions, or deletions therefrom). Includes:

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

[0023] In some embodiments, the second antigen-binding domain comprises: (i) a VH comprising the amino acid sequence of a VH of 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) an amino acid sequence of a VL 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); Contains VL Includes:

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

[0027] In some embodiments, the multispecific molecule comprises: 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 NKp30 (e.g., a second antibody molecule or ligand that binds NKp30); For example, a fourth polypeptide comprising, from N-terminus to C-terminus, a second VL and a second CL, 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 structure of Figure 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 comprises: a third antigen-binding domain that binds to a second calreticulin protein, e.g., the second calreticulin mutant protein comprises the amino acid sequence of SEQ ID NO: 6285 or 6286; and optionally further comprising: (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 comprises: (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) having the amino acid sequence of VHCDR1 in Table 4, Table 7A, or Table 17 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having the amino acid sequence of VHCDR2 in Table 4, Table 7A, or Table 17 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 having the amino acid sequence of VHCDR3 in Table 4, Table 7A, or Table 17 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); (ii) a light chain variable region (VL) comprising a VHCDR1 having the amino acid sequence of light chain complementarity determining region 1 (VLCDR1) in Table 5, Table 7A, or Table 18 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having the amino acid sequence of VLCDR2 in Table 5, Table 7A, or Table 18 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 having the amino acid sequence of VLCDR3 in Table 5, Table 7A, or Table 18 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); (iii) a VH comprising an amino acid sequence of a 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 an amino acid sequence of a VL in Table 7A or Table 16 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity thereto); (v) an amino acid sequence of heavy chain framework region 1 (VHFWR1) in Table 4 or Table 6 VHFWR1 having the amino acid sequence (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, or 9 or fewer mutations, e.g., substitutions, additions, or deletions) of VHFWR2 in Table 4 or Table 6; VHFWR2 having the amino acid sequence (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, or 9 or fewer mutations, e.g., substitutions, additions, or deletions) of VHFWR3 in Table 4 or Table 6; a VH comprising a VHFWR3 having the amino acid sequence (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, or 9 or fewer mutations, e.g., substitutions, additions, or deletions) of VHFWR3, and / or a VHFWR4 having the amino acid sequence (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, or 9 or fewer mutations, e.g., substitutions, additions, or deletions) of VHFWR4 in Table 4 or Table 6; and / or (vi) VLFWR1 having the amino acid sequence of light chain framework region 1 (VLFWR1) in Table 5 or Table 6 (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, or 9 or fewer mutations, e.g., substitutions, additions, or deletions), VLF having the amino acid sequence of VLFWR2 in Table 5 or Table 6 (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, or 9 or fewer mutations, e.g., substitutions, additions, or deletions). a VLFWR2 having the amino acid sequence of VLFWR3 in Table 5 or Table 6 (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, or 9 or fewer mutations, e.g., substitutions, additions, or deletions); and / or a VLFWR4 having the amino acid sequence of VLFWR4 in Table 5 or Table 6 (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, or 9 or fewer mutations, e.g., substitutions, additions, or deletions). Includes:

[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, e.g., 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 a VH and / or VL sequence listed in Table A or Table 20, for example. In some embodiments, the multifunctional molecule preferentially binds to myeloproliferative neoplastic cells over non-tumor cells, and optionally, the binding of the multifunctional molecule to myeloproliferative neoplastic cells is more than 10, 20, 30, 40, 50 times greater than the binding of the multifunctional molecule to non-tumor cells.

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

[0041] In some embodiments, the multispecific molecule further comprises a linker, eg, 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 helical linker, or a non-helical linker.

[0042] 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.

[0043] In another aspect, the disclosure provides nucleic acid molecules encoding the multifunctional molecules described herein. In another aspect, the disclosure provides vectors, e.g., expression vectors, comprising the nucleic acid molecules described herein.

[0044] In another aspect, the present 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, comprising culturing a host cell described herein under appropriate conditions, e.g., conditions suitable for gene expression and / or homo- or heterodimerization.

[0045] In another aspect, the present disclosure provides a pharmaceutical composition comprising a multifunctional molecule described herein and a pharmaceutically acceptable carrier, excipient, or stabilizer. In another aspect, the present disclosure provides a method of treating cancer, 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 the 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 for the 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 hematological cancer, optionally the cancer is a myeloproliferative neoplasm, such as primary or idiopathic myelofibrosis (MF), essential thrombocytosis (ET), polycythemia vera (PV), or chronic myeloid leukemia (CML), 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 administering a second therapeutic treatment. In some embodiments, the second therapeutic treatment comprises a therapeutic agent (eg, a chemotherapeutic agent, a biologic agent, a hormone therapy), radiation, or surgery.

[0051] In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent or a biologic agent. In another aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: (i) a first antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type or mutant calreticulin protein); (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) cytokine molecules; (c) a stromal-modified portion; or (d) a tumor targeting moiety that binds to a tumor antigen selected from, e.g., G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1. with one, two, or all of the The present invention features a multifunctional molecule (e.g., a polypeptide or nucleic acid encoding the same) comprising:

[0052] In one aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: (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 comprising an immune cell engager (e.g., a T cell engager, e.g., an antigen-binding domain that binds to TCRβV, e.g., as described herein); The present invention features a multifunctional molecule (e.g., a polypeptide or nucleic acid encoding the same) comprising:

[0053] In one aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: (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 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.

[0054] In one aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: (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 comprising an immune cell engager (e.g., a T cell engager, e.g., an antigen-binding domain that binds to a TCRβV, e.g., an anti-TCRβV antibody molecule 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; The present invention features a multifunctional molecule (e.g., a polypeptide or nucleic acid encoding the same) comprising:

[0055] In some embodiments, the multifunctional molecule is a cytokine molecule or a cytokine regulator. Further include anti-inflammatory drugs, eg, as described herein, eg, TGF-β inhibitors. In some embodiments, the multifunctional molecule further comprises an antigen-binding domain that binds to an NK cell engager, e.g., Nkp30, e.g., 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., a wild-type or mutant calreticulin protein) comprises an amino acid sequence selected from SEQ ID NOs: 6285-6312. In some embodiments, the 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 calreticulin protein (e.g., a wild-type or mutant calreticulin protein) is a calreticulin protein (e.g., a wild-type or mutant calreticulin protein) disclosed in Table 2 or 3. In some embodiments, the calreticulin protein (e.g., a wild-type or mutant calreticulin protein) comprises the amino acid sequence of SEQ ID NO: 6287. In some embodiments, the calreticulin protein (e.g., a wild-type or mutant calreticulin protein) comprises the amino acid sequence of SEQ ID NO: 6313. In some embodiments, the calreticulin protein (e.g., a wild-type or mutant calreticulin protein) comprises the amino acid sequence of SEQ ID NO: 6288. In some embodiments, the calreticulin protein (eg, a 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) comprises an amino acid sequence selected from SEQ ID NOs: 6313-6346. The second calreticulin protein (calreticulin protein) is a calreticulin protein disclosed in Table 2 or 3 (e.g., a wild-type or mutant calreticulin protein). 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., a wild-type or mutant calreticulin protein) is a type 1 calreticulin protein (e.g., a wild-type or mutant calreticulin protein), and the second calreticulin protein (e.g., a wild-type or mutant calreticulin protein) is a type 2 calreticulin protein (e.g., a 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 approximately the same affinity (e.g., equal affinity) for the first calreticulin protein (e.g., the mutant calreticulin protein) and the wild-type calreticulin protein.

[0062] In some embodiments, the second antigen-binding domain has approximately the same affinity (eg, equal affinity) for the second calreticulin protein (eg, the 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 for binding between the first antigen-binding domain and the first calreticulin mutant protein is D is the K for binding between the first antigen-binding domain and the wild-type calreticulin protein. DIn 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 a 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 for binding between the second antigen-binding domain and the second calreticulin mutant protein is D is the K for binding of the second antigen-binding domain to the wild-type calreticulin protein. D In some embodiments, the second antigen-binding domain binds to an epitope located within the C-terminus of the second calreticulin mutant protein. In some embodiments, the second antigen-binding domain binds to an epitope located within the amino acid sequence of SEQ ID NO: 6286. In some embodiments, the second antigen-binding domain binds to an epitope located within the amino acid sequence of SEQ ID NO: 6286. In some embodiments, the wild-type calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285.

[0065] In some embodiments, the multifunctional molecule preferentially binds to myeloproliferative neoplastic cells over non-tumor cells. In some embodiments, the binding between the multifunctional molecule and myeloproliferative neoplastic cells is 10, 20, 30, 40, or 50 times greater than the binding between the multifunctional molecule and non-tumor cells. In some embodiments, the myeloproliferative neoplastic cells are selected from myelofibrosis cells, essential thrombocythemia cells, polycythemia vera cells, or chronic myeloid cancer cells. In some embodiments, the myeloproliferative neoplastic cells do not contain a JAK2 V617F mutation. In some embodiments, the myeloproliferative neoplastic cells do not contain an MPL mutation.

[0066] 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: 6253 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 6254 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 6255 (or a sequence having no more than one, two, three, or four mutations, e.g., 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 no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), the VLCDR2 amino acid sequence of SEQ ID NO: 6260 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or the VLCDR3 amino acid sequence of SEQ ID NO: 6261 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions).

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

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

[0069] In some embodiments, the first and / or second antigen-binding domain comprises: (i) a VH comprising a VHCDR1 amino acid sequence of SEQ ID NO: 6253, a VHCDR2 amino acid sequence of SEQ ID NO: 6254, and a VHCDR3 amino acid sequence of SEQ ID NO: 6255; and (ii) 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; Includes:

[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 comprises: (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 Includes:

[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 no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions), the VHFWR2 amino acid sequence of SEQ ID NO: 6264 (or a sequence having no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions), the VHFWR3 amino acid sequence of SEQ ID NO: 6265 (or a sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 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 no more than one, two, or three mutations, e.g., substitutions, additions, or deletions), the VLFWR2 amino acid sequence of SEQ ID NO: 6278 (or a sequence having no more than one mutation, e.g., substitutions, additions, or deletions), the VLFWR3 amino acid sequence of SEQ ID NO: 6279 (or a sequence having no more than one mutation, 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 comprises: (i) a 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); a 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); a VHFWR3 amino acid sequence of SEQ ID NO: 6265 (or a sequence having one, two, three, four, five, or six or fewer mutations, e.g., substitutions, additions, or deletions); a VH comprising the VHFWR4 amino acid sequence of SEQ ID NO: 228, and / or a VHFWR4 sequence having one, seven, eight, nine, ten, or eleven or fewer mutations, e.g., substitutions, additions, or deletions; (ii) a VLFWR1 amino acid sequence of SEQ ID NO: 6277 (or a sequence having no more than one, two, or three mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6278 (or a sequence having no more than one mutation, e.g., substitutions, additions, or deletions), a VLFWR3 amino acid sequence of SEQ ID NO: 6279 (or a sequence having no more than one mutation, e.g., substitutions, additions, or deletions), and / or a VLFWR4 amino acid sequence of SEQ ID NO: 6280. Includes:

[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 comprises: (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 Includes:

[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 with 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 with 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 comprises: (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); Includes:

[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 with 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 with 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 with at least 70% or 75% sequence identity to SEQ ID NO: 6250, and (ii) a VL comprising an amino acid sequence with 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 no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 6257 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 6258 or 116 (or a sequence having no more than one, two, three, or four mutations, e.g., 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 no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), the VLCDR2 amino acid sequence of SEQ ID NO: 6260 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or the VLCDR3 amino acid sequence of SEQ ID NO: 6261 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions).

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

[0082] 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: 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. 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.

[0083] In some embodiments, the first and / or second antigen-binding domain comprises: (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 Includes:

[0084] In some embodiments, the first and / or second antigen-binding domain comprises a VH comprising the heavy chain framework 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6266 (or a sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), the VHFWR2 amino acid sequence of SEQ ID NO: 6267 (or a sequence having no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), the VHFWR3 amino acid sequence of SEQ ID NO: 6268 (or a sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., 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 domain comprises a VL comprising the VLFWR1 amino acid sequence of SEQ ID NO: 6277 (or a sequence having no more than one, two, or three mutations, e.g., substitutions, additions, or deletions), the VLFWR2 amino acid sequence of SEQ ID NO: 6278 (or a sequence having no more than one mutation, e.g., substitutions, additions, or deletions), the VLFWR3 amino acid sequence of SEQ ID NO: 6279 (or a sequence having no more than one mutation, e.g., 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 domain comprises: (i) a VH comprising the heavy chain framework 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6266 (or a sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), the VHFWR2 amino acid sequence of SEQ ID NO: 6267 (or a sequence having no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), the VHFWR3 amino acid sequence of SEQ ID NO: 6268 (or a sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and / or the VHFWR4 amino acid sequence of SEQ ID NO: 6269; and (ii) a VLFWR1 amino acid sequence of SEQ ID NO: 6277 (or a sequence having no more than one, two, or three mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6278 (or a sequence having no more than one mutation, e.g., substitutions, additions, or deletions), a VLFWR3 amino acid sequence of SEQ ID NO: 6279 (or a sequence having no more than one mutation, e.g., substitutions, additions, or deletions), and / or a VLFWR4 amino acid sequence of SEQ ID NO: 6280. Includes:

[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 with 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 with 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 comprises: (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); Includes:

[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 with 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 with 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 with at least 70% or 74% sequence identity to SEQ ID NO: 6251, and / or (ii) a VL comprising an amino acid sequence with 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, e.g., an effector cell. In some embodiments, the immune cell engager binds to but does not activate an immune cell, e.g., an effector cell.

[0091] In some embodiments, the immune cell engager is a T cell engager, e.g., a T cell engager that mediates binding to and activation of T cells, or a T cell engager that mediates binding to but not activation of T cells. 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, e.g., 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 but not activation of NK cells. In some embodiments, the NK cell engager is selected from the group consisting of 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, KI The NK cell engager is selected from an antibody molecule, e.g., an antigen-binding domain, or ligand, that binds to (e.g., activates) R2DS3, 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, optionally further comprising 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 of CD16, e.g., a CD16a / b ligand, e.g., a CD16a / b ligand further comprising an antibody Fc region. In some embodiments, the immune cell engager mediates binding to, or activation of, one or more of B cells, macrophages, and / or dendritic cells, or both.

[0093] In some embodiments, the immune cell engager comprises a B cell, macrophage, and / or dendritic cell engager selected from one or more of: 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 a combination thereof. In some embodiments, the immune cell engager is a B cell engager, e.g., 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, e.g., 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, e.g., a CD2 agonist, an OX40 antibody, OX40L, a 41BB agonist, a Toll-like receptor agonist or 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, e.g., cyclic di-GMP (cdGMP), cyclic di-AMP (cdAMP), or a combination thereof, optionally with 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, e.g., an IL15R alpha dimerization domain. In some embodiments, the cytokine molecule (e.g., IL-15) and the receptor dimerization domain (e.g., an IL15R alpha dimerization domain) are covalently linked. is not linked to, e.g., is non-covalently associated with,

[0095] In some embodiments, the modulator of a cytokine molecule comprises a TGF-β inhibitor. In some embodiments, the multifunctional molecule comprises a stroma-modifying moiety. In some embodiments, the stroma-modifying moiety causes one or more of the following: a decrease in the level or production of a stromal or extracellular matrix (ECM) component; a decrease in tumor desmoplasia; 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, e.g., a cancer therapeutic or cell therapy, into the tumor or tumor vasculature. In some embodiments, the decreased stromal or ECM component is selected from a glycosaminoglycan or an extracellular protein, or a combination 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 keratin sulfate. In some embodiments, the extracellular protein is selected from collagen, laminin, elastin, fibrinogen, fibronectin, or vitronectin. In some embodiments, the stromal-modifying portion comprises an enzyme molecule that degrades tumor stroma or extracellular matrix (ECM). In some embodiments, the enzyme molecule is selected from a hyaluronidase molecule, a collagenase molecule, a chondroitinase molecule, a matrix metalloproteinase molecule (e.g., macrophage metalloelastase), or a variant (e.g., fragment) of any of the foregoing. In some embodiments, the stromal-modifying portion reduces the level or production of hyaluronic acid. In some embodiments, the stromal-modifying portion 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 (e.g., fragment thereof). In some embodiments, the hyaluronan-degrading enzyme is active at neutral or acidic pH, for example, 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 (e.g., truncated form thereof) thereof.In some embodiments, the hyaluronidase molecule is selected from HYAL1, HYAL2, or PH-20 / SPAM1, or variants thereof (e.g., truncated forms thereof). In some embodiments, the truncated forms lack a C-terminal glycosylphosphatidylinositol (GPI) attachment site or a portion of the GPI attachment site. In some embodiments, the hyaluronidase molecule is glycosylated, e.g., comprises at least one N-linked glycan. In some embodiments, the hyaluronidase molecule comprises the amino acid sequence of SEQ ID NO: 6213, or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but no more than 5, 10, or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 6213). In some embodiments, the hyaluronidase molecule comprises amino acid residues 36 to 464 of SEQ ID NO: 6213. In some embodiments, the hyaluronidase molecule comprises amino acid residues 36-481, 36-482, or 36-483 of PH20, wherein PH20 has the amino acid sequence of SEQ ID NO: 6213. In some embodiments, the hyaluronidase molecule comprises an amino acid sequence having at least 95% to 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 comprises 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 comprises an amino acid sequence 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 substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than 5, 10, or 15 alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 6218). 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 PEGylated PH20 enzyme (PEGPH20). In some embodiments, the hyaluronan-degrading enzyme, e.g., hyaluronidase molecule, further comprises an immunoglobulin chain constant region (e.g., Fc region), e.g., selected from the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, more particularly the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the immunoglobulin constant region (e.g., Fc region) is linked, e.g., covalently linked, to the hyaluronan-degrading enzyme, e.g., hyaluronidase molecule. In some embodiments, the immunoglobulin chain constant region (e.g., Fc region) is altered, 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. In some embodiments, the hyaluronan-degrading enzyme, e.g., hyaluronidase molecule, forms a dimer. In some embodiments, the stromal-modifying moiety comprises an inhibitor of hyaluronan synthesis, e.g., HA synthase. In some embodiments, the inhibitor comprises a sense or antisense nucleic acid molecule against 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-modifying portion comprises a collagenase molecule, e.g., a mammalian collagenase molecule, or a variant (e.g., fragment) thereof. In some embodiments, the collagenase molecule is collagenase molecule IV, e.g., comprising the amino acid sequence of SEQ ID NO: 6219, or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change, but no more than 5, 10, or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 6219.

