Anti-HER2 / TROP2 antibodies and uses thereof

Abstract

The present disclosure relates to anti-HER2 antibodies or antigen-binding fragments thereof, anti-TROP2 antibodies or antigen-binding fragments thereof, antigen-binding protein constructs (e.g., bispecific antibodies or antigen-binding fragments thereof) that specifically bind to two different antigens (e.g., HER2 and TROP2), and antibody drug conjugates.

Description

[Technical field]

[0001] The present disclosure relates to antibodies or antigen-binding fragments thereof, antigen-binding protein constructs (e.g., bispecific antibodies), and antibody-drug conjugates.

[0002] Claiming priority This application claims the benefit of PCT Application No. PCT / CN2022 / 074078, filed January 26, 2022, PCT Application No. PCT / CN2022 / 110153, filed August 4, 2022, and PCT Application No. PCT / CN2022 / 128951, filed November 1, 2022. The entire contents of the foregoing are incorporated herein by reference. [Background technology]

[0003] Cancer is currently one of the diseases with the highest mortality rates among humans. According to statistics from the World Health Organization, the number of cancer cases and deaths worldwide reached 14 million and 8.2 million, respectively, in 2012. In China, the number of newly diagnosed cancer cases was 3.07 million, and the number of deaths was 2.2 million.

[0004] The recent clinical and commercial success of anti-cancer antibodies has generated considerable interest in antibody-based therapies, and further development of antibodies and antibody-drug conjugates is needed for the treatment of cancer. Summary of the Invention

[0005] The present disclosure relates to anti-HER2 antibodies or antigen-binding fragments thereof, anti-TROP2 antibodies or antigen-binding fragments thereof, antigen-binding protein constructs (e.g., bispecific antibodies or antigen-binding fragments thereof) that specifically bind to two different antigens (e.g., HER2 and TROP2), and antibody drug conjugates comprising these antibodies or antigen-binding fragments thereof.

[0006] In one aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VH CDR3 amino acid sequence; a light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VL CDR3 amino acid sequence; The selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are as follows: (1) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 7 to 9, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (2) the selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 10 to 12, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (3) the selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (4) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 16 to 18, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (5) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19 to 21, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (6) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 22 to 24, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively; (7) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 25 to 27, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively; (8) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 28 to 30, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively; (9) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 31 to 33, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively; (10) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 34 to 36, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively; The present invention relates to an antibody or an antigen-binding fragment thereof that binds to HER2 (human epidermal growth factor receptor 2), which is one of the receptors.

[0007] In some embodiments, according to the Kabat numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 7 to 9, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1 to 3, respectively.

[0008] In some embodiments, according to the Kabat numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 10 to 12, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1 to 3, respectively.

[0009] In some embodiments, according to the Kabat numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 13 to 15, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 1 to 3, respectively.

[0010] In some embodiments, according to the Kabat numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 16 to 18, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1 to 3, respectively.

[0011] In some embodiments, according to the Kabat numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 19 to 21, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 1 to 3, respectively.

[0012] In some embodiments, according to the Chothia numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 22 to 24, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 4 to 6, respectively.

[0013] In some embodiments, according to the Chothia numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 25 to 27, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 4 to 6, respectively.

[0014] In some embodiments, according to the Chothia numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 28 to 30, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 4 to 6, respectively.

[0015] In some embodiments, according to the Chothia numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 31 to 33, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 4 to 6, respectively.

[0016] In some embodiments, according to the Chothia numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 34 to 36, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 4 to 6, respectively.

[0017] In some embodiments, the antibody or antigen-binding fragment specifically binds to human HER2 or canine HER2.

[0018] In some embodiments, the antibody or antigen-binding fragment is a human or humanized antibody or antigen-binding fragment thereof.

[0019] In some embodiments, the antibody or antigen-binding fragment is a single chain variable fragment (scFv).

[0020] In one aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: a heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are selected from the following: (1) the selected VH sequence is SEQ ID NO:38 and the selected VL sequence is SEQ ID NO:37; (2) the selected VH sequence is SEQ ID NO:39 and the selected VL sequence is SEQ ID NO:37; (3) the selected VH sequence is SEQ ID NO:40 and the selected VL sequence is SEQ ID NO:37; (4) the selected VH sequence is SEQ ID NO:41 and the selected VL sequence is SEQ ID NO:37; (5) the selected VH sequence is SEQ ID NO: 42 and the selected VL sequence is SEQ ID NO: 37; The present invention relates to an antibody or an antigen-binding fragment thereof that binds to HER2, which is one of the antibodies.

[0021] In some embodiments, the VH comprises the sequence of SEQ ID NO:38 and the VL comprises the sequence of SEQ ID NO:37.

[0022] In some embodiments, the VH comprises the sequence of SEQ ID NO:39 and the VL comprises the sequence of SEQ ID NO:37.

[0023] In some embodiments, the VH comprises the sequence of SEQ ID NO:40 and the VL comprises the sequence of SEQ ID NO:37.

[0024] In some embodiments, the VH comprises the sequence of SEQ ID NO:41 and the VL comprises the sequence of SEQ ID NO:37.

[0025] In some embodiments, the VH comprises the sequence of SEQ ID NO:42 and the VL comprises the sequence of SEQ ID NO:37.

[0026] In some embodiments, the antibody or antigen-binding fragment specifically binds to human HER2 or canine HER2.

[0027] In some embodiments, the antibody or antigen-binding fragment is a human or humanized antibody or antigen-binding fragment thereof.

[0028] In some embodiments, the antibody or antigen-binding fragment is a single chain variable fragment (scFv).

[0029] In one aspect, the disclosure relates to antibodies, or antigen-binding fragments thereof, that cross-compete with the antibodies, or antigen-binding fragments thereof, described herein.

[0030] In one aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3 identical to the VH CDR1, a VH CDR2, and a VH CDR3 of a selected VH sequence; and a light chain variable region (VL) that comprises a VL CDR1, a VL CDR2, and a VL CDR3 identical to the VL CDR1, a VL CDR2, and a VL CDR3 of the selected VL sequence; The selected VH sequence and the selected VL sequence are (1) the selected VH sequence is SEQ ID NO:38 and the selected VL sequence is SEQ ID NO:37; (2) the selected VH sequence is SEQ ID NO:39 and the selected VL sequence is SEQ ID NO:37; (3) the selected VH sequence is SEQ ID NO:40 and the selected VL sequence is SEQ ID NO:37; (4) the selected VH sequence is SEQ ID NO:41 and the selected VL sequence is SEQ ID NO:37; (5) the selected VH sequence is SEQ ID NO: 42 and the selected VL sequence is SEQ ID NO: 37; The present invention relates to an antibody or an antigen-binding fragment thereof that binds to HER2, which is one of the antibodies.

[0031] In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific or multispecific antibody or antigen-binding fragment thereof.

[0032] In some embodiments, the antibody or antigen-binding fragment thereof further specifically binds to TROP2.

[0033] In one aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: (1) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) including complementarity determining regions (CDRs) 1, 2, and 3 each including an amino acid sequence as set forth in SEQ ID NO: 7 to 9, wherein the VH binds to HER2 when paired with a light chain variable region (VL) including an amino acid sequence as set forth in SEQ ID NO: 37; (2) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence shown in SEQ ID NO: 10 to 12, wherein the VH binds to HER2 when paired with a VL comprising the amino acid sequence shown in SEQ ID NO: 37; (3) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence shown in SEQ ID NO: 13 to 15, wherein the VH binds to HER2 when paired with a VL comprising the amino acid sequence shown in SEQ ID NO: 37; (4) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence shown in SEQ ID NO: 16 to 18, wherein the VH binds to HER2 when paired with a VL comprising the amino acid sequence shown in SEQ ID NO: 37; (5) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence shown in SEQ ID NO: 19 to 21, wherein the VH binds to HER2 when paired with a VL comprising the amino acid sequence shown in SEQ ID NO: 37; (6) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence shown in SEQ ID NO: 22 to 24, wherein the VH binds to HER2 when paired with a VL comprising the amino acid sequence shown in SEQ ID NO: 37; (7) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence shown in SEQ ID NO: 25 to 27, wherein the VH binds to HER2 when paired with a VL comprising the amino acid sequence shown in SEQ ID NO: 37; (8) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence shown in SEQ ID NO: 28 to 30, wherein the VH binds to HER2 when paired with a VL comprising the amino acid sequence shown in SEQ ID NO: 37; (9) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence shown in SEQ ID NO: 31 to 33, wherein the VH binds to HER2 when paired with a VL comprising the amino acid sequence shown in SEQ ID NO: 37; (10) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence shown in SEQ ID NO: 34 to 36, wherein the VH binds to HER2 when paired with a VL comprising the amino acid sequence shown in SEQ ID NO: 37; (11) An immunoglobulin light chain or a fragment thereof, comprising a light chain variable region (VL) comprising complementarity determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 1 to 3, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 38; (12) An immunoglobulin light chain or a fragment thereof, comprising a light chain variable region (VL) including complementarity determining regions (CDRs) 1, 2, and 3, each of which includes an amino acid sequence as set forth in SEQ ID NO: 1 to 3, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) including an amino acid sequence as set forth in SEQ ID NO: 39; (13) An immunoglobulin light chain or a fragment thereof, comprising a light chain variable region (VL) comprising complementarity determining regions (CDRs) 1, 2, and 3, each of which comprises the amino acid sequence set forth in SEQ ID NO: 1 to 3, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 40; (14) An immunoglobulin light chain or a fragment thereof, comprising a light chain variable region (VL) comprising complementarity determining regions (CDRs) 1, 2, and 3, each of which comprises the amino acid sequence set forth in SEQ ID NO: 1 to 3, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 41; (15) An immunoglobulin light chain or a fragment thereof, comprising a light chain variable region (VL) comprising complementarity determining regions (CDRs) 1, 2, and 3, each of which comprises the amino acid sequence set forth in SEQ ID NO: 1 to 3, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 42; (16) An immunoglobulin light chain or a fragment thereof, comprising a light chain variable region (VL) including complementarity determining regions (CDRs) 1, 2, and 3 each including an amino acid sequence as set forth in SEQ ID NO: 4 to 6, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) including an amino acid sequence as set forth in SEQ ID NO: 38; (17) An immunoglobulin light chain or a fragment thereof, comprising a light chain variable region (VL) including complementarity determining regions (CDRs) 1, 2, and 3 each including an amino acid sequence as set forth in SEQ ID NO: 4 to 6, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) including an amino acid sequence as set forth in SEQ ID NO: 39; (18) An immunoglobulin light chain or a fragment thereof, comprising a light chain variable region (VL) including complementarity determining regions (CDRs) 1, 2, and 3 each including an amino acid sequence as set forth in SEQ ID NO: 4 to 6, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) including an amino acid sequence as set forth in SEQ ID NO: 40; (19) An immunoglobulin light chain or a fragment thereof, comprising a light chain variable region (VL) including complementarity determining regions (CDRs) 1, 2, and 3 each including an amino acid sequence as set forth in SEQ ID NO: 4 to 6, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) including an amino acid sequence as set forth in SEQ ID NO: 41; or (20) An immunoglobulin light chain or a fragment thereof, comprising a light chain variable region (VL) including complementarity determining regions (CDRs) 1, 2, and 3 each including an amino acid sequence as set forth in SEQ ID NO: 4 to 6, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) including an amino acid sequence as set forth in SEQ ID NO: 42; The present invention relates to a nucleic acid comprising a polynucleotide encoding a polypeptide comprising:

[0034] In some embodiments, the VH, when paired with the VL, specifically binds human HER2 or canine HER2.

[0035] In some embodiments, the immunoglobulin heavy chain or fragment thereof is a human or humanized immunoglobulin heavy chain or fragment thereof.

[0036] In some embodiments, the nucleic acid encodes a single chain variable fragment (scFv).

[0037] In some embodiments, the nucleic acid is cDNA.

[0038] In one aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VH CDR3 amino acid sequence; a light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VL CDR3 amino acid sequence; The selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are as follows: (1) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 43 to 45, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (2) the selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 46 to 48, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (3) the selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 49 to 51, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (4) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 52 to 54, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively; (5) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 55 to 57, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively; (6) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 58 to 60, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively; The present invention relates to an antibody or an antigen-binding fragment thereof that binds to TROP2 (trophoblast surface antigen 2).

[0039] In some embodiments, according to the Kabat numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 43 to 45, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 1 to 3, respectively.

[0040] In some embodiments, according to the Kabat numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 46 to 48, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 1 to 3, respectively.

[0041] In some embodiments, according to the Kabat numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 49 to 51, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 1 to 3, respectively.

[0042] In some embodiments, according to the Chothia numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 52 to 54, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 4 to 6, respectively.

[0043] In some embodiments, according to the Chothia numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 55 to 57, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 4 to 6, respectively.

[0044] In some embodiments, according to the Chothia numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 58 to 60, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 4 to 6, respectively.

[0045] In some embodiments, the antibody or antigen-binding fragment specifically binds to human TROP2 or canine TROP2.

[0046] In some embodiments, the antibody or antigen-binding fragment is a human or humanized antibody or antigen-binding fragment thereof.

[0047] In some embodiments, the antibody or antigen-binding fragment is a single chain variable fragment (scFv).

[0048] In one aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90% identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are as follows: (1) the selected VH sequence is SEQ ID NO:61 and the selected VL sequence is SEQ ID NO:37; (2) the selected VH sequence is SEQ ID NO:62 and the selected VL sequence is SEQ ID NO:37; (3) the selected VH sequence is SEQ ID NO:63 and the selected VL sequence is SEQ ID NO:37; The present invention relates to an antibody or antigen-binding fragment thereof that binds to TROP2, which is one of the antibodies.

[0049] In some embodiments, the VH comprises the sequence of SEQ ID NO:61 and the VL comprises the sequence of SEQ ID NO:37.