[0096] In some embodiments, a 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, a 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-modifying moiety. In some embodiments, a multifunctional molecule comprises a cytokine molecule and a stromal-modifying moiety. In some embodiments, a 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-modifying 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 composition: A,B-[dimerization module]-C,-D For example, including the configurations shown in FIGS. 1A, 1B, and 1C, where: (1) The dimerization module is an immunoglobulin constant domain, e.g., a heavy chain constant domain. (e.g., a homodimeric or heterodimeric heavy chain constant region, e.g., an Fc region), or a constant domain of an immunoglobulin variable region (e.g., a Fab region); (2) A, B, C, and D are, 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, with the proviso that At least one, two, or three of A, B, C, and D comprise 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 are absent or contain one of an immune cell engager, a cytokine molecule, or a stromal-modifying moiety. This is subject to the following conditions.

[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, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule; (ii) 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 a cytokine molecule; (iii) 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 a stromal-modifying portion; (iv) A comprises 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; B comprises 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 C or D comprises an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule; (v) A comprises 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; B comprises 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 C or D comprises a cytokine molecule; (vi) A is a first calreticulin protein (e.g., a wild-type calreticulin A comprises a first antigen-binding domain that binds to a second 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, B comprises 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 C or D comprises a stromal-modifying portion; (vii) A comprises 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; C comprises 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 B or D comprises an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule; (viii) A comprises 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; C comprises 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 B or D comprises a cytokine molecule; (ix) A comprises 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; C comprises 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 B or D comprises a stromal-modifying moiety; (x) A comprises 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 B, 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; (xi) A comprises 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 B, 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; (xii) A comprises 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 B, C, or D comprises (a) a cytokine molecule and (b) a stroma-modifying moiety; (xiii) A is a first calreticulin protein (e.g., a wild-type calreticulin A comprises a first antigen-binding domain that binds to a 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, B comprises 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 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 comprises 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; B comprises 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 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 comprises 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; B comprises 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 C or D comprises (a) a cytokine molecule and (b) a stromal-modifying moiety; (xvi) A comprises 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; C comprises 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 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 comprises 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; C comprises 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 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 stromal-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 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 perreticulin protein comprises a second antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 6286, and B or D comprises (a) a cytokine molecule and (b) a stroma-modifying moiety; (xix) A comprises 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 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 stromal-modifying moiety; (xx) A comprises 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; B comprises 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 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-modifying moiety; or (xxi) A comprises 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; C comprises 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 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-modifying moiety.

[0099] In some embodiments, the dimerization module comprises one or more immunoglobulin chain constant regions (e.g., Fc regions) comprising one or more of paired holes and protrusions ("knobs-in-holes"), electrostatic interactions, or strand exchange. In some embodiments, the one or more immunoglobulin chain constant regions (e.g., Fc regions) comprise an amino acid substitution at one or more of 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, for example, a human IgG1. In some embodiments, one or more immunoglobulin chain constant regions (e.g., Fc regions) comprise an amino acid substitution selected from T366S, L368A, or Y407V (e.g., corresponding to a pore or hole), or T366W (e.g., corresponding to a protrusion or knob), or a combination thereof.

[0100] In some embodiments, the multifunctional molecule further comprises a linker, e.g., a linker between one or more of: the antigen binding domain and the immune cell engager, between the antigen binding domain and the cytokine molecule, between the antigen binding domain and the stromal modifying moiety, between the immune cell engager and the cytokine molecule, between the immune cell engager and the stromal modifying moiety, between the cytokine molecule and the stromal modifying moiety, between the antigen binding domain and the dimerization module, between the immune cell engager and the dimerization module, between the cytokine molecule and the dimerization module, or between the stromal modifying moiety and the dimerization module. In some embodiments, the linker is a cleavable linker, a non-cleavable linker, a peptide linker, The linker may be selected from 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 provides a method for producing a pharmaceutical composition comprising: (i) an antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), e.g., the calreticulin mutant protein comprises the amino acid sequence of SEQ ID NO: 6286; and (ii) a moiety that binds to CD3, e.g., an antibody molecule that binds to CD3; The present invention provides a multifunctional molecule comprising:

[0102] In some embodiments, the multifunctional molecule is 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 portion that binds CD3 (e.g., a first scFv that binds CD3); 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 portion that binds CD3 (e.g., a second scFv that binds CD3); For example, a fourth polypeptide comprising, from N-terminus to C-terminus, a second VL and a second CL, 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), and 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), wherein 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 structure of Figure 2A or 2B. In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) an antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), e.g., the calreticulin mutant protein comprises the amino acid sequence of SEQ ID NO: 6286; and (ii) a moiety that binds to a TCR (e.g., TCRβ), e.g., an antibody molecule that binds to a TCR (e.g., TCRβ); The present invention provides a multifunctional molecule comprising:

[0104] In some embodiments, the multifunctional molecule is For example, a first polypeptide comprising, from N-terminus to C-terminus, a first VL and a first CL; For example, from N-terminus to C-terminus, a first VH, a first CH1, a first dimerization domain (e.g., a first Fc), and a first portion (e.g., a a second polypeptide comprising a first scFv that binds to a TCR (e.g., TCRβ); 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 portion that binds to a TCR (e.g., TCRβ) (e.g., a second scFv that binds to a TCR (e.g., TCRβ)); For example, a fourth polypeptide comprising, from N-terminus to C-terminus, a second VL and a second CL, 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), and 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), wherein 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 structure of Figure 3A or 3B. In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) an antigen-binding domain that binds to a calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), e.g., the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286; and (ii) a moiety that binds to NKp30, such as an antibody molecule or a ligand that binds to (e.g., activates) NKp30; The present invention provides a multifunctional molecule comprising:

[0106] In some embodiments, the multifunctional molecule is 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 NKp30 (e.g., a second antibody molecule or ligand that binds NKp30); For example, a fourth polypeptide comprising, from N-terminus to C-terminus, a second VL and a second CL, 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), and 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), wherein 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 structure of Figure 4A or 4B. In another aspect, the present disclosure provides an isolated nucleic acid molecule encoding any of the multispecific or multifunctional molecules described herein. In another aspect, the present disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the multispecific or multifunctional molecules described herein, or a nucleotide sequence substantially homologous thereto (e.g., at least 80%, 90%, 95%, or 99.9% identical thereto). In another aspect, the present disclosure provides a host cell comprising the nucleic acid molecule or vector described herein.

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

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

[0110] In another aspect, the present disclosure provides a method of treating cancer, comprising administering to a subject in need thereof a multispecific or multifunctional molecule polypeptide described herein, wherein the multispecific antibody is administered in an amount effective to treat the 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, e.g., 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 hematological cancer, and optionally, the cancer is a myeloproliferative neoplasm, such as primary or idiopathic myelofibrosis (MF), essential thrombocytosis (ET), polycythemia vera (PV), or chronic myeloid 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 comprise myeloproliferative neoplastic cells. In embodiments, the myeloproliferative neoplastic cells are selected from myelofibrosis cells, essential thrombocythemia cells, polycythemia vera cells, or chronic myeloid cancer cells. In some embodiments, the myeloproliferative neoplastic cells are myelofibrosis cells. In some embodiments, the myeloproliferative neoplastic cells are essential thrombocythemia cells. In some embodiments, the myeloproliferative neoplastic cells are polycythemia vera cells. In some embodiments, the myeloproliferative neoplastic cells are chronic myeloid cancer cells. In some embodiments, the myeloproliferative neoplastic cells comprise a JAK2 mutation (e.g., a JAK2 V617F mutation). In some embodiments, the myeloproliferative neoplastic cells comprise a calreticulin mutation. In some embodiments, the myeloproliferative neoplastic cells comprise an MPL mutation.

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

[0113] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials similar or equivalent to those described herein can be used in the practice or testing of this invention, and suitable methods and materials are described below.All publications, patent applications, patents, and other references described herein are incorporated by reference in their entirety.In the event of any discrepancy, the present specification, including definitions, will prevail.In addition, materials, methods, and examples are merely illustrative and are not intended to be limiting.

[0114] Other features and advantages of the invention will be apparent from the following detailed description and claims. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with the color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0115] [Figure 1A] Figures 1A-1B show alignments of the mouse VH and VL framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4 regions of the antibody A source with their respective humanized sequences. Kabat CDRs are shown in bold, Chothia CDRs in italics, and combined CDRs in boxes. Backmutated framework positions are double underlined. Figure 1A shows the VH sequences of mouse antibody A (SEQ ID NO: 1) and humanized antibody AH (SEQ ID NO: 9). Figure 1B shows the VL sequences of mouse antibody A (SEQ ID NO: 2) and humanized antibody AH (SEQ ID NO: 10 and SEQ ID NO: 11). [Figure 1B] FIG. 1B shows the VL sequences of murine antibody A (SEQ ID NO: 2) and humanized antibody AH (SEQ ID NO: 10 and SEQ ID NO: 11). [Figure 2A] Figures 2A-2B show alignments of the mouse VH and VL framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4 regions of antibody B source with their respective humanized sequences. Kabat CDRs are shown in bold, Chothia CDRs in italics, and combined CDRs in boxes. Backmutated framework positions are double underlined. Figure 2A shows the VH sequences of mouse antibody B (SEQ ID NO: 15) and humanized VH sequences BH.1A-BH.1C (SEQ ID NOs: 23-25). Figure 2B shows the VL sequences of mouse antibody B (SEQ ID NO: 16) and humanized VL sequences BH.1D-BH.1H (SEQ ID NOs: 26-30). [Figure 2B-1] FIG. 2B shows the VL sequences of mouse antibody B (SEQ ID NO: 16) and humanized VL sequences BH.1D to BH.1H (SEQ ID NOs: 26 to 30). [Figure 2B-2] FIG. 2B shows the VL sequences of mouse antibody B (SEQ ID NO: 16) and humanized VL sequences BH.1D to BH.1H (SEQ ID NOs: 26 to 30). [Figure 3] FIG. 1 shows a phylogenetic tree of TCRBV gene families and subfamilies to which the corresponding antibodies were mapped. The subfamily identities are as follows: Subfamily A: TCRβ V6; Subfamily B: TCRβ V10; Subfamily C: TCRβ V12; Subfamily D: TCRβ V5; Subfamily E: TCRβ V7; Subfamily F: TCRβ V11; Subfamily G: TCRβ V14; Subfamily H: TCRβ V16; Subfamily I: TCRβ V18; Subfamily J: TCRβ V9; Subfamily K: TCRβ V13; Subfamily L: TCRβ V4; Subfamily M: TCRβ V3; Subfamily N: TCRβ V2; Subfamily O: TCRβ V15; Subfamily P: TCRβ V30; Subfamily Q: TCRβ V19; Subfamily R: TCRβ V27; Subfamily S: TCRβ V28; Subfamily T: TCRβ V24; Subfamily U: TCRβ V20; Subfamily V: TCRβ V25; and subfamily W: TCRβ V29 subfamily. Subfamily members are described in detail herein in the section entitled "TCR beta V (TCRβV)." [Figure 4A-1]Figures 4A-4C show human CD3+ T cells activated with anti-TCR Vβ13.1 antibody (AH.1) for 6 days. Human CD3+ T cells were isolated using magnetic bead separation (negative selection) and activated with immobilized (plate-coated) anti-TCR Vβ13.1 (AH.1) or anti-CD3ε (OKT3) antibodies at 100 nM for 6 days. Figure 4A shows two scatter plots (left: activated with OKT3; right: activated with AH.1) of expanded T cells assessed for TCR Vβ13.1 surface expression using anti-TCR Vβ13.1 (AH.1) followed by a secondary fluorochrome-conjugated antibody for flow cytometry analysis. Figure 4B shows the percentage (%) of TCR Vβ13.1-positive T cells activated by anti-TCR Vβ13.1 (AH.1) or anti-CD3e (OKT3) versus total T cells (CD3+). Figure 4C shows the relative cell counts obtained by counting the number of events in each T cell subset gate (CD3 or TCR Vβ13.1) at a constant rate of 60 μl / min for 20 seconds. Data are shown as average values ​​from three donors. [Figure 4A-2]Figures 4A-4C show human CD3+ T cells activated with anti-TCR Vβ13.1 antibody (AH.1) for 6 days. Human CD3+ T cells were isolated using magnetic bead separation (negative selection) and activated with immobilized (plate-coated) anti-TCR Vβ13.1 (AH.1) or anti-CD3ε (OKT3) antibodies at 100 nM for 6 days. Figure 4A shows two scatter plots (left: activated with OKT3; right: activated with AH.1) of expanded T cells assessed for TCR Vβ13.1 surface expression using anti-TCR Vβ13.1 (AH.1) followed by a secondary fluorochrome-conjugated antibody for flow cytometry analysis. Figure 4B shows the percentage (%) of TCR Vβ13.1-positive T cells activated by anti-TCR Vβ13.1 (AH.1) or anti-CD3e (OKT3) versus total T cells (CD3+). Figure 4C shows the relative cell counts obtained by counting the number of events in each T cell subset gate (CD3 or TCR Vβ13.1) at a constant rate of 60 μl / min for 20 seconds. Data are shown as average values ​​from three donors. [Figure 4B] FIG. 4B is a plot of the percentage (%) of TCR Vβ13.1 positive T cells activated by anti-TCR Vβ13.1 (AH.1) or anti-CD3e (OKT3) versus total T cells (CD3+). [Figure 4C] Figure 4C shows the relative cell numbers obtained by counting the number of events in each T cell subset gate (CD3 or TCR Vβ13.1) at a constant rate of 60 μl / min for 20 seconds. Data are shown as the average from three donors. [Figure 5A]Figures 5A-5B show the cytolytic activity of human CD3+ T cells activated with anti-TCR Vβ13.1 antibody (AH.1) against the transformed cell line RPMI8226. Figure 5A shows target cytolysis of human CD3+ T cells activated with AH.1 or OKT3. Human CD3+ T cells were isolated using magnetic bead separation (negative selection) and activated with immobilized (plate-coated) AH.1 or OKT3 at the indicated concentrations for 4 days before coculture with RPMI8226 cells at a 5:1 (E:T) ratio for 2 days. Samples were then analyzed for cytolysis of RPMI8226 cells using flow cytometry analysis by FACS staining for CFSE / CD138-labeled and membrane-impermeable DNA dye (DRAQ7). Figure 5B shows target cell lysis of human CD3+ T cells activated with AH.1 or OKT3 incubated with RPMI-8226 at a 5:1 (E:T) ratio for 6 days, followed by cytolysis analysis of RPMI8226 cells as described above. The percentage (%) of target cell lysis was determined by normalizing to basal target cell lysis (i.e., without antibody treatment) using the following formula: [(x-basal) / (100%-basal), where x is the cytolysis of the sample]. Data shown are representative of n=1 donor. [Figure 5B] Figure 5B shows target cell lysis of human CD3+ T cells activated with AH.1 or OKT3 incubated with RPMI-8226 at a 5:1 (E:T) ratio for 6 days, followed by cytolysis analysis of RPMI8226 cells as described above. The percentage (%) of target cell lysis was determined by normalizing to basal target cell lysis (i.e., without antibody treatment) using the following formula: [(x-basal) / (100%-basal), where x is the cytolysis of the sample]. Data shown are representative of n=1 donor. [Figure 6A]Figures 6A-6B show IFNg production by human PBMCs activated with the indicated antibodies. Human PBMCs were isolated from whole blood from the indicated number of donors, followed by solid-phase (plate-coated) stimulation with the indicated antibodies at 100 Nm. Supernatants were collected on days 1, 2, 3, 5, or 6. Figure 6A is a graph comparing IFNg production in human PBMCs activated with the indicated antibodies, activated with an anti-TCR Vβ13.1 antibody (AH.1 or AH.2) or an anti-CD3e antibody (OKT3 or SP34-2) on days 1, 2, 3, 5, or 6 after activation. Figure 6B shows IFNg production in human PBMCs activated with the indicated antibodies, activated with the indicated anti-TCR Vβ13.1 antibody or anti-CD3e antibody (OKT3) on days 1, 2, 3, 5, or 6 after activation. [Figure 6B] Figure 6B shows IFNg production in human PBMCs activated with the indicated anti-TCR Vβ13.1 antibodies or anti-CD3e antibody (OKT3) on days 1, 2, 3, 5, or 6 after activation. [Figure 7A] Figures 7A-7B show IL-2 production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described in Figures 6A-6B was used. [Figure 7B] Figures 7A-7B show IL-2 production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described in Figures 6A-6B was used. [Figure 8A] Figures 8A-8B show IL-6 production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described in Figures 6A-6B was used. [Figure 8B] Figures 8A-8B show IL-6 production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described in Figures 6A-6B was used. [Figure 9A]Figures 9A-9B show TNF-α production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described in Figures 6A-6B was used. [Figure 9B] Figures 9A-9B show TNF-α production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described in Figures 6A-6B was used. [Figure 10A] Figures 10A-10B show IL-1β production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described in Figures 6A-6B was used. [Figure 10B] Figures 10A-10B show IL-1β production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described in Figures 6A-6B was used. [Figure 11A] Figures 11A-11B are graphs showing the delayed kinetics of IFNg secretion in human PBMCs activated with the anti-TCR Vβ13.1 antibody AH.1 compared to PBMCs activated with the anti-CD3e antibody OKT3. Figure 11A shows IFNg secretion data from four donors. Figure 11B shows IFNg secretion data from an additional four donors. Data shown are representative of n=8 donors. [Figure 11B] Figure 11B shows IFNg secretion data from four additional donors. Data shown are representative of n=8 donors. [Figure 12] FIG. 1 shows an increase in CD8+ TSCM and Tempra T cell subsets in human PBMCs activated with anti-TCR Vβ13.1 antibodies (AH.1 or AH.2) compared to PBMCs activated with anti-CD3e antibodies (OKT3 or SP34-2). [Figure 13A]Figures 13A-13F show the characteristics of anti-TCRVb antibodies. Figure 13A is a graph showing the proliferation of T cells activated with anti-CD3 (OKT3) or anti-TCRVb antibodies. Figure 13B shows the selective expansion of CD45RA+ effector memory CD8+ and CD4+ T cells (TEMRA) using anti-TCRVb antibodies. Tn = naive T cells; Tscm = stem cell memory T cells; Tcm = central memory T cells; Tem = effector memory T cells; TEMRA = effector memory CD45RA+ T cells. Figure 13C is a graph showing IFN-γ secretion by PBMCs stimulated with anti-TCRVb or anti-CD3 antibodies. Figure 13D shows target cell lysis by T cells stimulated with anti-TCRVb or anti-CD3 antibodies. Cells were stimulated for 4 days, followed by 2 days of incubation with multiple myeloma target cells to assess cell killing. Figure 13E is a graph showing perforin secretion by T cells stimulated with anti-TCRVb antibody or anti-CD3 antibody. Perforin was analyzed by FACS staining in TCRVB-positive and TCRVB-negative T cells in PBMCs after stimulation with 100 ng / ml plate-bound antibody for 5 days. Figure 13F is a graph showing granzyme B secretion by T cells stimulated with anti-TCRVb antibody or anti-CD3 antibody. Granzyme B was analyzed by FACS staining in TCRVB-positive and TCRVB-negative T cells in PBMCs after stimulation with 100 ng / ml plate-bound antibody for 5 days. [Figure 13B] Figure 13B shows the selective expansion of CD45RA+ effector memory CD8+ and CD4+ T cells (TEMRA) cells using anti-TCRVb antibodies. Tn = naive T cells; Tscm = stem cell memory T cells; Tcm = central memory T cells; Tem = effector memory T cells; Temra = effector memory CD45RA+ T cells. [Figure 13C] Figure 13C is a graph showing IFN-g secretion by PBMCs stimulated with anti-TCRVb antibody or anti-CD3 antibody. [Figure 13D]Figure 13D shows target cell lysis by T cells stimulated with anti-TCRVb or anti-CD3 antibodies. Cells were stimulated for 4 days and then incubated with multiple myeloma target cells for 2 days to assess cell killing. [Figure 13E] Figure 13E is a graph showing perforin secretion by T cells stimulated with anti-TCRVb or anti-CD3 antibodies. Perforin was analyzed by FACS staining in TCRVb-positive and TCRVb-negative T cells in PBMCs after 5 days of stimulation with 100 ng / ml plate-bound antibody. [Figure 13F] Figure 13F is a graph showing granzyme B by T cells stimulated with anti-TCRVb or anti-CD3 antibodies. Granzyme B was analyzed by FACS staining in TCRVb-positive and TCRVb-negative T cells in PBMCs after stimulation with 100 ng / ml plate-bound antibody for 5 days. [Figure 14A] Figures 14A-14B show the production of IL-2 and IL-15 and the expansion of human NK cells following stimulation of PBMCs with anti-TCRVb antibody at a dose of 100 nM for 6 days. Figure 14A shows the secretion of IL-2 or IL-15 in T cells stimulated with anti-TCRVb antibody or anti-CD3 antibody. Figure 14B shows flow cytometry dot plots showing NKp46 staining versus CD56 antibody staining in cells stimulated with anti-TCRVb antibody or anti-CD3 antibody, or a control sample. [Figure 14B-1] Figure 14B shows flow cytometry dot plots showing NKp46 staining versus CD56 antibody staining in cells stimulated with anti-TCRVb or anti-CD3 antibodies, or control samples. [Figure 14B-2] Figure 14B shows flow cytometry dot plots showing NKp46 staining versus CD56 antibody staining in cells stimulated with anti-TCRVb or anti-CD3 antibodies, or control samples. [Figure 15A] 15A to 15C are diagrams showing cytokine secretion in PBMCs stimulated with anti-TCRVb antibody or anti-CD3 antibody. [Figure 15B] 15A to 15C are diagrams showing cytokine secretion in PBMCs stimulated with anti-TCRVb antibody or anti-CD3 antibody. [Figure 15C] 15A to 15C are diagrams showing cytokine secretion in PBMCs stimulated with anti-TCRVb antibody or anti-CD3 antibody. [Figure 16A] Figures 16A-16B show killing of MM cells by dual-targeting BCMA-TCRvb antibody molecules. Figure 16A shows in vitro killing by one of the following dual-targeting antibody molecules: BCMA-TCRVb, BCMA-CD3, or control-TCRVb; or an isotype control. Figure 16B shows in vivo killing of MM cells by dual-targeting BCMA-TCRVb antibodies. [Figure 16B] Figure 16B shows in vivo killing of MM cells by dual-targeting BCM-TCRVb antibodies. [Figure 17] FIG. 1 shows lysis of MM target cells using a dual-targeting antibody that recognizes FcRH5 on one arm and TCRVb on the other arm. [Figure 18A]18A-18C are schematic diagrams of exemplary formats and configurations of functional moieties attached to dimerization modules, e.g., immunoglobulin constant domains. FIG. 18A shows moieties A, B, C, and D covalently linked to a heterodimeric Fc domain. FIG. 18B shows moieties A, B, C, and D covalently linked to a homodimeric Fc domain. FIG. 18C shows moieties A, B, C, and D covalently linked to heterodimeric heavy and light chain constant domains (e.g., Fab CH1 and Fab CL). In some embodiments, the functional moieties are antigen-binding domains that bind to a calreticulin protein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein). In some embodiments, the functional moieties are antigen-binding domains that bind to a wild-type calreticulin protein and a calreticulin mutant protein with approximately the same affinity. In some embodiments, the functional moiety is an antigen-binding domain that preferentially binds to a calreticulin mutant protein over a wild-type calreticulin protein, e.g., a first calreticulin mutant protein comprises the amino acid sequence of SEQ ID NO: 6286 and the wild-type calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285. In some embodiments, the functional moiety is 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 functional moiety is a cytokine molecule. In some embodiments, the functional moiety is a stromal-modifying moiety. [Figure 18B] FIG. 18B shows moieties A, B, C, and D covalently linked to a homodimeric Fc domain. [Figure 18C] FIG. 18C shows moieties A, B, C, and D covalently linked to heterodimeric heavy and light chain constant domains (eg, Fab CH1 and Fab CL). [Figure 19A]Figures 19A and 19B are schematic diagrams of exemplary formats and configurations of multifunctional molecules that include a first antigen-binding domain (e.g., a first Fab) that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), a second antigen-binding domain (e.g., a second Fab) that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), and one or more moieties that bind to CD3 (e.g., an scFv that binds to CD3). In one embodiment, the first antigen-binding domain (e.g., a first Fab) binds to a calreticulin protein disclosed herein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), e.g., a calreticulin mutant protein disclosed in Table 2 or 3, e.g., a type 1 or type 2 calreticulin mutant protein disclosed in Table 2 or 3, e.g., a calreticulin mutant protein comprising the amino acid sequence of SEQ ID NO: 6113 or 6314. In one embodiment, the second antigen-binding domain (e.g., a second Fab) binds to a calreticulin protein disclosed herein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), e.g., a calreticulin mutant protein disclosed in Table 2 or 3, e.g., a type 1 or type 2 calreticulin mutant protein disclosed in Table 2 or 3, e.g., a calreticulin mutant protein comprising the amino acid sequence of SEQ ID NO: 6313 or 6314. [Figure 19B]Figures 19A and 19B are schematic diagrams of exemplary formats and configurations of multifunctional molecules that include a first antigen-binding domain (e.g., a first Fab) that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), a second antigen-binding domain (e.g., a second Fab) that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), and one or more moieties that bind to CD3 (e.g., an scFv that binds to CD3). [Figure 20A]Figures 20A and 20B are schematic diagrams of exemplary formats and configurations of multifunctional molecules that include a first antigen-binding domain (e.g., a first Fab) that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), a second antigen-binding domain (e.g., a second Fab) that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), and one or more moieties that bind to a TCR (e.g., TCRβ) (e.g., an scFv that binds to a TCR (e.g., TCRβ)). In one embodiment, the first antigen-binding domain (e.g., a first Fab) binds to a calreticulin protein disclosed herein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), e.g., a calreticulin mutant protein disclosed in Table 2 or 3, e.g., a type 1 or type 2 calreticulin mutant protein disclosed in Table 2 or 3, e.g., a calreticulin mutant protein comprising the amino acid sequence of SEQ ID NO: 6313 or 6314. In one embodiment, the second antigen-binding domain (e.g., a second Fab) binds to a calreticulin protein disclosed herein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), e.g., a calreticulin mutant protein disclosed in Table 2 or 3, e.g., a type 1 or type 2 calreticulin mutant protein disclosed in Table 2 or 3, e.g., a calreticulin mutant protein comprising the amino acid sequence of SEQ ID NO: 6313 or 6314. [Figure 20B]Figures 20A and 20B are schematic diagrams of exemplary formats and configurations of multifunctional molecules that include a first antigen-binding domain (e.g., a first Fab) that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), a second antigen-binding domain (e.g., a second Fab) that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), and one or more moieties that bind to a TCR (e.g., TCRβ) (e.g., an scFv that binds to a TCR (e.g., TCRβ)). [Figure 21A]Figures 21A and 21B are schematic diagrams of exemplary formats and configurations of multifunctional molecules that include a first antigen-binding domain (e.g., a first Fab) that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), a second antigen-binding domain (e.g., a second Fab) that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), and one or more moieties that bind to NKp30 (e.g., an antibody molecule or a ligand that binds to NKp30). In one embodiment, the first antigen-binding domain (e.g., a first Fab) binds to a calreticulin protein disclosed herein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), e.g., a calreticulin mutant protein disclosed in Table 2 or 3, e.g., a type 1 or type 2 calreticulin mutant protein disclosed in Table 2 or 3, e.g., a calreticulin mutant protein comprising the amino acid sequence of SEQ ID NO: 6313 or 6314. In one embodiment, the second antigen-binding domain (e.g., a second Fab) binds to a calreticulin protein disclosed herein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), e.g., a calreticulin mutant protein disclosed in Table 2 or 3, e.g., a type 1 or type 2 calreticulin mutant protein disclosed in Table 2 or 3, e.g., a calreticulin mutant protein comprising the amino acid sequence of SEQ ID NO: 6313 or 6314. [Figure 21B]Figures 21A and 21B are schematic diagrams of exemplary formats and configurations of multifunctional molecules that include a first antigen-binding domain (e.g., a first Fab) that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), a second antigen-binding domain (e.g., a second Fab) that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and / or a calreticulin mutant protein), and one or more moieties that bind to NKp30 (e.g., an antibody molecule or a ligand that binds to NKp30). [Figure 22] Figure 22 is a graph showing binding of NKp30 antibodies to NK92 cells. Data was calculated as percent AF747 positive population. [Figure 23] Figure 23 is a graph showing activation of NK92 cells by NKp30 antibodies. Data was generated using a hamster anti-NKp30 mAb. [Figure 24A]Figures 24A-24D are schematic diagrams depicting exemplary multispecific molecules comprising a TGFβ inhibitor. In some embodiments, the TGFβ inhibitor comprises a TGF-beta receptor ECD homodimer. In some embodiments, the TGFβ inhibitor comprises a TGFBR2 ECD heterodimer. In Figures 24A and 24B, two TGFBR ECD domains are linked to the C-terminus of two Fc regions. In some embodiments, the CH1-Fc-TGFBR ECD region depicted in Figure 24A or 24B comprises the amino acid sequence of SEQ ID NO: 6405 or 3193. In some embodiments, the Fc-TGFBR ECD region depicted in Figure 24A or 24B comprises the amino acid sequence of SEQ ID NO: 6407 or 6408. In Figures 24C and 24D, two TGFBR ECD domains are linked to CH1 and CL, respectively. In some embodiments, the TGFBR ECD-CH1-Fc region shown in Figure 24C or 24D comprises the amino acid sequence of SEQ ID NO: 6409 or 6410. In some embodiments, the TGFBR ECD-CL region shown in Figure 24C or 24D comprises the amino acid sequence of SEQ ID NO: 6411 or 6412. In some embodiments, the multispecific molecule comprises binding moiety A and binding moiety B. In some embodiments, binding moiety A or binding moiety B is a calreticulin-targeting antigen-binding domain disclosed herein. [Figure 24B] In Figures 24A and 24B, two TGFBR ECD domains are linked to the C-terminus of two Fc regions. In some embodiments, the CH1-Fc-TGFBR ECD region shown in Figure 24A or 24B comprises the amino acid sequence of SEQ ID NO: 6405 or 3193. In some embodiments, the Fc-TGFBR ECD region shown in Figure 24A or 24B comprises the amino acid sequence of SEQ ID NO: 6407 or 6408. [Figure 24C]In Figures 24C and 24D, the two TGFBR ECD domains are linked to a CH1 and a CL, respectively. In some embodiments, the TGFBR ECD-CH1-Fc region shown in Figure 24C or 24D comprises the amino acid sequence of SEQ ID NO: 6409 or 6410. In some embodiments, the TGFBR ECD-CL region shown in Figure 24C or 24D comprises the amino acid sequence of SEQ ID NO: 6411 or 6412. [Figure 24D] In Figures 24C and 24D, the two TGFBR ECD domains are linked to a CH1 and a CL, respectively. In some embodiments, the TGFBR ECD-CH1-Fc region shown in Figure 24C or 24D comprises the amino acid sequence of SEQ ID NO: 6409 or 6410. In some embodiments, the TGFBR ECD-CL region shown in Figure 24C or 24D comprises the amino acid sequence of SEQ ID NO: 6411 or 6412. DETAILED DESCRIPTION OF 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), e.g., the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285 or 6286; 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; Disclosed herein are multifunctional molecules (also referred to herein as "multispecific molecules") comprising multiple (e.g., two or more) functionalities (or binding specificities), including one, two, or all of: (a) a stroma-modifying moiety; and (b) a tumor-targeting moiety (e.g., 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). In some embodiments, the antigen-binding domain binds to a calreticulin protein (e.g., a wild-type calreticulin protein or, e.g., 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 a type 1 and type 2 calreticulin mutant protein disclosed in Table 2 or 3. In some embodiments, the T cell engager comprises an additional antigen-binding domain that binds to a 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), trispecific (or trifunctional), or tetraspecific (or tetrafunctional) molecule. In one embodiment, the multispecific or multifunctional molecule is a bispecific molecule.