[0050] In some embodiments, the VH comprises the sequence of SEQ ID NO:62 and the VL comprises the sequence of SEQ ID NO:37.

[0051] In some embodiments, the VH comprises the sequence of SEQ ID NO:63 and the VL comprises the sequence of SEQ ID NO:37.

[0052] In some embodiments, the antibody or antigen-binding fragment specifically binds to human TROP2 or canine TROP2.

[0053] In some embodiments, the antibody or antigen-binding fragment is a human or humanized antibody or antigen-binding fragment thereof.

[0054] In some embodiments, the antibody or antigen-binding fragment is a single chain variable fragment (scFv).

[0055] In one aspect, the disclosure relates to antibodies, or antigen-binding fragments thereof, that cross-compete with the antibodies, or antigen-binding fragments thereof, described herein.

[0056] In one aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3 identical to the VH CDR1, a VH CDR2, and a VH CDR3 of a selected VH sequence; and a light chain variable region (VL) that comprises a VL CDR1, a VL CDR2, and a VL CDR3 identical to the VL CDR1, a VL CDR2, and a VL CDR3 of the selected VL sequence; The selected VH sequence and the selected VL sequence are (1) the selected VH sequence is SEQ ID NO:61 and the selected VL sequence is SEQ ID NO:37; (2) the selected VH sequence is SEQ ID NO:62 and the selected VL sequence is SEQ ID NO:37; (3) the selected VH sequence is SEQ ID NO:63 and the selected VL sequence is SEQ ID NO:37; The present invention relates to an antibody or antigen-binding fragment thereof that binds to TROP2, which is one of the antibodies.

[0057] In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific or multispecific antibody or antigen-binding fragment thereof.

[0058] In some embodiments, the antibody or antigen-binding fragment thereof further specifically binds to HER2.

[0059] In one aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: (1) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) including complementarity determining regions (CDRs) 1, 2, and 3, each of which includes an amino acid sequence as set forth in SEQ ID NO: 43 to 45, wherein the VH binds to TROP2 when paired with a light chain variable region (VL) including an amino acid sequence as set forth in SEQ ID NO: 37; (2) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence shown in SEQ ID NO: 46 to 48, wherein the VH binds to TROP2 when paired with a VL comprising the amino acid sequence shown in SEQ ID NO: 37; (3) An immunoglobulin heavy chain or a fragment thereof, comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence shown in SEQ ID NO: 49 to 51, wherein the VH binds to TROP2 when paired with a VL comprising the amino acid sequence shown in SEQ ID NO: 37; or (4) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence shown in SEQ ID NO: 52 to 54, wherein the VH binds to TROP2 when paired with a VL comprising the amino acid sequence shown in SEQ ID NO: 37; (5) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence shown in SEQ ID NO: 55 to 57, wherein the VH binds to TROP2 when paired with a VL comprising the amino acid sequence shown in SEQ ID NO: 37; (6) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence shown in SEQ ID NO: 58 to 60, wherein the VH binds to TROP2 when paired with a VL comprising the amino acid sequence shown in SEQ ID NO: 37; (7) An immunoglobulin light chain or a fragment thereof, comprising a light chain variable region (VL) comprising complementarity determining regions (CDRs) 1, 2, and 3, each of which comprises the amino acid sequence set forth in SEQ ID NO: 1 to 3, wherein the VL binds to TROP2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 61; (8) An immunoglobulin light chain or a fragment thereof, comprising a light chain variable region (VL) comprising complementarity determining regions (CDRs) 1, 2, and 3, each of which comprises the amino acid sequence set forth in SEQ ID NO: 1 to 3, wherein the VL binds to TROP2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 62; (9) An immunoglobulin light chain or a fragment thereof, comprising a light chain variable region (VL) comprising complementarity determining regions (CDRs) 1, 2, and 3, each of which comprises the amino acid sequence set forth in SEQ ID NO: 1 to 3, wherein the VL binds to TROP2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 63; (10) An immunoglobulin light chain or a fragment thereof, comprising a light chain variable region (VL) comprising complementarity determining regions (CDRs) 1, 2, and 3, each of which comprises the amino acid sequence set forth in SEQ ID NO: 4 to 6, wherein the VL binds to TROP2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 61; (11) An immunoglobulin light chain or a fragment thereof, comprising a light chain variable region (VL) comprising complementarity determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 4 to 6, wherein the VL binds to TROP2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 62; (12) An immunoglobulin light chain or a fragment thereof, comprising a light chain variable region (VL) comprising complementarity determining regions (CDRs) 1, 2, and 3, each comprising the amino acid sequence set forth in SEQ ID NO: 4 to 6, wherein the VL binds to TROP2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 63; The present invention relates to a nucleic acid comprising a polynucleotide encoding a polypeptide comprising:

[0060] In some embodiments, the VH, when paired with the VL, specifically binds human TROP2 or canine TROP2.

[0061] In some embodiments, the immunoglobulin heavy chain or fragment thereof is a human or humanized immunoglobulin heavy chain or fragment thereof.

[0062] In some embodiments, the nucleic acid encodes a single chain variable fragment (scFv).

[0063] In some embodiments, the nucleic acid is cDNA.

[0064] In one aspect, the present disclosure relates to an antigen binding protein construct comprising a first antigen binding domain that specifically binds to HER2 and a second antigen binding domain that specifically binds to TROP2.

[0065] In some embodiments, the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0066] In some embodiments, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to a selected VH1 CDR3 amino acid sequence, and the first light chain variable region (VL1) comprises CDR1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VL1 CDR3 amino acid sequence; The selected VH1 CDR1, 2, and 3 amino acid sequences and the selected VL1 CDR1, 2, and 3 amino acid sequences are as follows: (1) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 7 to 9, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (2) the selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 10 to 12, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (3) the selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (4) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 16 to 18, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (5) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19 to 21, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (6) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 22 to 24, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively; (7) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 25 to 27, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively; (8) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 28 to 30, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively; (9) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 31 to 33, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively; (10) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 34 to 36, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively; It is one of them.

[0067] In some embodiments, the second heavy chain variable region (VH2) comprises CDR1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VL2 CDR2 amino acid sequence, the VH2 CDR3 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VH2 CDR3 amino acid sequence, and a second light chain variable region (VL2) comprising CDR1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a selected VL2 CDR3 amino acid sequence, and wherein the selected VH2 CDR1, 2, and 3 amino acid sequence and the selected VL2 CDR1, 2, and 3 amino acid sequence are selected from the following: (1) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 43 to 45, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (2) the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 46 to 48, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (3) the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 49 to 51, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (4) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 52 to 54, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively; (5) the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 55 to 57, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively; (6) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 58 to 60, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively; It is one of them.