[0118] Without being bound by theory, it is predicted that the multispecific or multifunctional molecules disclosed herein localize (e.g., cross-link) and / or activate immune cells (e.g., immune effector cells selected from T cells, NK cells, B cells, dendritic cells, or macrophages) in the presence of cells that express calreticulin protein, e.g., on their surface. Using the multispecific or multifunctional molecules described herein to increase the proximity and / or activity of immune cells in the presence of cells that express calreticulin protein is predicted to enhance the immune response against the target cells, thereby providing a more effective therapy.

[0119] Novel multifunctional, e.g., multispecific, molecules are disclosed that include (i) a stroma-modifying moiety 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), e.g., the calreticulin protein includes the amino acid sequence of SEQ ID NO: 6285 or 6286. Without being bound by theory, the multifunctional molecules disclosed herein are believed to, inter alia, target (e.g., localize to) cancer sites and alter the tumor stroma, e.g., alter 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, provided herein are, inter alia, multifunctional, e.g., multispecific molecules comprising the foregoing moieties, nucleic acids encoding the same, methods of producing the foregoing molecules, and methods of treating cancer using the foregoing molecules.

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

[0121] definition In some embodiments, the multifunctional molecule comprises an immune cell engager. "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 (e.g., a full-length ligand, a ligand fragment, or a fusion thereof (e.g., a ligand-Fc fusion)) 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). In embodiments, the immune cell engager specifically binds to a target immune cell, e.g., preferentially binds to a 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, an NK cell antigen, a B cell antigen, a dendritic cell antigen, and / or a 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 interchangeably 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 TCRRVβ or TCRβV includes isoforms, mammalian, e.g., human TCRβV, human species homologs, and analogs that share at least one common epitope with TCRβV. Human TCRβV includes, but is not limited to, the TCRβV6 subfamily, TCRβV10 subfamily, TCRβV12 subfamily, TCRβV5 subfamily, TCRβV7 subfamily, TCRβV11 subfamily, TCRβV14 subfamily, TCRβV2 subfamily, TCRβV3 subfamily, TCRβV4 subfamily, TCRβV5 subfamily, TCRβV6 subfamily, TCRβV7 subfamily, TCRβV11 subfamily, TCRβV14 subfamily, TCRβV6 subfamily, TCRβV7 subfamily, TCRβV12 subfamily, TCRβV14 subfamily, TCRβV14 subfamily, TCRβV2 subfamily, TCRβV3 subfamily, TCRβV4 subfamily, TCRβV5 subfamily, TCRβV6 subfamily, TCRβV7 subfamily, TCRβV11 subfamily, TCRβV14 subfamily, TCRβV14 subfamily, TCRβV2 subfamily, TCRβV3 subfamily, TCRβV4 subfamily, TCRβV5 subfamily, TCRβV6 subfamily, TCRβV10 subfamily, TCRβV12 subfamily, TCRβV5 subfamily, TCRβV7 subfamily, TCRβV11 subfamily, TCRβV14 subfamily, TCRβV14 subfamily, TCRβV2 subfamily, TCRβV3 subfamily, TCRβV4 subfamily, TCRβV5 subfamily, TCRβ In some embodiments, the TCRβ V6 subfamily includes a gene family comprising a subfamily including the 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. * 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 * In some embodiments, the TCRβ V includes TCRβ V6-5. * Includes 01. TCRβ V6-5 *TCRβ V6-5 is also known as TRBV65; TCRBV6S5; TCRBV13S1, or TCRβ V13.1. * 01, for example, human TCRβ V6-5 * The amino acid sequence of TCRβ V6-5 is known in the art and is provided, for example, by IMGT ID L36092. * 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. * 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 a full-length, fragment, or variant of a cytokine; a cytokine further comprising a receptor domain, e.g., a cytokine receptor dimerization domain; or an agonist of a cytokine receptor, e.g., an antibody molecule (e.g., an agonist antibody) against a cytokine receptor, which induces activation of at least one naturally occurring cytokine. 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 gamma, or a fragment or variant thereof, or a combination of any of the aforementioned cytokines. The cytokine molecule may be a monomer or a dimer. In embodiments, the cytokine molecule may further comprise a cytokine receptor dimerization domain. In other embodiments, the cytokine molecule is an agonist of a cytokine receptor, for example, an antibody molecule (eg, an agonist antibody) against a cytokine receptor selected from IL-15Ra or IL-21R.

[0124] As used herein, the term "molecule," as used in, e.g., antibody molecules, cytokine molecules, receptor molecules, includes full-length naturally occurring molecules as well as variants, e.g., functional variants (e.g., truncations, fragments, mutations (e.g., substantially similar sequences) or derivatized forms thereof), so 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 comprises a stromal-modifying moiety. As used herein, "stromal-modifying moiety" refers to an agent, e.g., a protein (e.g., an enzyme), that can alter, e.g., degrade, a component of the stroma. In embodiments, the stromal component is selected from, for example, an ECM component, e.g., a glycosaminoglycan such as hyaluronan (also known as hyaluronic acid or HA), chondroitin sulfate, chondroitin, dermatan sulfate, heparin sulfate, heparin, entactin, tenascin, aggrecan, and keratin sulfate; or an extracellular protein 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 to more than one, e.g., at least one, of the grammatical object of the article. The use of the terms "a" or "an," when used with the term "comprising" herein, can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

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

[0128] As used herein, an "antibody molecule" refers to a protein, e.g., an immunoglobulin chain or fragment thereof, that contains at least one immunoglobulin variable domain sequence. Antibody molecules encompass antibodies (e.g., full-length antibodies) and antibody fragments. In certain embodiments, an antibody molecule comprises 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 the recombinant process of normal immunoglobulin gene fragments. In embodiments, an antibody molecule refers to an immunologically active antigen-binding portion of an immunoglobulin molecule, e.g., an antibody fragment. An antibody fragment, e.g., a functional fragment, is a portion of an antibody, e.g., a Fab, Fab', F(ab')2, F(ab)2, variable fragment (Fv), domain antibody (dAb), or single-chain variable fragment (scFv). A functional antibody fragment binds to the same antigen recognized by an intact (e.g., full-length) antibody. The term "antibody fragment" or "functional fragment" also includes isolated fragments consisting of the 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 does not include a portion of an antibody that does not have antigen-binding activity, e.g., an Fc fragment or a single amino acid residue. Exemplary antibody molecules include full-length antibodies and antibody fragments, e.g., dAb (domain antibody), single-chain, Fab, Fab', and F(ab')2 fragments, and single-chain variable fragments (scFv).

[0129] As used herein, "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 part 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 compatible with forming a protein structure.

[0130] In embodiments, the antibody molecule is monospecific, e.g., comprises binding specificity for a single epitope. In some embodiments, the antibody molecule is multispecific, e.g., comprises multiple immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence has binding specificity for a first epitope and a 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 that has specificity for more than one (e.g., two, three, four, or more) epitopes and / or antigens.

[0131] As used herein, "antigen" (Ag) refers to a molecule capable of eliciting an immune response, e.g., involving activation of certain immune cells and / or antibody production. Any macromolecule, including almost any protein or peptide, 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 eliciting an immune response encodes an "antigen." In embodiments, an antigen need not be encoded solely by the full-length nucleotide sequence of a gene, nor need it be encoded by a gene at all. In embodiments, antigens can be synthesized or derived from a biological sample, e.g., a tissue sample, a tumor sample, cells, or a fluid containing other biological components. As used herein, "tumor antigen" or, synonymously, "cancer antigen" includes any molecule present on or associated with a cancer, e.g., a cancer cell, or the tumor microenvironment, capable of eliciting an immune response. As used herein, "immune cell antigen" includes any molecule present on or associated with an immune cell, capable of eliciting an immune response.

[0132] An "antigen-binding site" or "binding site" of an antibody molecule refers to the portion of the antibody molecule that is involved in antigen binding, For example, it refers to a portion 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 (H) and light (L) chains. Three highly divergent stretches within the variable regions of the heavy and light chains, called hypervariable regions, are located between more conserved adjacent regions called "framework regions" (FR). FRs are amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. 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 a bound antigen. The three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions" or "CDRs." Framework regions and CDRs are defined and described, for example, in Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDapartment 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 from amino terminus to carboxy terminus in the following amino acid order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0133] As used herein, "cancer" can encompass all types of oncogenic processes and / or cancerous growths. In embodiments, cancer includes primary tumors as well as metastatic tissues, or malignantly transformed cells, tissues, or organs. In embodiments, cancer encompasses all histopathologies and stages of cancer, e.g., invasive / severe stages. In embodiments, cancer includes recurrent and / or resistant cancers. The terms "cancer" and "tumor" can be used interchangeably. For example, both terms encompass solid tumors and liquid tumors. As used herein, the term "cancer" or "tumor" includes precancers, 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 infection and foreign agents. In embodiments, the term includes leukocytes, e.g., neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Innate leukocytes include phagocytes (e.g., macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer cells. Innate leukocytes 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 specialized type of leukocyte 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 that is involved in an immune response, e.g., promoting an immune effector response. Examples of immune effector cells include, but are not limited to, T cells, e.g., 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 a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines.

[0137] The compositions and methods of the present invention are directed to specific sequences, or sequences substantially identical or Similar sequences include polypeptides and nucleic acids having sequences that are at least 80%, 85%, 90%, 95% identical, or more identical to a particular sequence. In the context of amino acid sequences, the term "substantially identical" is used herein to refer to a first amino acid sequence that contains a sufficient or minimal number of amino acid residues that are i) identical to a second amino acid sequence, or ii) are conservative substitutions for aligned amino acid residues in a second amino acid sequence, such that the first and second amino acid sequences may have a common structural domain and / or a common functional activity. 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] The term "substantially identical" as used herein in the context of nucleotide sequences refers to a first nucleic acid sequence that contains a sufficient number or a minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode polypeptides having a common functional activity, or encode a common polypeptide structural domain or common polypeptide functional activity. For example, a nucleotide sequence that 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.

[0139] The term "variant" refers to a polypeptide having substantially the same amino acid sequence as a reference amino acid sequence or encoded by substantially the same 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 substantially identical to a reference amino acid sequence, or a polypeptide encoded by a substantially identical nucleotide sequence, which may have one or more activations of the reference amino acid sequence.

[0141] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a 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 equivalent to amino acid or nucleic acid "homology").

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

[0143] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using a Blossum62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and gap weights of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 10 or a length weight of 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, 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 gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and those that should be used unless otherwise specified) is the Blossum62 scoring 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 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 a 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 as "query sequences" to conduct searches against public databases, for example, 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, word length=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, word length=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 BLAST and Gapped BLAST programs, the default parameters of the respective programs (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 which do not substantially affect their function. The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that contain both amino and acid functionalities and can be included in naturally occurring amino acid polymers. Exemplary amino acids include naturally occurring amino acids; their analogs, derivatives and homologs; amino acid analogs with 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] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include those with 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), and the like. These include amino acids with 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" (when single-chain) are used interchangeably herein to refer to polymers of amino acids of any length. The polymers can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. Polypeptides can be isolated from natural sources, produced by recombinant techniques from eukaryotic or prokaryotic hosts, or can be the product of synthetic techniques.

[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 their analogs. A polynucleotide can be either single-stranded or double-stranded, and if single-stranded, can be the coding strand or the non-coding (antisense) strand. A polynucleotide can comprise 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 does not occur in nature or is linked to another polynucleotide in a non-naturally occurring sequence.

[0150] The term "isolated" as used herein refers to a material that is removed from its original or natural environment (e.g., the natural environment if it occurs in nature). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but an identical 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 may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, and such a vector or composition may still be isolated in that it is not part of the environment in which it is found in nature.

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

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

[0153] As used herein, the term "transforming growth factor beta-3 (TGF-beta3)" refers to a gene expressed in humans by the gene TGFB3, or its orthologues. The amino acid sequence of the TGF-beta 3 gene is SEQ ID NO: 6397. ...80. The amino acid sequence of the TGF-beta 3 gene is SEQ ID NO: 6380. The amino acid sequence of the TGF-beta 3 gene is SEQ ID NO: 6397. The amino acid sequence of the TGF-beta 3 gene is SEQ ID NO: 6380. The amino acid sequence of the TGF-beta 3 gene is SEQ ID NO: 6397. The amino acid sequence of the TGF-beta 3 gene is SEQ ID NO: 6380. The amino acid sequence of the TGF-beta 3 gene is SEQ ID NO: 6397. The amino acid sequence of the TGF-beta 3 gene is SEQ ID NO: 6380. The amino acid sequence of the TGF-beta 3 gene is SEQ ID NO: 6380. The amino acid sequence of the TGF-beta 3 gene is SEQ

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

[0155] As used herein, the term "transforming growth factor beta receptor type 1 (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 No. P36897 provides an exemplary human TGFBR1 amino acid sequence. Exemplary immature human TGFBR1 amino acid sequences are provided in SEQ ID NOs: 6381, 6382, and 6383. Exemplary mature human TGFBR1 amino acid sequences are provided in 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 2 (TGFBR2)" refers to the protein encoded in humans by the gene TGFBR2, or its ortholog. Swiss-Prot Accession No. P37173 provides an exemplary human TGFBR2 amino acid sequence. Exemplary immature human TGFBR2 amino acid sequences are provided in SEQ ID NOs: 6384 and 6385. Exemplary mature human TGFBR2 amino acid sequences are provided in SEQ ID NOs: 6401 and 6402. As used herein, "TGFBR2 polypeptide" refers to TGFBR2 or a fragment or variant thereof.

[0157] As used herein, the term "transforming growth factor beta receptor type 3 (TGFBR3)" refers to the protein encoded by the gene TGFBR3, or its ortholog, in humans. Swiss-Prot Accession No. Q03167 provides an exemplary human TGFBR3 amino acid sequence. Exemplary immature human TGFBR3 amino acid sequences are provided in SEQ ID NOs: 6392 and 6393. Exemplary mature human TGFBR3 amino acid sequences are provided in SEQ ID NOs: 6403 and 6404. As used herein, "TGFBR3 polypeptide" refers to TGFBR3 or a fragment or variant thereof.

[0158] Various aspects of the 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 comprise antibody molecules. In one embodiment, the antibody molecule binds to a cancer antigen, e.g., a tumor antigen or a stromal antigen. In some embodiments, the cancer antigen is, for example, a mammalian, e.g., human, cancer antigen. In other embodiments, the antibody molecule binds to an immune cell antigen, e.g., a mammalian, e.g., human, immune cell antigen. For example, the antibody molecule specifically binds to an epitope, e.g., a linear epitope or a conformational epitope, on the cancer antigen or immune cell antigen.

[0160] In one embodiment, the antibody molecule is a monospecific antibody molecule, binding to a single epitope. For example, a monospecific antibody molecule has multiple immunoglobulin variable domain sequences that each bind to the same epitope.

[0161] In one embodiment, the antibody molecule is a multispecific or multifunctional antibody molecule, e.g., it comprises multiple immunoglobulin variable domain sequences, wherein a first of the multiple immunoglobulin variable domain sequences has binding specificity for a first epitope and a second of the multiple immunoglobulin variable domain sequences has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., 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, e.g., different proteins (or different subunits of a multimeric protein). In one embodiment, the multispecific antibody molecule comprises 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 tetraspecific antibody molecule.