[0068] In some embodiments, (1) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 10 to 12, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 49 to 51, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (2) the selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19 to 21, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 49 to 51, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (3) the selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 7 to 9, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 49 to 51, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (4) the selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 7 to 9, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 46 to 48, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (5) the selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 10 to 12, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 46 to 48, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (6) the selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19 to 21, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 46 to 48, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (7) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 7 to 9, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 43 to 45, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (8) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 10 to 12, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 43 to 45, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (9) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19 to 21, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 43 to 45, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (10) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 49 to 51, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (11) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 46 to 48, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (12) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 43 to 45, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (13) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 16 to 18, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 49 to 51, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (14) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 16 to 18, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 46 to 48, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (15) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 16 to 18, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 43 to 45, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively;

[0069] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:39, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:63, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:37.

[0070] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:42, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:63, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:37.

[0071] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:38, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:63, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:37.

[0072] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:38, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:62, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:37.

[0073] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:39, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:62, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:37.

[0074] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:42, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:62, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:37.

[0075] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:38, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:61, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:37.

[0076] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:39, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:61, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:37.

[0077] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:42, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:61, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:37.

[0078] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:40, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:63, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:37.

[0079] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:41, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:63, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:37.

[0080] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:40, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:62, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:37.

[0081] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:41, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:62, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:37.

[0082] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:40, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:61, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:37.

[0083] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:41, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:61, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:37.

[0084] In some embodiments, the antigen binding protein construct is a bispecific antibody.

[0085] In some embodiments, the first light chain variable region and the second light chain variable region are identical.

[0086] In one aspect, the disclosure relates to a vector comprising one or more of the nucleic acids described herein, or a nucleic acid encoding an antibody or antigen-binding fragment thereof described herein, or a nucleic acid encoding an antigen-binding protein construct described herein.

[0087] In one aspect, the disclosure relates to a cell comprising the vector described herein.

[0088] In some embodiments, the cells are CHO cells.

[0089] In one aspect, the disclosure relates to a cell comprising one or more of the nucleic acids described herein, or a nucleic acid encoding an antibody or antigen-binding fragment thereof described herein, or a nucleic acid encoding an antigen-binding protein construct described herein.

[0090] In one aspect, the present disclosure provides a method for producing an antibody or antigen-binding fragment thereof, or an antigen-binding protein construct, the method comprising: (a) culturing a cell described herein under conditions sufficient for the cell to produce an antibody or antigen-binding fragment, or an antigen-binding protein construct; (b) harvesting the antibody or antigen-binding fragment thereof, or the antigen-binding protein construct produced by the cells; and The present invention relates to a method comprising the steps of:

[0091] In one aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: (a) an antibody or antigen-binding fragment thereof described herein; or (b) the antigen-binding protein construct described herein The present invention relates to an antibody drug conjugate comprising a therapeutic agent covalently attached to the antibody.

[0092] In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

[0093] In some embodiments, the therapeutic agent is MMAE or MMAF.

[0094] In one aspect, the present disclosure relates to a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein, an antigen-binding protein construct described herein, or an antibody drug conjugate described herein.

[0095] In some embodiments, the subject has a solid tumor.

[0096] In some embodiments, the cancer is thyroid cancer, urothelial cancer, breast cancer, colorectal cancer, renal cancer, cervical cancer, ovarian cancer, lung cancer, uterine cancer, skin cancer, gastric cancer, pancreatic cancer, prostate cancer, liver cancer, lymphoma, or glioma.

[0097] In some embodiments, the cancer is cervical cancer, prostate cancer, thyroid cancer, urothelial cancer, head and neck cancer, uterine cancer, ovarian cancer, lung cancer, breast cancer, carcinoid, skin cancer, liver cancer, or testicular cancer.

[0098] In some embodiments, the cancer is multiple myeloma or renal carcinoma.

[0099] In some embodiments, the subject is a human.

[0100] In some embodiments, the subject is a non-human animal.

[0101] In one aspect, the disclosure provides a method of reducing tumor growth rate, the method comprising: The present invention relates to a method comprising contacting a tumor cell with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein, an antigen-binding protein construct described herein, or an antibody drug conjugate described herein.

[0102] In one aspect, the present disclosure relates to a method of killing a tumor cell, the method comprising contacting the tumor cell with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein, an antigen-binding protein construct described herein, or an antibody drug conjugate described herein.

[0103] In one aspect, the present disclosure provides a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and (a) an antibody or antigen-binding fragment thereof described herein; (b) an antigen-binding protein construct as described herein, or (c) an antibody drug conjugate as described herein and a pharmaceutical composition comprising:

[0104] In one aspect, the present disclosure relates to an antigen binding protein construct comprising a first antigen binding domain that specifically binds to HER2 and a second antigen binding domain that specifically binds to TROP2.

[0105] As used herein, the term "antibody" refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementarity determining regions (CDRs) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody can contain an Fc region of a human antibody. The term antibody also includes derivatives, such as bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0106] As used herein, the term "human antibody" refers to an antibody encoded by endogenous nucleic acid derived from a human (e.g., a rearranged human immunoglobulin heavy or light chain locus). In some embodiments, a human antibody is recovered from a human or produced in human cell culture (e.g., in human hybridoma cells). In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line). In some embodiments, a human antibody is produced in a bacterial cell or a yeast cell. In some embodiments, a human antibody is produced in a transgenic non-human animal (e.g., a cow) that contains unrearranged or rearranged human immunoglobulin loci (e.g., a heavy or light chain human immunoglobulin locus).

[0107] As used herein, the term "chimeric antibody" refers to an antibody that contains sequences present in at least two different species (e.g., an antibody from two different mammalian species, such as a human and a murine antibody). A non-limiting example of a chimeric antibody is an antibody that contains variable domain sequences (e.g., all or a portion of the light and / or heavy chain variable domain sequences) of a non-human (e.g., murine) antibody and the constant domain of a human antibody. Further examples of chimeric antibodies are described herein and are well known in the art.

[0108] As used herein, the term "humanized antibody" refers to a non-human antibody that contains minimal sequence derived from non-human (e.g., murine) immunoglobulin and contains sequence derived from human immunoglobulin. In a non-limiting example, a humanized antibody is a human antibody (recipient antibody) in which residues from hypervariable (e.g., CDR) regions of the recipient antibody are replaced by residues from hypervariable (e.g., CDR) regions of a non-human antibody (e.g., donor antibody), such as a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity. In some embodiments, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., murine) immunoglobulin residues. In some embodiments, humanized antibodies can contain residues that are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance. In some embodiments, a humanized antibody contains nearly all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops (CDRs) corresponding to those of a non-human (e.g., murine) immunoglobulin and all or substantially all of the framework regions being those of a human immunoglobulin sequence. The humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Humanized antibodies can be produced using molecular biology techniques well known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.

[0109] As used herein, the term "antigen-binding protein construct" refers to (i) a single polypeptide comprising at least one antigen-binding domain, or (ii) a complex of two or more polypeptides (e.g., the same or different polypeptides) that together form at least one or more different antigen-binding domains. Non-limiting examples and aspects of antigen-binding protein constructs are described herein. Further examples and aspects of antigen-binding protein constructs are known in the art. In some embodiments, the antigen-binding protein construct has 1, 2, 3, 4, 5, 6, 7, 8, or more than 9 antigen-binding domains.

[0110] As used herein, the term "antigen-binding domain" refers to one or more protein domains (e.g., formed from amino acids from a single polypeptide, or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides) that can specifically bind to one or more different antigens). In some examples, an antigen-binding domain can bind to an antigen or epitope with specificity and affinity similar to that of a naturally occurring antibody. In some embodiments, the antigen-binding domain can be an antibody or a fragment thereof. One example of an antigen-binding domain is an antigen-binding domain formed by a VH-VL dimer. In some embodiments, the antigen-binding domain is a VHH. Non-limiting examples of antigen-binding domains are described herein. Further examples of antigen-binding domains are well known in the art. In some examples, an antigen-binding domain can bind to a single antigen.

[0111] As used herein, the term "bispecific antibody" refers to an antibody that binds to two different epitopes, which can be on the same antigen or on different antigens.

[0112] As used herein, the term "multispecific antibody" refers to an antibody that binds to two or more different epitopes, which can be on the same antigen or on different antigens.

[0113] As used herein, "VHH" refers to the variable domain of a heavy chain antibody. In some embodiments, the VHH is a humanized VHH.

[0114] As used herein, the term "common light chain" refers to a light chain capable of interacting with two or more different heavy chains to form different antigen-binding domains, which can specifically bind to different antigens or epitopes. Similarly, the term "common light chain variable region" refers to a light chain variable region capable of interacting with two or more different heavy chain variable regions to form different antigen-binding domains, which can specifically bind to different antigens or epitopes. In some embodiments, the antigen-binding constructs can have a common light chain. In some embodiments, the antigen-binding constructs can have a common light chain variable region.

[0115] As used herein, the phrases "specifically bind" and "specifically bind" when referring to an antibody mean that the antibody interacts with its target molecule (e.g., HER2) preferably in preference to other molecules because the interaction is dependent on the presence of a particular structure (i.e., an antigenic determinant or epitope) on the target molecule, in other words, because the reagent generally recognizes and binds to a molecule that contains a particular structure rather than to the entire molecule. An antibody that specifically binds to a target molecule may also be referred to as a target-specific antibody. For example, an antibody that specifically binds to a HER2 molecule may also be referred to as a HER2-specific antibody or an anti-HER2 antibody.

[0116] 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 for use in the present invention are described herein; other suitable methods and materials known in the art can also be used.Materials, methods, and examples are illustrative only and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control.

[0117] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0118] [Figure 1] FIG. 1 is a schematic diagram showing the structure of an exemplary anti-HER2 / TROP2 bispecific antibody. [Diagram 2] The results of endocytosis of purified antibodies in NCI-N87 cells are shown. [Diagram 3] The results of endocytosis of purified antibodies in NCI-N87 cells are shown. [Figure 4] 1 shows the results of endocytosis of purified antibodies in NCI-H292 cells. [Diagram 5] 1 shows the results of endocytosis of purified antibodies in NCI-H292 cells. [Figure 6] Figure 1 shows accelerated stability of anti-HER2 / TROP2 bispecific antibodies H-1H2-T-6F7, H-2B2-T-6F7, T-6F7-H-1H2, and H-3C8-T-6F7. ND: Not detectable. Freeze 1: 1 freeze-thaw; Freeze 10: 10 freeze-thaw cycles. [Figure 7] 1 is a graph showing the mean tumor volume in various groups of mice injected with lung adenocarcinoma cells and treated with various antibody drug conjugates (ADCs). [Figure 8]The CDR sequences of anti-HER2 antibodies H-1H2, H-2B2, H-3E5, H-3C6, and H-3C8, as defined by Kabat numbering, are listed. [Figure 9] The CDR sequences of anti-HER2 antibodies H-1H2, H-2B2, H-3E5, H-3C6, and H-3C8, as defined by Chothia numbering, are listed. [Figure 10] The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-HER2 antibody are listed below. [Figure 11] The CDR sequences of the anti-TROP2 antibodies T-3A4, T-4B9, T-4C12, T-5C8, and T-6F7, as defined by the Kabat numbering, are listed. [Figure 12] The CDR sequences of anti-TROP2 antibodies T-3A4, T-4B9, T-4C12, T-5C8, and T-6F7, as defined by Chothia numbering, are listed. [Figure 13] The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TROP2 antibody are listed below. [Figure 14-1] The amino acid sequences described in this disclosure are listed below. [Figure 14-2] The amino acid sequences described in this disclosure are listed below. [Figure 14-3] The amino acid sequences described in this disclosure are listed below. [Figure 14-4] The amino acid sequences described in this disclosure are listed below. [Figure 14-5] The amino acid sequences described in this disclosure are listed below. [Figure 15] 1 is a graph showing the mean tumor volume in various groups of mice injected with lung adenocarcinoma cells and treated with PBS, MMAE, H-2B2-T-6F7, or H-2B2-T-6F7-ADC. [Figure 16] 1 is a graph showing the mean tumor volume in various groups of mice injected with lung adenocarcinoma cells and treated with PBS, H-2B2-T-6F7-ADC, sacituzumab govitecan analog, dicitamab vedotin, or trastuzumab deruxtecan. [Figure 17]FIG. 13 is a graph showing the mean tumor volume in various groups of mice injected with ovarian cancer cells and treated with PBS, isotype control-ADC, H-2B2-T-6F7-ADC, H-2B2-IgG1-ADC, T-6F7-IgG1-SI-ADC, trastuzumab deruxtecan analog, or DS-1062 analog. [Figure 18] FIG. 13 is a graph showing the mean tumor volume in various groups of mice injected with breast cancer cells and treated with PBS, isotype control-ADC, H-2B2-T-6F7-ADC, H-2B2-IgG1-ADC, T-6F7-IgG1-SI-ADC, sacituzumab govitecan analog, dicitamab vedotin, or trastuzumab deruxtecan. [Figure 19] FIG. 13 is a graph showing the mean tumor volume in various groups of mice injected with patient-derived pancreatic tumor fragments and treated with 5 mg / kg of PBS, H-2B2-T-6F7-ADC, T-6F7-IgG1-SI-ADC, sacituzumab govitecan analog, dicitamab vedotin, or trastuzumab deruxtecan. [Figure 20] FIG. 13 is a graph showing the mean tumor volume in various groups of mice injected with patient-derived pancreatic tumor fragments and treated with 3 mg / kg of PBS, H-2B2-T-6F7-ADC, T-6F7-IgG1-SI-ADC, sacituzumab govitecan analog, dicitamab vedotin, or trastuzumab deruxtecan. [Figure 21A] 1 is a graph showing the average body weight in various groups of mice administered saline or H-2B2-T-6F7-ADC. [Figure 21B] 1 is a graph showing the mean body weight change in various groups of mice administered saline or H-2B2-T-6F7-ADC. [Figure 22] FIG. 13 shows the mean tumor volumes in various groups of mice injected with patient-derived colon tumor fragments and treated with PBS, 6 mg / kg H-2B2-T-6F7-ADC, 10 mg / kg sacituzumab govitecan analog, 6 mg / kg dicitamab vedotin, or 6 mg / kg trastuzumab deruxtecan. [Figure 23]FIG. 13 shows the mean tumor volumes in various groups of mice injected with patient-derived lung tumor fragments and treated with PBS, 3 mg / kg or 6 mg / kg H-2B2-T-6F7-ADC, 3 mg / kg H-2B2-IgG1-ADC, 3 mg / kg T-6F7-IgG1-SI-ADC, 10 mg / kg sacituzumab govitecan analog, 6 mg / kg dicatimab vedotin, or 6 mg / kg trastuzumab deruxtecan. [Figure 24] FIG. 13 shows the mean tumor volumes in various groups of mice injected with patient-derived gastric tumor fragments and treated with PBS, 3 mg / kg or 6 mg / kg H-2B2-T-6F7-ADC, 10 mg / kg sacituzumab govitecan analog, 6 mg / kg dicitamab vedotin, or 6 mg / kg trastuzumab deruxtecan. [Diagram 25] 1 shows the results of a plasma stability study for the H-2B2-T-6F7-ADC of the disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0119] An antibody or antigen-binding fragment thereof is an artificial protein that can simultaneously bind to two different epitopes (e.g., on two different antigens). In some embodiments, a bispecific antibody or antigen-binding fragment thereof can have two arms (arms A and B). Each arm has one heavy chain variable region and one light chain variable region, forming an antigen-binding domain (or antigen-binding region). In some embodiments, a bispecific antibody has a common light chain.