[0162] In one embodiment, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for only two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., 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, e.g., different proteins (or different subunits of a multimeric protein). In one embodiment, a bispecific antibody molecule comprises heavy and light chain variable domain sequences that have binding specificity for a first epitope and heavy and light chain variable domain sequences that have binding specificity for a second epitope. In one embodiment, a 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, a bispecific antibody molecule comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In one embodiment, a bispecific antibody molecule comprises an scFv or Fab, or fragment thereof, having binding specificity for a first epitope and an scFv or Fab, or 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), as well as 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 comprises 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 the modification of full-length antibodies or synthesized de novo using recombinant DNA technology. These functional antibody fragments retain the ability to selectively bind with their respective antigen or receptor. Antibodies and antibody fragments can be from any class of antibodies, including, but not limited to, IgG, IgA, IgM, IgD, and IgE, as well as any subclass of antibodies (e.g., The antibody molecules may be monoclonal or polyclonal. The antibody molecules may also be human, humanized, CDR-grafted, or in vitro generated. The antibody may have a heavy chain constant region chosen from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody may also have a light chain chosen from, for example, kappa or lambda. The term "immunoglobulin" (Ig) is used interchangeably with the term "antibody" herein.

[0164] Examples of antigen-binding fragments of antibody molecules include (i) Fab fragments, which are monovalent fragments consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments, which consist of the VH and CH1 domains; (iv) Fv fragments, which consist of the VL and VH domains of a single arm of an antibody; (v) diabody (dAb) fragments, which consist of a VH domain; (vi) camel or camelized variable domains; (vii) single-chain Fvs (scFvs), see, e.g., 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 are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0165] Antibody molecules include intact molecules as well as functional fragments thereof. The constant regions of an antibody molecule 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, the number of cysteine ​​residues, effector cell function, or complement function).

[0166] The antibody molecule may also be a single-domain antibody. Single-domain antibodies can include antibodies whose complementary determining regions are portions of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, antibodies naturally lacking 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 may be any known in the art or any future single-domain antibody. The single-domain antibody may 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 light chains. Such single-domain antibodies are disclosed, for example, in WO9404678. For clarity, this variable domain derived from a heavy-chain antibody naturally lacking light chains is known herein as a VHH or nanobody to distinguish it from the conventional VH of four-chain immunoglobulins. Such VHH molecules can be derived from antibodies produced in Camelidae species, such as camel, llama, dromedary, alpaca and guanaco. Other non-Camelidae species may produce heavy chain antibodies that are naturally devoid of light chains, and such VHHs are within the scope of the present invention.

[0167] The VH and VL regions can be further divided into regions of hypervariability called "complementarity-determining regions" (CDRs), interspersed with regions that are more conserved called "framework regions" (FR or FW).

[0168] The extent of the framework regions and CDRs has been precisely defined in a number of ways (Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, N IH 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, e.g., "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 "complementarity determining region" and "CDR" as used herein refer to the amino acid sequences in an antibody variable region 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 by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National The CDRs defined according to the "Chothia" numbering scheme may be determined using any of a number of known schemes, including those described in Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme), Institutes of Health, Bethesda, MD ("Kabat" numbering scheme). As used herein, CDRs defined according to the "Chothia" numbering scheme may also be referred to as "hypervariable loops."

[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 the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).

[0172] Each VH and VL typically comprises 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 may be a polyclonal or monoclonal antibody. The terms "monoclonal antibody" or "monoclonal antibody composition," as used herein, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays 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 recombinantly produced, 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. Pat. No. 5,223,409; Kang et al., International Publication No. WO 92 / 18619; Dower et al., International Publication No. WO 91 / 17271; Winter et al., International Publication No. WO 92 / 20791; Markland et al., International Publication No. WO 92 / 20791). WO92 / 15679; Breitling et al., International Publication No. WO93 / 01288; McCafferty et al., International Publication No. WO92 / 01047; Garrard et al., International Publication No. WO92 / 09690; Ladner et al., International Publication No. 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; Griffths 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 contents of all of which are hereby incorporated by reference.

[0175] In one embodiment, the antibody is a fully human antibody (e.g., an antibody made in a mouse genetically engineered to produce antibodies from human immunoglobulin sequences), or a non-human antibody, such as a rodent (mouse or rat), goat, primate (e.g., monkey), or 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 carrying the human immunoglobulin genes rather than the mouse system. Spleen cells from these transgenic mice immunized with an antigen of interest are used to produce hybridomas secreting human mAbs with specific affinity for epitopes from human proteins (see, e.g., Wood et al., International Application WO 91 / 00906; Kucherlapati et al., PCT International Publication WO 91 / 10741; Lonberg et al., International Application WO 92 / 03918; Kay et al., International Application 92 / 03917; Lonberg, N. et al., 1994 Nature 368:856-859; Green, LL et al., 1994 Nature Genet. 7:13-21; Morrison, SL 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 may be one in which the variable region, or a portion thereof, such as the CDR, is generated in a non-human organism, such as a rat or a mouse. Chimeric, CDR-grafted, and humanized antibodies are within the scope of the present invention. Antibody molecules generated in a non-human organism, such as a rat or a mouse, and then modified, for example, in the variable framework or constant region to reduce antigenicity in humans are within the scope of the present invention.

[0178] An "effectively human" protein is one that does not substantially elicit a neutralizing antibody response, e.g., a human anti-mouse antibody (HAMA) response. HAMA can be problematic in many situations, for example, when antibody molecules are administered repeatedly, for example, in the treatment of chronic or recurring disease states. HAMA responses can be a concern due to increased antibody clearance from serum (see, e.g., Saleh et al., Cancer Immunol. Immunother., 32:180-190 (1990)), and also due to potential allergic reactions (see, e.g., LoBuglio et al., Hybridoma, 5:5117-5123 (1990)). 86)), making repeated antibody administration potentially ineffective.

[0179] Chimeric antibodies can be produced by recombinant DNA techniques known in the art (Robinson et al., International Publication No. 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 WO 86 / 01533; Cabilly et al., U.S. Pat. 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, but generally all three, recipient CDRs (of the heavy and / or light immunoglobulin chains) replaced with donor CDRs. The antibody may have at least a portion of a non-human CDR replaced, 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 antigen binding. Preferably, the donor is a rodent antibody, e.g., a rat or mouse antibody, and the recipient is a human framework or human consensus framework. Typically, the immunoglobulin providing the CDRs is referred to as the "donor," and the immunoglobulin providing the framework is referred to as 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 more identical thereto, 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. If two amino acids occur equally frequently, either can be included in the consensus sequence. "Consensus framework" refers to the framework region in the consensus immunoglobulin sequence.

[0182] Antibody molecules can be humanized by methods known in the art (see, e.g., Morrison, SL, 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; and Queen et al., U.S. Pat. Nos. 5,585,089, 5,693,761, and 5,693,762, the 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 CDRs of an immunoglobulin chain are replaced. DR can be replaced. See, for example, 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 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 antibody of the present invention (UK Patent Application No. 2188638A, filed March 26, 1987; Winter, U.S. Patent No. 5,225,539) (the contents of which are hereby expressly incorporated by reference).

[0184] Humanized antibody molecules in which specific amino acids are substituted, deleted, or added are also within the scope of the present invention. Criteria for selecting amino acids from donors 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., EP 519596 A1, published December 23, 1992.

[0185] The antibody molecule may be a single chain antibody. Single chain antibodies (scFV) may be engineered (see, e.g., Colcher, D. et al. (1999) Ann NY Acad Sci 880:263-80; and Reiter, Y., (1996) Clin (See Cancer Res 2:245-52.) Single-chain antibodies can dimerize or multimerize to generate multivalent antibodies with specificities 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, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE heavy chain constant regions, particularly, for example, IgG1, IgG2, IgG3, and IgG4 (e.g., human) heavy chain constant regions. In another embodiment, the antibody molecule has a light chain constant region selected from, for example, kappa or lambda (e.g., human) 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, the number of cysteine ​​residues, effector cell function, and / or complement function). In one embodiment, the antibody has effector function and can fix complement. In another embodiment, the antibody does not recruit effector cells and does not fix complement. In another embodiment, the antibody has reduced or no ability to bind to Fc receptors. For example, it may be an isotype or subtype, fragment or other mutant that does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.

[0187] Methods for modifying antibody constant regions are known in the art. Antibodies with altered functions, such as altered affinity for effector ligands, such as FcR on cells, 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, for example, EP388,151A1, U.S. Pat. No. 5,624,821, and U.S. Pat. No. 5,648,260, the contents of all of which are hereby incorporated by reference). Similar types of modifications can be described that reduce or eliminate these functions when applied to murine or other species' immunoglobulins.

[0188] An antibody molecule can be derivatized or linked 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 fluorescent moieties, radionucleotides, toxins, enzymes, or affinity ligands, such as biotin. Thus, the antibody molecules of the present invention are intended to include derivatized and otherwise modified forms of the antibodies described herein, including immunoadhesion molecules. For example, an antibody molecule can be functionally linked (by chemical coupling, genetic fusion, noncovalent 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 of the antibody or antibody portion with another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0189] One type of derivatized antibody molecule is produced by crosslinking two or more antibodies (of the same or different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, with two independently 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 multispecific and multifunctional molecules as defined herein are described throughout. Exemplary structures are found in Weidle U et al. (2013) The Intriguing Options of Multispecific Antibodies. Formats for Treatment of 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, a multispecific antibody molecule can contain more than one antigen-binding site, with different sites specific for different antigens. In embodiments, a multispecific antibody molecule can bind to more than one (e.g., two or more) epitopes on the same antigen. In embodiments, a multispecific antibody molecule contains 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, a multispecific antibody molecule is a bispecific, trispecific, or tetraspecific antibody molecule. In one embodiment, a 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 additional antigen-binding moieties appended, (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-hole assembly, charge pair, Fab arm exchange, SEED body, triomab, LUZ-Y, Fcab, κλ body, and orthogonal Fab. Spiess et al., Mol. Immunol. 67(2015):95-106. Exemplary BsIgGs include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm), which contains an anti-CD3 arm and an anti-EpCAM arm, and Examples include ertumaxomab (Neovii Biotech, Fresenius Biotech), which targets HER2. In some embodiments, the BsIgG comprises a heavy chain engineered for heterodimerization. For example, the heavy chain can be engineered for heterodimerization using a "knobs-into-holes" strategy, the SEED platform, a common heavy chain (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, duobodies, azymetrics, charge pairs, HA-TF, SEED bodies, and differential Protein A affinity. See ibid. BsIgG can be produced by separate expression of component antibodies in different host cells followed by purification / assembly into BsIgG. BsIgG can also be produced by expression of component antibodies in a single host cell. BsIgG can be purified using affinity chromatography, for example, using Protein A and sequential pH elution.

[0193] IgG with an additional antigen-binding moiety added 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 chains or variable light chains), engineered protein scaffolds, and paired antibody variable domains (e.g., single-chain variable fragments or variable fragments). See ibid. 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 an IgG-scFv is MM-141 (Merrimack Pharmaceuticals), which binds to IGF-1R and HER3. Examples of DVD-Igs include ABT-981 (AbbVie), which binds to IL-1α and IL-1β, and ABT-122 (AbbVie), which binds to TNF and IL-17A.

[0194] Bispecific antibody fragments (BsAbs) are a format of bispecific antibody molecule that lacks some or all of the antibody constant domains. For example, some BsAbs lack the Fc region. In embodiments, bispecific antibody fragments comprise heavy and light chain regions connected by a peptide linker that allows efficient expression of the BsAb in a single host cell. Exemplary bispecific antibody fragments include, but are not limited to, nanobodies, nanobody-HAS, BiTEs, diabodies, DARTs, TandAbs, scDiabodies, scDiabody-CH3s, diabody-CH3s, triplebodies, miniantibodies, minibodies, TriBiminibodies, scFv-CH3 KIHs, Fab-scFvs, scFv-CH-CL-scFvs, F(ab')2, F(ab')2-scFv2s, scFv-KIHs, Fab-scFv-Fc, tetravalent HCAbs, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, and intrabodies. See ibid. For example, the BiTE format comprises tandem scFvs, where the component scFvs bind to CD3 on T cells and to a surface antigen on cancer cells.

[0195] 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 an anti-CD30 antibody linked to an affinity-matured T cell receptor that recognizes an HLA-presented peptide. The immTAC comprises three scFvs. In embodiments, the dock-and-lock (DNL) method can be used to generate bispecific antibody molecules with higher valency. Fusion to albumin-binding proteins or human serum albumin can also extend the serum half-life of antibody fragments. See Id.

[0196] In embodiments, chemical conjugation, e.g., chemical conjugation of antibodies and / or antibody fragments, can be used to generate BsAb molecules. See ibid. An exemplary bispecific antibody conjugate includes 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 an antibody or its fragment. 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 ibid.

[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, such as CHO cells, or insect cells, such as 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 using, for example, 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 scaffolds In some embodiments, the antibody molecule is a CDR-grafted scaffold domain. In some embodiments, the scaffold domain is based on a fibronectin domain, for example, 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 an antibody heavy chain. There are three loops at the end 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 does not have disulfide bonds, and therefore, unlike antibodies and their fragments, Fn3 is stable under reducing conditions (see, for example, 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 domains that bind to, for example, the antigens / markers / cells described herein.

[0199] In embodiments, 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). A "minibody" can be used to display two hypervariable loops. In embodiments, the scaffold domain is a V-like domain (see, e.g., Coia et al., WO 99 / 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 used to select domains that bind to, for example, markers / antigens / cells as described herein. , can be modified or altered (e.g., using the CDRs or hypervariable loops). 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, ecotin, BPTI, etc.), TPR repeats, trifoil structures, zinc finger domains, DNA-binding proteins, particularly monomeric DNA-binding proteins, RNA-binding proteins, enzymes, e.g., proteases (particularly inactivating proteases), RNases, chaperones, e.g., thioredoxin, and heat shock proteins, and intracellular signaling domains (e.g., SH2 and SH3 domains). See, e.g., U.S. Patent Application Publication No. 20040009530 and U.S. Patent No. 7,501,121, incorporated herein by reference.

[0201] In embodiments, scaffold domains are evaluated and selected, for example, by one or more of the following criteria: (1) amino acid sequence, (2) sequence 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, scaffold domains are small, stable protein domains, e.g., proteins of less than 100, 70, 50, 40, or 30 amino acids. The domains may contain one or more disulfide bonds or may chelate a metal, e.g., zinc.

[0202] Antibody-Based Fusions A variety of antibody formats can be generated containing additional binding entities attached to the N- or C-terminus of the antibody. These fusions with single-chain or disulfide-stabilized Fv or Fab result in the generation of tetravalent molecules with bivalent binding specificities for each antigen. The combination of scFv and scFab with IgG allows the production of molecules capable of recognizing three or more different antigens.

[0203] Antibody-Fab fusion An antibody-Fab fusion is a bispecific antibody that contains 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 Fab share a common light chain. Antibody fusions 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, antibody-scFv fusions 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 Antibody-scFv fusions are bispecific antibodies containing 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 of the scFv heavy chain either directly or via a linker peptide. Antibody fusions 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, antibody-scFv fusions 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), which consists of a VH and VL domain of a second specificity positioned N-terminal to the V domain by a shorter linker sequence.

[0206] Other exemplary multispecific antibody formats include, for example, those described in 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. 20150203591 ... WO20150203591A1, U. 40322221A1, 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. 9,359,437, U.S. Patent Application Publication No. 20150018529, WO2016115274A1, WO2016087416A1, U.S. Patent Application Publication No. 20080069820A1, U.S. Patent No. 9,145,588B, U.S. Patent No. 7,919,257, and U.S. Patent Application Publication No. 20150232560A1. Exemplary multispecific molecules utilizing a full-length antibody-Fab / scFab format include those described in the following: U.S. Patent No. 9,382,323 B2, U.S. Patent Application Publication No. 20140072581 A1, U.S. Patent Application Publication No. 20140308285 A1, U.S. Patent Application Publication No. 20130165638 A1, U.S. Patent Application Publication No. 20130267686 A1, U.S. Patent Application Publication No. 20140377269 A1, U.S. Patent No. 7,741,446 B2, and WO1995009917 A1.Exemplary multispecific molecules utilizing the domain-swapping format include those described in the following: 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, U.S. Patent Application Publication No. 20130078249.