[0120] The present disclosure relates to anti-HER2 antibodies or antigen-binding fragments thereof, anti-TROP2 antibodies or antigen-binding fragments thereof, antigen-binding protein constructs (e.g., bispecific antibodies or antigen-binding fragments thereof) that specifically bind to two different antigens (e.g., HER2 and TROP2), and antibody drug conjugates.

[0121] Anti-HER2 antibodies and antigen-binding fragments Human epidermal growth factor receptor 2 (HER2), also known as ERBB2, is a transmembrane receptor that belongs to the epidermal growth factor receptor subfamily of receptor protein tyrosine kinases.

[0122] HER2 is reported to be overexpressed in various types of cancer, such as breast cancer and gastric cancer, and to be a negative prognostic factor in breast cancer. Trastuzumab, trastuzumab emtansine, pertuzumab, lapatinib, and other anti-HER2 drugs that are effective against HER2-overexpressing cancers are known.

[0123] The present disclosure provides several antibodies and antigen-binding fragments thereof that specifically bind to HER2. In some embodiments, anti-HER2 / TROP2 antigen-binding protein constructs (e.g., bispecific antibodies) can comprise antigen-binding regions derived from these antibodies.

[0124] The antibodies and antigen-binding fragments described herein are capable of binding to HER2. The disclosure provides, for example, anti-HER2 antibodies H-1H2 ("1H2"), H-2B2 ("2B2"), H-3E5 ("3E5"), H-3C6 ("3C6"), H-3C8 ("3C8"), and antibodies derived therefrom.

[0125] CDR sequences for 1H2 and antibodies derived from 1H2 (e.g., humanized antibodies) include the heavy chain variable domain CDRs SEQ ID NOs: 7-9 and the light chain variable domain CDRs SEQ ID NOs: 1-3, as defined by Kabat numbering. CDRs can also be defined by the Chothia system. According to Chothia numbering, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOs: 22-24 and the light chain variable domain CDR sequences are set forth in SEQ ID NOs: 4-6.

[0126] Similarly, CDR sequences for 2B2 and antibodies derived from 2B2 include the heavy chain variable domain CDRs SEQ ID NOs: 10-12 and the light chain variable domain CDRs SEQ ID NOs: 1-3 as defined by Kabat numbering. According to Chothia numbering, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOs: 25-27 and the light chain variable domain CDRs are set forth in SEQ ID NOs: 4-6.

[0127] CDR sequences for 3E5 and antibodies derived from 3E5 include the heavy chain variable domain CDRs SEQ ID NOs: 13-15 and the light chain variable domain CDRs SEQ ID NOs: 1-3 as defined by Kabat numbering. According to Chothia numbering, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOs: 28-30 and the light chain variable domain CDRs are set forth in SEQ ID NOs: 4-6.

[0128] CDR sequences for 3C6 and antibodies derived from 3C6 include the heavy chain variable domain CDRs SEQ ID NOs: 16-18 and the light chain variable domain CDRs SEQ ID NOs: 1-3, as defined by Kabat numbering. According to Chothia numbering, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOs: 31-33 and the light chain variable domain CDRs are set forth in SEQ ID NOs: 4-6.

[0129] CDR sequences for 3C8 and antibodies derived from 3C8 include the heavy chain variable domain CDRs SEQ ID NOs: 19-21 and the light chain variable domain CDRs SEQ ID NOs: 1-3 as defined by Kabat numbering. According to Chothia numbering, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOs: 34-36 and the light chain variable domain CDRs are set forth in SEQ ID NOs: 4-6.

[0130] Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may also contain one, two, or three heavy chain variable region CDRs selected from the group consisting of SEQ ID NOs: 7 to 9, SEQ ID NOs: 10 to 12, SEQ ID NOs: 13 to 15, SEQ ID NOs: 16 to 18, SEQ ID NOs: 19 to 21, SEQ ID NOs: 22 to 24, SEQ ID NOs: 25 to 27, SEQ ID NOs: 28 to 30, SEQ ID NOs: 31 to 33, and SEQ ID NOs: 34 to 36; and / or one, two, or three light chain variable region CDRs selected from the group consisting of SEQ ID NOs: 1 to 3, and SEQ ID NOs: 4 to 6.

[0131] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR3 amino acid sequence, and a light chain variable region (VL) comprising CDR1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VL CDR1 amino acid sequence, and the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VL CDR3 amino acid sequence. The light chain variable region (VL) may comprise or consist of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to the CDR2 amino acid sequence, and said CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VL CDR3 amino acid sequence. Selected VH CDR1, 2, 3 amino acid sequences and selected VL CDR1, 2, 3 amino acid sequences are shown in Figure 8 (Kabat CDRs) and Figure 9 (Chothia CDRs).

[0132] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the CDRs of SEQ ID NO:7 with 0, 1 or 2 amino acid insertions, deletions or substitutions, SEQ ID NO:8 with 0, 1 or 2 amino acid insertions, deletions or substitutions, or SEQ ID NO:9 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0133] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO:10 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO:11 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO:12 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0134] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 13 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 15 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0135] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 16 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 18 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0136] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the CDRs of SEQ ID NO: 19 with 0, 1 or 2 amino acid insertions, deletions or substitutions, SEQ ID NO: 20 with 0, 1 or 2 amino acid insertions, deletions or substitutions, or SEQ ID NO: 21 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0137] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the CDRs of SEQ ID NO: 22 with 0, 1 or 2 amino acid insertions, deletions or substitutions, SEQ ID NO: 23 with 0, 1 or 2 amino acid insertions, deletions or substitutions, or SEQ ID NO: 24 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0138] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 25 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 26 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 27 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0139] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO:28 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO:29 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO:30 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0140] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 31 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 32 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 33 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0141] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the CDRs of SEQ ID NO: 34 with 0, 1 or 2 amino acid insertions, deletions or substitutions, SEQ ID NO: 35 with 0, 1 or 2 amino acid insertions, deletions or substitutions, or SEQ ID NO: 36 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0142] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the CDRs of SEQ ID NO:1 with 0, 1 or 2 amino acid insertions, deletions or substitutions, SEQ ID NO:2 with 0, 1 or 2 amino acid insertions, deletions or substitutions, or SEQ ID NO:3 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0143] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the CDRs of SEQ ID NO:4 with 0, 1 or 2 amino acid insertions, deletions or substitutions, SEQ ID NO:5 with 0, 1 or 2 amino acid insertions, deletions or substitutions, or SEQ ID NO:6 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0144] The insertions, deletions and substitutions can be within the CDR sequences or at either or both termini of the CDR sequences.