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

[0208] Fc-containing multispecific molecules In some embodiments, the multispecific molecules disclosed herein comprise 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., Fc region) is altered, 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 interface of the first and second immunoglobulin chain constant regions (e.g., first and second Fc regions) is altered, e.g., mutated, to increase or decrease dimerization, e.g., compared to an unengineered interface, e.g., a naturally occurring interface. For example, dimerization of immunoglobulin chain constant regions (e.g., Fc regions) can be enhanced by providing one or more of paired holes and protrusions ("knobs-in-holes"), electrostatic interactions, or strand exchange at the Fc interface of the first and second Fc regions, thereby forming a higher ratio of heteromultimers:homomultimers, e.g., compared to an unengineered interface.

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

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

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

[0214] Heterodimerized bispecific antibodies are based on the natural IgG structure, with the two binding arms recognizing different antigens. IgG-derived formats that allow for 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 chains). Forced heavy chain heterodimerization can be achieved, for example, using knobs-in-holes or strand-exchange engineered domains (SEEDs).

[0215] Knob-in-Hole Knobs-in-holes, as described in U.S. Patent Nos. 5,731,116, 7,476,724, and Ridgway, J. et al. (1996) Prot. Engineering 9(7):617-621, generally involve (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 parent antibody with a larger one (e.g., T366Y or T366W), while the "hole" or "pore" is created by replacing a larger residue in the parent antibody with a smaller one (e.g., Y407T, T366S, L368A, and / or Y407V).

[0216] For bispecific antibodies containing Fc domains, the introduction of specific mutations into the constant region of the heavy chain to promote correct heterodimerization of the Fc portion can be utilized. Some such techniques are described in Klein et al. (mAb (2012) 4:6, pp. 1-11) (their The contents of which are hereby incorporated by reference in their entirety. These techniques include the "knob-into-hole" (KiH) approach, which involves 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 correct pairing of the heavy chains (see, e.g., U.S. Pat. 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, along with additional optional stabilizing Fc cysteine ​​mutations, are provided in Table 1.

[0218] [Table 1]

[0219] Other Fc mutations were provided by Igawa and Tsunoda, who identified three negatively charged residues in the CH3 domain of one chain that paired with three positively charged residues in the CH3 domain of the other chain. These specific pairs of charged residues were E356-K439, E357-K370, D399-K409, and vice versa. Introducing three mutations in chain A: E356K, E357K, and D399K, plus at least two of K370E, K409D, and K439E in chain B, alone or in combination with newly identified disulfide bridges, could favor highly 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 maximal heterodimerization, defining the combination 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 to promote heterodimerization of multispecific antibodies include those described in the following references, the contents of each of which are hereby incorporated by reference: WO2016071377A1, U.S. Patent Application Publication No. 20140079689A1, U.S. Patent Application Publication No. 20160194389A1, U.S. Patent Application Publication No. 20160257763 No. 2016071376A2, WO2015107026A1, WO2015107025A1, WO2015107015A1, U.S. Patent Application Publication No. 20150353636A1, U.S. Patent Application Publication No. 20140199294A1, U.S. Patent No. 7750128B2, U.S. Patent Application Publication No. 20160229915A1, U.S. Patent Application Publication No. 2015034 4570A1, U.S. Patent No. 8003774A1, U.S. Patent Application Publication No. 20150337049A1, U.S. Patent Application Publication No. 20150175707A1, U.S. Patent Application Publication No. 20140242075A1, U.S. Patent Application Publication No. 20130195849A1, U.S. Patent Application Publication No. 20120149876A1, U.S. Patent Application Publication No. 20140200331A1 No. 9,309,311 B2, U.S. Pat. No. 8,586,713, U.S. Patent Application Publication No. 20140037621 A1, U.S. Patent Application Publication No. 20130178605 A1, U.S. Patent Application Publication No. 20140363426 A1, U.S. Patent Application Publication No. 20140051835 A1, and U.S. Patent Application Publication No. 20110054151 A1.

[0221] Stabilizing cysteine ​​mutations have also been used in combination with KiH and other Fc heterodimerization-promoting variants (see, e.g., U.S. Patent No. 7,183,076). Other exemplary cysteine ​​modifications include those disclosed in, e.g., U.S. Patent Application Publication No. 20140348839A1, U.S. Patent No. 7,855,275B2, and U.S. Patent No. 9,000,130B2.

[0222] Strand exchange engineering domain (SEED) Heterodimeric Fc platforms are known that support the design of bispecific and asymmetric fusion proteins by engineering strand exchange engineered domain (SEED) C(H)3 heterodimers. These derivatives of human IgG and IgA C(H)3 domains create complementary human SEED C(H)3 heterodimers composed of alternating segments of 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 consist of [IgG1 hinge]-C(H)2-[SEED C(H)3], which may be genetically linked to one or more fusion partners (see, e.g., Davis JH et al., "SEEDbodies: fusion proteins based on strand exchange engineered domain (SEED) C(H)3 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.

[0223] Duo Body "Duobody" technology for producing bispecific antibodies with correct heavy chain pairing is known. Duobody technology involves three basic steps to generate stable bispecific human IgG1 antibodies in a post-production exchange reaction. In the first step, two IgG1s, each containing a single matching mutation in the third constant (CH3) domain, are produced separately using a standard mammalian recombinant cell system. 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 produce very high yields ( Typically >95%) resulting in a bispecific antibody product (see, e.g., 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).

[0224] electrostatic interactions A method for producing a multispecific antibody has been disclosed in which CH3 amino acid changes are made with charged amino acids so that homodimer formation is electrostatically unfavorable. EP1870459 and WO2009089004 describe other strategies for favoring heterodimer formation during co-expression of different antibody domains in a host cell. In these methods, one or more residues that make up the CH3-CH3 interface in the heavy chain constant domain 3 (CH3), both CH3 domains, are replaced with charged amino acids so that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable. Additional methods of generating multispecific molecules using electrostatic interactions are described in references including U.S. Patent Application Publication No. 20100015133, U.S. Patent No. 8,592,562 B2, U.S. Patent No. 9,200,060 B2, U.S. Patent Application Publication No. 20140154254 A1, and U.S. Patent No. 9,358,286 A1, the contents of each of which are hereby incorporated by reference herein.

[0225] Common light chain Light chain mispairing must be avoided to produce a homogeneous preparation of bispecific IgG. One way to achieve this is through the use of the common light chain principle, i.e., 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 to interact with each of the heteromeric variable heavy chain regions of the bispecific antibody. Compositions of bispecific antibodies with a common light chain and methods for their production are disclosed, for example, in U.S. Pat. No. 7,183,076 B2, U.S. Patent Application Publication No. 20110177073 A1, EP2847231 A1, WO2016079081 A1, and EP3055329 A1 (the contents of each of which are hereby incorporated by reference).

[0226] CrossMab Another option for reducing light chain mispairing is the CrossMab technology, which avoids nonspecific light chain mispairing by exchanging the CH1 and CL domains in one half of the Fab of a bispecific antibody. Such crossover variants retain binding specificity and affinity, but the two arms are different to prevent light chain mispairing. CrossMab technology involves domain swapping between heavy and light chains to promote correct pairing (as reviewed in Klein et al., supra). Briefly, a two-step modification process is applied to construct bispecific IgG-like CrossMab antibodies capable of binding to two antigens by using two distinct light-heavy chain pairs. First, a dimerization interface is engineered into the C-terminus of each heavy chain using a heterodimerization approach, e.g., knob-into-hole (KiH) technology, to ensure that only heterodimers of two distinct heavy chains from one antibody (e.g., antibody A) and a second antibody (e.g., antibody B) form efficiently. Next, the constant heavy chain 1 (CH1) and constant light chain (CL) domains of one antibody are swapped (Antibody A), while keeping the variable heavy chain (VH) and variable light chain (VL) domains consistent. The swapping of the CH1 and CL domains ensured that only the desired bispecific CrossMab was efficiently formed, because the modified antibody (Antibody A) light chain dimerized efficiently only with the modified antibody (Antibody A) heavy chain, and the unmodified antibody (Antibody B) light chain dimerized efficiently only with the unmodified antibody (Antibody B) heavy chain (see, e.g., Cain, C., SciBX 4(28); doi:10.1038 / scibx.2011). 783, the contents of which are hereby incorporated by reference).

[0227] Common heavy chain An exemplary method for enhancing the formation of a desired bispecific antibody from a mixture of monomers is by providing a common variable heavy chain for interaction 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.

[0228] Amino acid modifications Alternative compositions of multispecific antibodies with correct light chain pairing and their production methods include various amino acid modifications.For example, Zymeworks describes a heterodimer that has one or more amino acid modifications in CH1 and / or CL domain, one or more amino acid modifications in VH and / or VL domain, or a combination thereof, which are the interface between light chain and heavy chain, and create preferential pairing between each heavy chain and desired light chain, so that when the 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 other (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 and kappa light chain polypeptides can be used to enable heterodimerization. Methods for generating bispecific antibody molecules comprising lambda 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, multispecific molecules include multispecific antibody molecules, e.g., antibody molecules that comprise two binding specificities, e.g., bispecific antibody molecules. Multispecific antibody molecules include: Lambda light chain polypeptide 1 (LLCP1) specific for the 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 a second epitope Includes:

[0231] "Lambda light chain polypeptide 1 (LLCP1)," as that term is used herein, refers to a polypeptide comprising sufficient light chain (LC) sequence that, when combined with a cognate heavy chain variable region, is capable of mediating specific binding to its epitope and complexing with HCP1. In one embodiment, it comprises 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 sufficient sequences therefrom to mediate specific binding of the epitope and complex with HCP1. LLCP1, together with its HCP1, provides specificity for a first epitope (KLCP2, together with its HCP2, provides specificity for a second epitope). As described elsewhere herein, LLCP1 has a higher affinity for HCP1 than for HCP2. do.

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

[0234] "Heavy chain polypeptide 2 (HCP2)," as that term is used herein, refers to a polypeptide that includes sufficient heavy chain (HC) sequence, e.g., HC variable region sequence, that, when combined with its cognate LLCP1, is capable of mediating specific binding to its epitope and complexing with HCP1. 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 sequences therefrom sufficient to (i) mediate specific binding of its epitope and complex with KLCP2, (ii) preferentially complex to KLCP2 as opposed to LLCP1, as described herein, and (iii) preferentially complex to HCP1 as opposed to another molecule of HCP2, as described herein. HCP2, together with its KLCP2, provides specificity for the second epitope (LLCP1, together with its HCP1, provides specificity for the 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 greater than its affinity for HCP2, such that under preselected conditions, e.g., in an aqueous buffer, e.g., at pH 7, e.g., in saline, e.g., at pH 7, or under physiological conditions, at least 75, 80, 90, 95, 98, 99, 99.5, or 99.9% of the multispecific antibody molecule has LLCP1 complexed or associated with 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 greater than its affinity for a second molecule, such that under preselected conditions, e.g., in an aqueous buffer, e.g., at pH 7, e.g., in saline, e.g., 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 or associated with HCP2.

[0239] In another aspect, disclosed herein is a method for generating or producing a multispecific antibody molecule, the method comprising: (i) providing 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), a first CH1, a first heavy chain constant region (e.g., a first CH2, a first CH3, or both)); (ii) providing 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), a second CH1, a second heavy chain constant region (e.g., a second CH2, a second CH3, or both)); (iii) providing a lambda chain polypeptide (e.g., a lambda light chain variable region (VLλ), a lambda light chain constant chain (VLλ), or both) that preferentially associates with a first heavy chain polypeptide (e.g., a first VH); and (iv) providing a kappa chain polypeptide (e.g., a lambda light chain variable region (VLκ), a lambda light chain constant chain (VLκ), or both) that preferentially associates with a second heavy chain polypeptide (e.g., a second VH). Includes:

[0240] In embodiments, the first and second heavy chain polypeptides form an Fc interface that enhances heterodimerization. In embodiments, (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 embodiments, (i)-(iv) are expressed in the cell.

[0241] In embodiments, (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 embodiments, (i)-(iv) are expressed in the cells.

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

[0243] In embodiments, the method further comprises evaluating the cell-expressed multispecific antibody molecules. For example, the purified cell-expressed multispecific antibody molecules can be analyzed by techniques known in the art, including mass spectrometry. In one embodiment, the purified cell-expressed antibody molecules are cleaved, for example, digested with papain, to produce Fab portions, which are evaluated using mass spectrometry.

[0244] In embodiments, the method provides a high yield, e.g., at least 75%, 80%, 90%, 95 ... 8, 99, 99.5 or 99.9% of the time, they produce correctly paired kappa / lambda polyspecific, e.g., bispecific, antibody molecules.

[0245] In other embodiments, the multispecific, e.g., bispecific, antibody molecule comprises: (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)), e.g., HCP1, which binds to a first epitope; (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)), e.g., an HCP2 that binds to a second epitope; (iii) a lambda light chain polypeptide (LLCP1) (e.g., a lambda light chain variable region (VL1), a lambda light chain constant chain (VL1), or both) that preferentially associates with a first heavy chain polypeptide (e.g., a first VH), e.g., LLCP1 that binds a first epitope; 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), e.g., KLCP2 that binds a second epitope. Includes:

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

[0247] An exemplary wild-type human calreticulin is set forth as SEQ ID NO:6285. EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGLQTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNSLDQ TDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKDDEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASKPEDWDER AKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEPPVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGVLGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQDEEQRLKEEEEDKKRKEEEEAEDKEDDEDKDEDEEDEEDKEEDEEEDVPGQAKDEL (SEQ ID NO: 6285)

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

[0249] The main mutations in calreticulin are type 1 and type 2 mutations (see Tables 2 and 3). Type 1 mutations are 52 bp deletions (c.1092_1143del), whereas type 2 mutations are 5 bp insertions (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 calreticulin-targeting 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 that includes one, two, or three CDRs derived from the murine 16B11.1 antibody, e.g., as described in Table 4. For example, in some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH that includes the heavy chain complementarity-determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6358 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 6360 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 227 (or a sequence having no more than one, two, three, or four mutations, e.g., 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 no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 6360 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 227 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions). In some embodiments, the calreticulin-targeting antigen-binding domain comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6358 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6360 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 227 (or a sequence having no more than one, two, three, or four mutations, e.g., 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 with 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 with 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 with one, two, three, or four or fewer mutations, e.g., substitutions, additions, or deletions).

[0261] Alternatively, or in combination with a calreticulin-targeting antigen-binding domain comprising a VH comprising one, two, or three CDRs derived from the murine 16B11.1 antibody, the calreticulin-targeting antigen-binding domain comprises a VL comprising one, two, or three CDRs derived from, for example, the murine 16B11.1 antibody described in Table 4. For example, 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: 251 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), the VLCDR2 amino acid sequence of SEQ ID NO: 246 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or the VLCDR3 amino acid sequence of SEQ ID NO: 248 (or a sequence having no more than one, two, three, or four 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: 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-targeting antigen-binding domain comprises a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 279, a VLCDR2 amino acid sequence of SEQ ID NO: 281, and a 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 no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 6254 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 6255 (or a sequence having no more than one, two, three, or four mutations, e.g., 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 light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6259 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 6260 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VLCDR3 amino acid sequence of SEQ ID NO: 6270 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VLCDR4 amino acid sequence of SEQ ID NO: 6271 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VLCDR5 amino acid sequence of SEQ ID NO: 6272 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VLCDR6 amino acid sequence of SEQ ID NO: 6273 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VLCDR7 amino acid sequence of SEQ ID NO: 6274 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VLCDR8 amino acid sequence of SEQ ID NO: 6275 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VLCDR9 amino acid sequence of SEQ ID NO: 6276 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VLCDR10 amino In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 6259, a VLCDR2 amino acid sequence of SEQ ID NO: 6260, and a VLCDR3 amino acid sequence of SEQ ID NO: 6261 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions).

[0264] In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VH comprising one, two, three, or four framework regions derived from a humanized 16B11.1 antibody, e.g., as 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-targeting antigen-binding domain is SEQ ID NO: 62 24, 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-targeting antigen-binding domain comprises a VL comprising a 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-targeting antigen-binding domain comprises a VH comprising the VHFWR1 amino acid sequence of SEQ ID NO: 6263 (or a sequence having no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions), the VHFWR2 amino acid sequence of SEQ ID NO: 6264 (or a sequence having no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions), the VHFWR3 amino acid sequence of SEQ ID NO: 6265 (or a sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and / or the VHFWR4 amino acid sequence of SEQ ID NO: 228. In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VL comprising the VLFWR1 amino acid sequence of SEQ ID NO: 6277 (or a sequence having no more than one, two, or three mutations, e.g., substitutions, additions, or deletions), the VLFWR2 amino acid sequence of SEQ ID NO: 6278 (or a sequence having no more than one mutation, e.g., substitutions, additions, or deletions), the VLFWR3 amino acid sequence of SEQ ID NO: 6279 (or a sequence having no more than one mutation, e.g., substitutions, additions, or deletions), and / or the VLFWR4 amino acid sequence of SEQ ID NO: 6280. In some embodiments, the calreticulin-targeting 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-targeting 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-targeting 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-targeting 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-targeting 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-targeting 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-targeting 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-targeting 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-targeting 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-targeting 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-targeting 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-targeting 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-targeting 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-targeting 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-targeting antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6247. In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6249. In some embodiments, the calreticulin-targeting 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 Domains In some embodiments, the calreticulin-targeting antigen-binding domain comprises any CDR amino acid sequence or variable region amino acid sequence disclosed in Tables 16-19. 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 no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 243 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 6255 (or a sequence having no more than one, two, three, or four mutations, e.g., 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: 243, and / or the VHCDR3 amino acid sequence of SEQ ID NO: 6255. 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 no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), the VLCDR2 amino acid sequence of SEQ ID NO: 6260 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or the VLCDR3 amino acid sequence of SEQ ID NO: 6261 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions). In some embodiments, the calreticulin-targeting antigen-binding domain comprises a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 6259, a VLCDR2 amino acid sequence of SEQ ID NO: 6260, and a VLCDR3 amino acid sequence of SEQ ID NO: 6261. In some embodiments, the calreticulin-targeting antigen-binding domain comprises an amino acid sequence of SEQ ID NO: 244. In some embodiments, the calreticulin-targeting 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-targeting 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-targeting 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-targeting 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-targeting 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 with 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 (or an amino acid sequence with 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 with 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: 238. 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: 238 (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: 238. 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: 238 (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: 235 and a VL comprising the amino acid sequence of SEQ ID NO: 238. In some embodiments, the calreticulin-targeting 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-targeting 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-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: 238 (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: 238.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 an 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: 6372 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: 234 (or an amino acid sequence with 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 with 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: 239. 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: 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: 235 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: 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-targeting 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-targeting antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 and a VH 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: 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-targeting 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. 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: 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: 237 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: 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: 241 (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: 241. 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: 241 (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: 241.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: 241 (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: 235 and a VL comprising the amino acid sequence of SEQ ID NO: 241. In some embodiments, the calreticulin-targeting 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-targeting 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-targeting antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 (or an amino acid sequence with 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 with 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: 241. In some embodiments, the calreticulin-targeting antigen-binding domain comprises an VH comprising the amino acid sequence of SEQ ID NO: 2372 (or an amino acid sequence with 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 (or an amino acid sequence with 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 with 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 (or an amino acid sequence with 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 with 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: 242. 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 with 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 with 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: 235 and a VL comprising the amino acid sequence of SEQ ID NO: 242. In some embodiments, the calreticulin-targeting 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-targeting 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-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: 242 (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: 242.

[0276] In some embodiments, the calreticulin-targeting 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 bind to and / or mediate the activation of immune cells, e.g., immune effector cells. In some embodiments, the immune cells are selected from T cells, NK cells, B cells, dendritic cells, or macrophage cell engagers, or combinations thereof. In some embodiments, the immune cell engagers are selected from one, two, three, or all of T cell engagers, NK cell engagers, B cell engagers, dendritic cell engagers, or macrophage cell engagers, 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, e.g., an Fc region), a small molecule, or a nucleotide molecule. T Cell Engager The present disclosure provides, inter alia, multispecific (e.g., bi-, tri-, tetra-specific) or multifunctional molecules that are 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 an antigen-binding domain or ligand that binds to (e.g., in some embodiments, activates) 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 an antigen-binding domain or ligand that binds to, but does not activate, 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. In some embodiments, the T cell engager binds to TCRβV. Human T cell receptor (TCR) complex T cell receptors (TCRs) can be found on the surface of T cells. TCRs recognize antigens, e.g., peptides, bound to major histocompatibility complex (MHC) molecules, presented on the surface of cells, e.g., antigen-presenting cells. TCRs are heterodimeric molecules and can include an alpha chain, a beta chain, a gamma chain, or a delta chain. TCRs containing an alpha chain and a beta chain are 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 online, University of South Carolina School of Medicine). The TCRα chain consists of the V, J, and C regions. Rearrangement of the T cell receptor (TCR) by somatic recombination of the V (variable), D (diverse), J (joining), and C (constant) regions is a critical event in the development and maturation of T cells. TCR gene rearrangement occurs in the thymus.

[0283] A TCR may comprise a receptor complex known as a TCR complex, which comprises a TCR heterodimer comprising an alpha chain and a beta chain, and a dimeric signaling molecule, e.g., a CD3 co-receptor, e.g., CD3δ / ε and / or CD3γ / ε.

[0284] TCR beta V (TCRβV) Diversity in the immune system allows for defense against a vast array of pathogens. Because the size of the germline genome is limited, diversity is achieved not only through the process of V(D)J recombination, but also through deletion of nucleotide junctions (the junctions between the VD and DJ segments) and pseudorandom, non-templated addition of nucleotides. TCR beta genes undergo 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 functional gene segments and large insertion / deletion-associated polymorphisms encompassing two V beta gene segments. The present disclosure provides, inter alia, antibody molecules and fragments thereof that bind to, e.g., specifically bind to, e.g., human TCR beta V chains (TCRβV), e.g., TCRβV gene families (also referred to as groups), e.g., TCRβV subfamilies (also referred to as subgroups), e.g., as described herein. TCR beta V families and subfamilies are known in the art and are described, for example, in Yassai et al. (2009) Immunogenet ics, Vol. 61 (No. 7), pp. 493-502; Wei, S. and Concannon P. (1994) Human Immunology 41(3) pp. 201-206. The antibodies described herein can be recombinant antibodies, e.g., recombinant non-murine antibodies, e.g., recombinant human or humanized antibodies.

[0286] In certain aspects, the present disclosure provides anti-TCRβ antibody molecules that bind to a human TCRβV, e.g., a TCRβV family, e.g., a gene family or variant thereof. In some embodiments, the TCRβV gene family includes one or more subfamilies, e.g., as described herein, e.g., as described in Figure 3, Table 8A or 8B. In some embodiments, the TCRβV gene family includes one or more subfamilies, e.g., a TCRβV V6 subfamily, a TCRβV V10 subfamily, a TCRβV V12 subfamily, a TCRβV V5 subfamily, a TCRβV V7 subfamily, a TCRβV V11 subfamily, a TCRβV V14 subfamily, a TCRβV V16 subfamily, a TCRβV V20 subfamily, a TCRβV V30 subfamily, a TCRβV V40 subfamily, a TCRβV V51 subfamily, a TCRβV V62 subfamily, a TCRβV V73 subfamily, a TCRβV V11 subfamily, a TCRβV V14 subfamily, a TCRβV V16 subfamily, a TCRβV V18 subfamily, a TCRβV V20 subfamily, a TCRβV V22 subfamily, a TCRβV V24 subfamily, a TCRβV V26 subfamily, a TCRβV V28 subfamily, a TCRβV V29 subfamily, a TCRβV V30 subfamily, a TCRβV V31 subfamily, a TCRβV V32 subfamily, a TCRβV V33 subfamily, a TCRβV V34 subfamily, a TCRβV V35 subfamily, a TCRβV V36 subfamily, a TCRβV V37 subfamily, a TCRβV V38 subfamily, a TCRβV V39 subfamily, a TCRβV V40 subfamily, a TCRβV 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 V1 subfamily, TCRβ V17 subfamily, TCRβ V21 subfamily, TCRβ V23 subfamily, or 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 also known as 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 a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-4 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-4 * 02, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-9 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-8 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-5 *01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-6 * 02, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-6 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβV6-2 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-3 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-1 * 01, or any 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 also includes 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 V8.1. V12-4 * 01. TCRβ V12-3 * 01, or TCRβ V12-5 * 01, or variants thereof. In some embodiments, TCR β V12 is recognized by, e.g., bound by, SEQ ID NO: 15 and / or SEQ ID NO: 16. In some embodiments, TCR β V12 is recognized by, 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 any variant thereof.

[0293] In some embodiments, the TCRβ V11 subfamily is TCRβ V11-1 * 01. TCRβ V11-2 * 01 or TCRβ V11-3 * 01, or any variant thereof.

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

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

[0296] In some embodiments, the TCRβ V13 subfamily is TCRβ V13 * 01, or any variant thereof. In some embodiments, the TCRβ V4 subfamily is TCRβ V4-2 * 01. TCRβ V4-3 * 01, or TCRβ V4-1 * 01, or any variant thereof.

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

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

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

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

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

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

[0303] [Table 12]

[0304] [Table 13]

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

[0306] In some embodiments, the anti-TCRβV antibody molecules disclosed herein do not recognize, eg, 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 TCRβV region is JOVI.1, described in 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 (e.g., specifically bind) to the TCRβV region. In some embodiments, binding of the anti-TCRβV antibody molecules disclosed herein results in a cytokine profile that differs from the cytokine profile of a T cell engager that binds to a receptor or molecule other than the TCRβV region (a "non-TCRβV-binding T cell engager"). In some embodiments, the non-TCRβV-binding T cell engager comprises 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 the human TCRβVs, e.g., TCRβV gene families, e.g., TCRβV subfamilies, e.g., as described herein, e.g., in Figure 3, Table 8A, or Table 8B. In some embodiments, the anti-TCRβV antibody molecule binds to one or more of the following: TCRβV6 subfamily, TCRβV10 subfamily, TCRβV12 subfamily, TCRβV5 subfamily, TCRβV7 subfamily, TCRβV11 subfamily, TCRβV14 subfamily, TCRβV16 subfamily, TCRβV18 subfamily, TCRβV9 subfamily, TCRβV13 subfamily, TCRβV4 subfamily, TCRβV3 subfamily, TCRβV2 subfamily, TCRβV15 subfamily, TCRβV30 subfamily, TCRβV19 subfamily. The antibody binds to one or more TCRβ V subfamilies, or variants thereof, selected from the group consisting of 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, and TCRβ V26 subfamily.

[0310] In some embodiments, the anti-TCRβ antibody molecule 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 * In some embodiments, the TCRβ V6 subfamily comprises TCRβ V6-5, TCRβ V6-6, or a variant thereof. * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-4 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-4 * 02, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-9 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-8 * 01, or a variant thereof. V6 is TCRβ V6-5 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-6 *02, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-6 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-2 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-3 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-1 * 01, or any variant thereof.

[0311] In some embodiments, the anti-TCRβ antibody molecule is TCRβ V10-1 * 01. TCRβ V10-1 * 02. TCRβ V10-3 * 01 or TCRβ V10-2 * 01, or its variants, and bind to the TCRβ V10 subfamily.

[0312] In some embodiments, the anti-TCRβ antibody molecule is TCRβ V12-4 * 01. TCRβ V12-3 * 01 or TCRβ V12-5 * 01, or its variants, and bind to the TCRβ V12 subfamily.

[0313] In some embodiments, the anti-TCRβ antibody molecule is TCRβ V5-5 * 01. TCRβ V5-6 * 01. TCRβ V5-4 * 01. TCRβ V5-8 * 01. TCRβ V5-1 * 01, or its variants, and bind to the TCRβ V5 subfamily.

[0314] In some embodiments, the anti-TCRβ antibody molecule does not bind to TCRβ V12 or binds to TCRβ V12 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-fold lower) than the affinity and / or binding specificity of the 16G8 murine antibody or a humanized version thereof described in U.S. Pat. No. 5,861,155.

[0315] In some embodiments, the anti-TCRβ 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 murine antibody or a humanized version thereof described in U.S. Pat. No. 5,861,155.

[0316] In some embodiments, the anti-TCRβ antibody molecule binds to a TCRβ region other than TCRβ V12 (e.g., a TCRβ V region described herein, e.g., a TCRβ V region of the TCRβ V6 subfamily (e.g., TCRβ V6-5)) 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-fold higher) than the affinity and / or binding specificity of the 16G8 murine antibody or a humanized version thereof described in U.S. Pat. No. 5,861,155. * 01).

[0317] In some embodiments, the anti-TCRβ antibody molecule is TCRβ V5-5 * TCRβ V5-1 in 01 * or binds to TCRβ V5-5 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 murine antibody C or a humanized version thereof described in U.S. Pat. No. 5,861,155. * 01 or TCRβ V5-1* Combine with 01.

[0318] In some embodiments, the anti-TCRβ 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 murine antibody C or a humanized version thereof described in U.S. Pat. No. 5,861,155. * 01 or TCRβ V5-1 * Combine with 01.

[0319] In some embodiments, the anti-TCRβ 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 murine antibody C or a humanized version thereof described in U.S. Pat. No. 5,861,155. * 01 or TCRβ V5-1 * TCRβ V regions other than TCRβ V region 01 (e.g., TCRβ V regions described herein, e.g., TCRβ V6 subfamily (e.g., TCRβ V6-5 * 01).

[0320] Anti-TCRβ V6 antibody Thus, in one aspect, the present disclosure provides a method for producing a 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 *In some embodiments, the TCRβ V6 subfamily includes TCRβ V6-5. * 01 or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-4 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises the TCRβ V6-4 * 02, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-9 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-8 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-5 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-6 * 02, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-6 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-2 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-3 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-1 * 01, or any 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 ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACC CAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACTC

[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 more identity thereto. SEQ ID NO: 44 MSIGLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSY In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule is a non-murine antibody molecule, e.g., a human or humanized antibody molecule. In some embodiments, the antibody molecule is an anti-TCRβ V antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule is a human antibody molecule. In some embodiments, the antibody molecule is an anti-TCRβ V antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule is a humanized antibody molecule.

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

[0324] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises at least one, two, three, or four variable regions from an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.68, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1A, or encoded by a nucleotide sequence in Table 1A, or a sequence 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, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule is an antibody described herein, e.g., AH.1 to AH.68, e.g., AH.1, AH.2 or AH.68, or an antibody molecule encoded by a nucleotide sequence set forth in Table 1A, or a sequence 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, an anti-TCRβ antibody molecule, such as 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 described herein, e.g., an antibody selected from any one of AH.1-AH.68, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1A, or encoded by a nucleotide sequence in Table 1A, or a sequence 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, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule comprises a heavy chain constant region of IgG4, e.g., human IgG4. In yet another embodiment, an anti-TCRβ V antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) The antibody molecule comprises a heavy chain constant region of IgG1, e.g., human IgG1. In one embodiment, the heavy chain constant region comprises an amino acid sequence set forth in Table 3A, or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).

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

[0329] In some embodiments, an anti-TCRβ 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 heavy chain variable region (VH) of an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.68, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1A, or encoded by a nucleotide sequence in Table 1A, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0330] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) The antibody molecule comprises at least one, two, o...

Claims

1. (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, e.g., 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, e.g., an anti-NKp30 antigen-binding domain disclosed in Tables 7-10 or 18; A multifunctional molecule comprising:

2. The multifunctional molecule of claim 1 , wherein the second antigen-binding domain binds to a TCRβV.

3. The multifunctional molecule of claim 2 , wherein the second antigen-binding domain activates T cells or the second antigen-binding domain does not activate T cells.

4. The multifunctional molecule of claim 2 or 3, wherein the second antigen-binding domain binds to TCRβV12 or TCRβV6 (e.g., comprising the amino acid sequence of SEQ ID NO: 1044).

5. The multifunctional molecule of any one of claims 2 to 4, wherein 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.