[0145] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to HER2. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) that comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) that comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 38, 39, 40, 41, or 42, and the selected VL sequence is SEQ ID NO: 37.

[0146] In some embodiments, an antibody or antigen-binding fragment thereof may have three VH CDRs identical to the CDRs of any VH sequence described herein. In some embodiments, an antibody or antigen-binding fragment thereof may have three VL CDRs identical to the CDRs of any VL sequence described herein.

[0147] The disclosure also provides a nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises the CDRs shown in Figure 8 or Figure 9, or has the sequence shown in Figure 10. When the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to HER2 (e.g., human HER2).

[0148] The anti-HER2 antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments, as well as multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multispecific (multimeric, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibodies or antigen-binding fragments thereof are IgG antibodies or antigen-binding fragments thereof.

[0149] Antibody fragments are suitable for use in the methods provided, so long as they retain the desired affinity and specificity of the full-length antibody. Thus, antibody fragments that bind to HER2 retain the ability to bind to HER2. Fv fragments are antibody fragments that contain a complete antigen recognition and binding site. This region is composed of a tightly associated dimer of one heavy chain variable domain and one light chain variable domain, which can be essentially covalent, for example in scFv. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Overall, the six CDRs or a subset thereof collectively confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) can have the ability to recognize and bind to an antigen, but usually with a lower affinity than the entire binding site.

[0150] Anti-TROP2 antibodies and antigen-binding fragments Trophoblast surface antigen 2 (TROP2), also known as tumor-associated calcium signaling substance 2 (TACSTD2), is a cell surface glycoprotein that is encoded and expressed by the TACSTD2 gene. It has a structural sequence very similar to that of the epithelial adhesion molecule Epcam. TROP2 is a protein closely related to tumors. TROP2 mainly promotes tumor cell growth, proliferation, and metastasis by controlling calcium ion signaling pathways, cyclin expression, and reducing fibronectin adhesion. Studies have found that TROP2 protein is highly expressed in breast cancer, colon cancer, bladder cancer, gastric cancer, oral squamous cell carcinoma, and ovarian cancer. The protein can promote tumor cell proliferation, invasion, metastasis, spreading, and other processes. In addition, it has also been found that high expression of TROP2 in breast cancer and other cancers is closely related to more aggressive disease of tumors and poor clinical prognosis.

[0151] The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to TROP2. Anti-HER2 / TROP2 antigen-binding protein constructs (e.g., bispecific antibodies) can comprise antigen-binding regions derived from these antibodies.

[0152] The antibodies and antigen-binding fragments described herein are capable of binding to TROP2. The disclosure provides anti-TROP2 antibodies T-3A4 ("3A4"), T-4B9 ("4B9"), and T-6F7 ("6F7"), and antibodies derived therefrom.

[0153] CDR sequences for 3A4 and antibodies derived from 3A4 (e.g., humanized antibodies) include the heavy chain variable domain CDRs SEQ ID NOs: 43-45 and the light chain variable domain CDRs SEQ ID NOs: 1-3, as defined by Kabat numbering. CDRs can also be defined by the Chothia system. According to Chothia numbering, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOs: 52-54 and the light chain variable domain CDR sequences are set forth in SEQ ID NOs: 4-6.

[0154] Similarly, CDR sequences for 4B9 and antibodies derived from 4B9 include the heavy chain variable domain CDRs SEQ ID NOs: 46-48 and the light chain variable domain CDRs SEQ ID NOs: 1-3 as defined by Kabat numbering. According to Chothia numbering, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOs: 55-57 and the light chain variable domain CDRs are set forth in SEQ ID NOs: 4-6.

[0155] CDR sequences for 6F7 and 6F7-derived antibodies include the heavy chain variable domain CDRs SEQ ID NOs: 49-51 and the light chain variable domain CDRs SEQ ID NOs: 1-3 as defined by Kabat numbering. According to Chothia numbering, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOs: 58-60 and the light chain variable domain CDRs are set forth in SEQ ID NOs: 4-6.

[0156] Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may also contain one, two, or three heavy chain variable region CDRs selected from the group consisting of SEQ ID NOs: 43 to 45, SEQ ID NOs: 46 to 48, SEQ ID NOs: 49 to 51, SEQ ID NOs: 52 to 54, SEQ ID NOs: 55 to 57, and SEQ ID NOs: 58 to 60, and / or one, two, or three light chain variable region CDRs selected from the group consisting of SEQ ID NOs: 1 to 3 and SEQ ID NOs: 4 to 6.

[0157] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR3 amino acid sequence, and a light chain variable region (VL) comprising CDR1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VL CDR1 amino acid sequence, and the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VL CDR3 amino acid sequence. The antibody may have a light chain variable region (VL) that comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to the CDR2 amino acid sequence, and said CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VL CDR3 amino acid sequence. Selected VH CDR1, 2, 3 amino acid sequences and selected VL CDR1, 2, 3 amino acid sequences are shown in Figure 11 (Kabat CDRs) and Figure 12 (Chothia CDRs).

[0158] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the CDRs of SEQ ID NO: 43 with 0, 1 or 2 amino acid insertions, deletions or substitutions; SEQ ID NO: 44 with 0, 1 or 2 amino acid insertions, deletions or substitutions; or SEQ ID NO: 45 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0159] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the CDRs of SEQ ID NO: 46 with 0, 1 or 2 amino acid insertions, deletions or substitutions; SEQ ID NO: 47 with 0, 1 or 2 amino acid insertions, deletions or substitutions; or SEQ ID NO: 48 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0160] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the CDRs of SEQ ID NO: 49 with 0, 1 or 2 amino acid insertions, deletions or substitutions, SEQ ID NO: 50 with 0, 1 or 2 amino acid insertions, deletions or substitutions, or SEQ ID NO: 51 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0161] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the CDRs of SEQ ID NO: 52 with 0, 1 or 2 amino acid insertions, deletions or substitutions, SEQ ID NO: 53 with 0, 1 or 2 amino acid insertions, deletions or substitutions, or SEQ ID NO: 54 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0162] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the CDRs of SEQ ID NO: 55 with 0, 1 or 2 amino acid insertions, deletions or substitutions, SEQ ID NO: 56 with 0, 1 or 2 amino acid insertions, deletions or substitutions, or SEQ ID NO: 57 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0163] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the CDRs of SEQ ID NO: 58 with 0, 1 or 2 amino acid insertions, deletions or substitutions, SEQ ID NO: 59 with 0, 1 or 2 amino acid insertions, deletions or substitutions, or SEQ ID NO: 60 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0164] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the CDRs of SEQ ID NO:1 with 0, 1 or 2 amino acid insertions, deletions or substitutions, SEQ ID NO:2 with 0, 1 or 2 amino acid insertions, deletions or substitutions, or SEQ ID NO:3 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0165] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the CDRs of SEQ ID NO:4 with 0, 1 or 2 amino acid insertions, deletions or substitutions, SEQ ID NO:5 with 0, 1 or 2 amino acid insertions, deletions or substitutions, or SEQ ID NO:6 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0166] The insertions, deletions and substitutions can be within the CDR sequences or at either or both termini of the CDR sequences.