6. The second antigen-binding domain comprises: (a) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) VH comprises a VHCDR1 having the amino acid sequence of 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 no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having the amino acid sequence of VHCDR2 in Table 1A, Table 2A, Table 10A, Table 11A, Table 12A, or Table 13A (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 having the amino acid sequence of VHCDR3 in Table 1A, Table 2A, Table 10A, Table 11A, Table 12A, or Table 13A (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); (ii) VL comprises a VLCDR1 having an amino acid sequence of 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 no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VLCDR2 having an amino acid sequence of VLCDR2 in Table 1A, Table 2A, Table 10A, Table 11A, Table 12A, or Table 13A (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VLCDR3 having an amino acid sequence of VLCDR3 in Table 1A, Table 2A, Table 10A, Table 11A, Table 12A, or Table 13A (or a sequence having no more than one, two, three, or four mutations, e.g., 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), (i) VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 3 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 4 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 5 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or (ii) VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 7 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 8 (or a sequence having no more than one, two, three, or four mutations, e.g., 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), (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 45 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 46 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 47 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); and / or (ii) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 51 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 52 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 53 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); and / or (d) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 48 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 49 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 50 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or (ii) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 54 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 55 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 56 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); The multifunctional molecule of any one of claims 2 to 5, comprising:

7. The second antigen-binding domain comprises: (a) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) the VH comprises an amino acid sequence of a 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 comprises an amino acid sequence of a 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 comprises an 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 comprises an 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), (i) the VH comprises an 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) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein VL comprises 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); and / or (c) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) the VH comprises 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 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); Heavy chain variable region (VH) and / or light chain variable region (VL) The multifunctional molecule of any one of claims 2 to 5, comprising:

8. The second antigen-binding domain comprises: (a) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 17 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 18 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 19 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or (ii) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 20 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 21 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 22 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); (b) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 57 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 58 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 59 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or (ii) VL has the light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 63 ( a heavy chain variable region (VH) and / or a light chain variable region (VL) comprising the VHCDR2 amino acid sequence of SEQ ID NO: 64 (or a sequence having no more than one, two, three or four mutations, e.g., substitutions, additions or deletions), the VHCDR2 amino acid sequence of SEQ ID NO: 64 (or a sequence having no more than one, two, three or four mutations, e.g., substitutions, additions or deletions), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 65 (or a sequence having no more than one, two, three or four mutations, e.g., substitutions, additions or deletions); and / or (c) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 60 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 61 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 62 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or (ii) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 66 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 67 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 68 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); The multifunctional molecule of any one of claims 2 to 5, comprising:

9. The second antigen-binding domain comprises: (a) a heavy chain variable region (VH) and / or a light chain variable region (VL), (i) the VH comprises an 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) the 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), (i) VH is an 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 comprises 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 is 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 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); Heavy chain variable region (VH) and / or light chain variable region (VL) The multifunctional molecule of any one of claims 2 to 5, comprising:

10. 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 portion that binds a TCR (e.g., TCRVβ) (e.g., a first scFv that binds a TCR (e.g., TCRVβ)); a third polypeptide comprising, e.g., 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 portion that binds a TCR (e.g., TCRVβ) (e.g., a second scFv that binds a TCR (e.g., TCRVβ)); For example, a fourth polypeptide comprising, from N-terminus to C-terminus, a second VL and a second CL. Including, 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 FIG. 3A or 3B . A multifunctional molecule according to any one of claims 2 to 9.

11. The multifunctional molecule of claim 1 , wherein the second antigen-binding domain binds to NKp30.

12. The multifunctional molecule of claim 11, wherein 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, e.g., the second antigen-binding domain is an antibody molecule or a ligand that binds to (e.g. activates) NKp30.

13. The second antigen-binding domain comprises: (i) a VHCDR1 having an amino acid sequence of heavy chain complementarity determining region 1 (VHCDR1) of Table 7, Table 9, Table 10, or Table 18 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having an amino acid sequence of VHCDR2 of Table 7, Table 9, Table 10, or Table 18 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or an amino acid sequence of VHCDR3 of Table 7, Table 9, Table 10, or Table 18 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or (ii) a VLCDR1 having an amino acid sequence of a VLCDR2 of Table 8, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, or four or less mutations, e.g., substitutions, additions, or deletions); and / or a light chain variable region (VL) comprising a VLCDR3 having an amino acid sequence of a VLCDR3 of Table 8, Table 9, Table 10, or Table 18 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions). The multifunctional molecule of claim 11 or 12, comprising:

14. The second antigen-binding domain comprises: (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 7313 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6001 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 7315 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); and / or (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 7326 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 7327 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VLCDR3 amino acid sequence of SEQ ID NO: 7329 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions). The multifunctional molecule of claim 13 , comprising:

15. The second antigen-binding domain comprises: (i) a VH comprising an amino acid sequence of any of SEQ ID NOs: 7298 or 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 NOs: 7298 or 7300-7304); and / or (ii) a VL comprising an amino acid sequence of any one of SEQ ID NOs: 7299 or 7305-7309 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 7299 or 7305-7309). The multifunctional molecule of claim 13 or 14, comprising:

16. The second antigen-binding domain comprises: (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 an 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 SEQ ID NO: 7302), and a VL comprising an 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 SEQ ID NO: 7309). The multifunctional molecule of any one of claims 13 to 15, comprising:

17. The second antigen-binding domain comprises: (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 at least about 75% , 80%, 85%, 90%, 95%, or 99% sequence identity) The multifunctional molecule of any one of claims 13 to 16, comprising:

18. The second antigen-binding domain comprises: (i) a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6000, the VHCDR2 amino acid sequence of SEQ ID NO: 6001, and / or the VHCDR3 amino acid sequence of SEQ ID NO: 6002; and (ii) a light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6063, the VLCDR2 amino acid sequence of SEQ ID NO: 6064, and / or the VLCDR3 amino acid sequence of SEQ ID NO: 7293 The multifunctional molecule of claim 11 or 12, comprising:

19. The second antigen-binding domain comprises: (1) VHFWR1 having the amino acid sequence of heavy chain framework region 1 (VHFWR1) of Table 7, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, four, five, or six or fewer mutations, e.g., substitutions, additions, or deletions therefrom); VHFWR2 having the amino acid sequence of VHFWR2 of Table 7, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, four, five, or six or fewer mutations, e.g., substitutions, additions, or deletions therefrom); a heavy chain variable region (VH) comprising a VHFWR3 having the amino acid sequence of VHFWR3 of Table 9, Table 10, or Table 18 (or a sequence having no more than one, two, three, four, five, or six mutations, e.g., substitutions, additions, or deletions therefrom), or a VHFWR4 having the amino acid sequence of VHFWR4 of Table 7, Table 9, Table 10, or Table 18 (or a sequence having no more than one, two, three, four, five, or six mutations, e.g., substitutions, additions, or deletions therefrom); and / or (2) VLFWR1 having an amino acid sequence of light chain framework region 1 (VLFWR1) of Table 8, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, four, five, or six or fewer mutations, e.g., substitutions, additions, or deletions therefrom); VLFWR2 having an amino acid sequence of VLFWR2 of Table 8, Table 9, Table 10, or Table 18 (or a sequence having one, two, three, four, five, or six or fewer mutations, e.g., substitutions, additions, or deletions therefrom); a light chain variable region (VL) comprising a VLFWR3 having the amino acid sequence of VLFWR3 of Table 8, Table 9, Table 10, or Table 18 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom); or a VLFWR4 having the amino acid sequence of VLFWR4 of Table 8, Table 9, Table 10, or Table 18 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom).

20. The multifunctional molecule of claim 11, 12, or 18, comprising:

20. The second antigen-binding domain comprises: (1) A heavy chain variable region (VH) comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6003, the VHFWR2 amino acid sequence of SEQ ID NO: 6004, the VHFWR3 amino acid sequence of SEQ ID NO: 6005, or the VHFWR4 amino acid sequence of SEQ ID NO: 6006; and (3) The multifunctional molecule of claim 19, comprising a light chain variable region (VL) comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6066, the VLFWR2 amino acid sequence of SEQ ID NO: 6067, the VLFWR3 amino acid sequence of SEQ ID NO: 7292, or the VLFWR4 amino acid sequence of SEQ ID NO: 6069.

21. The second antigen-binding domain comprises: (i) a VH comprising an amino acid sequence of a VH of 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 an amino acid sequence of a VL 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).

21. The multifunctional molecule of any one of claims 11, 12, or 18 to 20, comprising:

22. 22. The multifunctional molecule of any of claims 11, 12, or 18-21, wherein the second antigen-binding domain comprises a heavy chain comprising an amino acid sequence of a heavy chain in Table 10 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

23. 23. The multifunctional molecule of any of claims 11, 12, or 18-22, wherein the second antigen-binding domain comprises a light chain comprising an amino acid sequence of a light chain in Table 10 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

24. 24. The multifunctional molecule of any of claims 11, 12, or 18-23, wherein the second antigen-binding domain comprises a heavy chain comprising an amino acid sequence of a heavy chain in 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 an amino acid sequence of a light chain in Table 10 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

25. 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 portion 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 NKp30 (e.g., a second antibody molecule or ligand that binds NKp30); For example, a fourth polypeptide comprising, from N-terminus to C-terminus, a second VL and a second CL, 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 FIG. 3A or 3B . A multifunctional molecule according to any one of claims 11 to 24.

26. The multifunctional molecule of any one of claims 1 to 25, wherein the calreticulin protein comprises an amino acid sequence selected from SEQ ID NOs: 6285 to 6312, and optionally, the calreticulin protein comprises an amino acid sequence selected from SEQ ID NOs: 6313 to 6346. child.

27. The multifunctional molecule of any one of claims 1 to 26, wherein the calreticulin protein comprises the amino acid sequence of SEQ ID NO:6285.

28. The multifunctional molecule of any one of claims 1 to 27, wherein the calreticulin protein comprises the amino acid sequence of SEQ ID NO:6286.

29. 29. The multifunctional molecule of any one of claims 1 to 28, wherein 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.

30. and optionally further comprising a third antigen-binding domain that binds to a second calreticulin protein, e.g., the second calreticulin mutant protein comprises the amino acid sequence of SEQ ID NO: 6285 or 6286, (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; The multifunctional molecule according to any one of claims 1 to 29.

31. The multifunctional molecule of claim 30 , wherein the second calreticulin molecule is the same as the calreticulin molecule bound by the first antigen-binding domain.

32. The multifunctional molecule of claim 30 , wherein the second calreticulin molecule is different from the calreticulin molecule bound by the first antigen-binding domain.

33. 33. The multifunctional molecule of any of claims 30 to 32, wherein 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.

34. The multifunctional molecule of claim 33, wherein 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.

35. A multifunctional molecule described in any of claims 30 to 34, wherein 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.

36. The first antigen-binding domain comprises: (i) a heavy chain variable region (VH) comprising a VHCDR1 having the amino acid sequence of heavy chain complementarity determining region 1 (VHCDR1) in Table 4, Table 7A, or Table 17 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having the amino acid sequence of VHCDR2 in Table 4, Table 7A, or Table 17 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 having the amino acid sequence of VHCDR3 in Table 4, Table 7A, or Table 17 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); (ii) a light chain complementarity determining region 1 (VLCDR1) in Table 5, Table 7A, or Table 18 a light chain variable region (VL) comprising a VHCDR1 having an amino acid sequence of (or a sequence with no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having an amino acid sequence of a VLCDR2 in Table 5, Table 7A, or Table 18 (or a sequence with no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 having an amino acid sequence of a VLCDR3 in Table 5, Table 7A, or Table 18 (or a sequence with no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions); (iii) a VH comprising an amino acid sequence of a 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 an amino acid sequence of a VL in Table 7A or Table 16 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity thereto); (v) VHFWR1 having the amino acid sequence of heavy chain framework region 1 (VHFWR1) in Table 4 or Table 6 (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, or 9 or less mutations, e.g., substitutions, additions, or deletions); VHFWR2 having the amino acid sequence of VHFWR2 in Table 4 or Table 6 (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, or 9 or less mutations, e.g., substitutions, additions, or deletions); or a VHFWR3 having the amino acid sequence of VHFWR3 in Table 6 (or a sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), and / or a VHFWR4 having the amino acid sequence of VHFWR4 in Table 4 or Table 6 (or a sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), and / or (vi) VLFWR1 having an amino acid sequence of light chain framework region 1 (VLFWR1) in Table 5 or Table 6 (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, or 9 or less mutations, e.g., substitutions, additions, or deletions), VLF having an amino acid sequence of VLFWR2 in Table 5 or Table 6 (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, or 9 or less mutations, e.g., substitutions, additions, or deletions). VLFWR2, VLFWR3 having the amino acid sequence of VLFWR3 in Table 5 or Table 6 (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, or 9 or less mutations, e.g., substitutions, additions, or deletions), and / or VLFWR4 having the amino acid sequence of VLFWR4 in Table 5 or Table 6 (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, or 9 or less mutations, e.g., substitutions, additions, or deletions). The multifunctional molecule according to any one of claims 1 to 35, comprising:

37. The multifunctional molecule of any one of claims 1 to 36, further comprising a tumor targeting moiety.

38. The multifunctional molecule of claim 37 , wherein the tumor targeting moiety binds to a tumor antigen.

39. 39. The multifunctional molecule of claim 38, wherein 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.

40. The tumor targeting moiety can be, for example, G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or The multifunctional molecule of claim 37, comprising an antibody molecule that binds to a tumor antigen selected from TM4SF1.

41. 41. The multifunctional molecule of claim 40, wherein the tumor targeting moiety comprises a VH and / or VL sequence, e.g., listed in Table A or Table 20.

42. 42. The multifunctional molecule of any one of claims 1 to 41, which preferentially binds to myeloproliferative neoplastic cells over non-tumor cells, and optionally, the binding of the multifunctional molecule to myeloproliferative neoplastic cells is greater than 10, 20, 30, 40, 50 fold greater than the binding of the multifunctional molecule to non-tumor cells.

43. the myeloproliferative neoplastic cells are selected from myelofibrosis cells, essential thrombocythemia vera cells, or chronic myeloid cancer cells; and optionally the myeloproliferative neoplastic cells do not contain a JAK2 V617F mutation, or the myeloproliferative neoplastic cells do not contain the MPL mutation; The multifunctional molecule of claim 42.

44. The multifunctional molecule of any one of claims 1 to 43, further comprising a linker, for example a linker between the first and second antigen-binding domains.

45. 45. The multifunctional molecule of claim 44, wherein 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.

46. The multifunctional molecule of claim 44 or 45, wherein the linker is a peptide linker.

47. The multifunctional molecule of claim 46, wherein the peptide linker comprises Gly and Ser.

48. 47. The multifunctional molecule of claim 46, wherein the peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 6214-6217, or 6220-6221 and 77-78.

49. A nucleic acid molecule encoding a multifunctional molecule according to any one of claims 1 to 48.

50. 50. A vector, e.g., an expression vector, comprising the nucleic acid molecule of claim 49.

51. 51. A cell comprising the nucleic acid molecule of claim 49 or the vector of claim 50.

52. A method for making, e.g. producing, a multifunctional molecule according to any one of claims 1 to 48, comprising culturing a cell according to claim 51 under suitable conditions, e.g. conditions suitable for gene expression and / or homo- or heterodimerization.

53. 49. A pharmaceutical composition comprising a multifunctional molecule according to any one of claims 1 to 48 and a pharma- ceutically acceptable carrier, excipient or stabilizer.

54. 50. A method of treating cancer, comprising administering to a subject in need thereof a multifunctional molecule of any one of claims 1 to 48, wherein the multifunctional molecule is administered in an amount effective to treat cancer.

55. 50. Use of a multifunctional molecule according to any one of claims 1 to 48 for the manufacture of a medicament for treating cancer.

56. 56. The method of claim 54 or the use of claim 55, wherein the subject has cancer cells that express the first and / or second calreticulin protein.

57. 57. The method of claim 54 or 56 or the use of claim 55 or 56, wherein the subject has a JAK2 V617F mutation.

58. 57. The method of claim 54 or 56 or the use of claim 55 or 56, wherein the subject does not have a JAK2 V617F mutation.

59. 59. The method of any one of claims 54 or 56 to 58 or the use of any one of claims 55 to 58, wherein the subject has an MPL mutation.

60. 59. The method of any one of claims 54 or 56 to 58 or the use of any one of claims 55 to 58, wherein the subject does not have an MPL mutation.

61. 61. The method of any one of claims 54 or 56 to 60 or the use of any one of claims 55 to 60, wherein the cancer is a hematological cancer, optionally wherein the cancer is a myeloproliferative neoplasm, such as primary or idiopathic myelofibrosis (MF), essential thrombocytosis (ET), polycythemia vera (PV), or chronic myelogenous leukemia (CML), optionally wherein the cancer is myelofibrosis.

62. 61. The method of any one of claims 54 or 56 to 60 or the use of any one of claims 55 to 60, wherein the cancer is a solid tumor cancer.

63. 63. The method of any of claims 54 or 56-62 or the use of any of claims 55-62, further comprising the step of administering a second therapeutic treatment.

64. 64. The method of claim 63 or the use of claim 63, wherein the second therapeutic treatment comprises a therapeutic agent (e.g. a chemotherapeutic agent, a biological agent, a hormonal therapy), radiation, or surgery.

65. 65. The method of claim 64 or the use of claim 64, wherein the therapeutic agent is selected from a chemotherapeutic agent or a biological agent.

Images