[0167] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to TROP2. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) that comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) that comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO:61, 62, or 63, and the selected VL sequence is SEQ ID NO:37.

[0168] In some embodiments, an antibody or antigen-binding fragment thereof may have three VH CDRs identical to the CDRs of any VH sequence described herein. In some embodiments, an antibody or antigen-binding fragment thereof may have three VL CDRs identical to the CDRs of any VL sequence described herein.

[0169] The disclosure also provides a nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain comprising the CDRs shown in Figure 11 or Figure 12, or having the sequence shown in Figure 13. When the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to TROP2.

[0170] Anti-TROP2 antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments, as well as multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multispecific (multimeric, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibodies or antigen-binding fragments thereof are IgG antibodies or antigen-binding fragments thereof.

[0171] Antibody fragments are suitable for use in the methods provided, so long as they retain the desired affinity and specificity of the full-length antibody. Thus, antibody fragments that bind to TROP2 retain the ability to bind to TROP2. Fv fragments are antibody fragments that contain a complete antigen recognition and binding site. This region is composed of a tightly associated dimer of one heavy chain variable domain and one light chain variable domain, which can essentially be covalently linked, for example in scFv. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Overall, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) can have the ability to recognize and bind to an antigen, but usually with a lower affinity than the entire binding site.

[0172] Antibodies, antigen-binding fragments, and antigen-binding protein constructs The present disclosure provides an antibody, an antigen-binding fragment thereof, or an antigen-binding protein construct (e.g., a bispecific antibody). The antigen-binding protein construct (e.g., a bispecific antibody) can include an anti-HER2 antibody or an antigen-binding fragment thereof, and an anti-TROP2 antibody or an antigen-binding fragment thereof. These antigen-binding protein constructs (e.g., a bispecific antibody), anti-HER2 antibody, anti-TROP2 antibody, and antigen-binding fragment thereof can have various forms.

[0173] Generally, antibodies (also called immunoglobulins) can be composed of two classes of polypeptide chains, light chains and heavy chains. A non-limiting antibody of the present disclosure can be an intact four immunoglobulin chain antibody, including two heavy chains and two light chains. The heavy chain of the antibody can be of any isotype, including IgM, IgG, IgE, IgA, or IgD, or a subisotype, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. An antibody can include two identical copies of a light chain and / or two identical copies of a heavy chain. The heavy chains, each of which contains one variable domain (or variable region, VH) and multiple constant domains (or constant regions), are bound to each other via disulfide bonds within their constant domains to form the "stem" of the antibody. Each light chain, which contains one variable domain (or variable region, VL) and one constant domain (or constant region), is bound to one heavy chain via disulfide bonds. The variable region of each light chain aligns with the variable region of the heavy chain to which it is bound. The variable regions of both the light and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR).

[0174] The hypervariable regions, known as complementarity determining regions (CDRs), form the loops that comprise the principal antigen-binding surface of an antibody. The four framework regions largely conform to a β-sheet structure, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs of each chain are held in close proximity by the framework regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding region.

[0175] Methods for identifying CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and several definitions of CDR are commonly used: the Kabat definition is based on sequence variability, and the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described, for example, in Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan, et al., "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular immunology 45.14 (2008): 3832-3839; Wu, TT and Kabat, EA (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991); Morea et al., Biophys Chem. 68 (1-3): 9-16 (Oct. 1997); Morea et al., J Mol Biol. 275 (2): 269-94 (Jan. .1998), Chothia et al., Nature 342(6252):877-83 (Dec.1989), and Ponomarenko and Bourne, BMC Structural Biology 7:64(2007), each of which is incorporated by reference in its entirety.

[0176] CDRs are important for recognizing the epitope of an antigen. As used herein, an "epitope" is the smallest part of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be about 3, 4, 5, 6, or 7 amino acids, but these amino acids do not need to be in a contiguous linear sequence in the primary structure of the antigen, since the epitope can depend on the three-dimensional structure of the antigen based on the secondary and tertiary structure of the antigen.

[0177] In some embodiments, antibodies are intact immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved and differ in their constant regions, particularly the hinge and upper CH2 domains. The sequences and differences of IgG subclasses are well known in the art and are described, for example, in Vidarsson, et al., "IgG subclasses and allotypes: from structure to effector functions." Frontiers in immunology 5 (2014); Irani, et al., "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases." Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016, each of which is incorporated by reference herein in its entirety.

[0178] An antibody can also be an immunoglobulin molecule from any species (e.g., human, rodent, mouse, rat, camel). Antibodies disclosed herein include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies, and chimeric antibodies comprising an immunoglobulin binding domain fused to another polypeptide. An antigen-binding domain or antigen-binding fragment is any portion of an antibody that retains the specific binding activity of the intact antibody, i.e., that is capable of specifically binding to an epitope on the target molecule of the intact antibody. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, an antibody or antigen-binding fragment thereof can be, for example, an scFv, Fv, Fd, dAb, bispecific antibody, bispecific scFv, diabody, linear antibody, single chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide that includes a binding domain that is an antibody binding domain, or is homologous thereto. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs derived from either the heavy or light chain of an intact antibody.

[0179] In some embodiments, an scFv has two heavy chain variable domains and two light chain variable domains, and in some embodiments, an scFv has two antigen-binding regions (antigen-binding regions: A and B), each capable of binding to a target antigen with different affinities.

[0180] In some embodiments, the antigen-binding fragment can form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single chain variable fragment (scFv) described herein fused to the CD3ζ transmembrane and endodomain. In some embodiments, the chimeric antigen receptor also comprises intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD28-41BB, or CD3z-CD28-OX40, for increased potency. Thus, in one aspect, the present disclosure further provides a cell (e.g., a T cell) expressing a chimeric antigen receptor described herein.

[0181] In some embodiments, an antibody, antigen-binding fragment thereof, or antigen-binding protein construct (eg, a bispecific antibody) is capable of binding to two different antigens or two different epitopes.

[0182] In some embodiments, an antibody, antigen-binding fragment thereof, or antigen-binding protein construct (e.g., a bispecific antibody) can comprise one, two, or three heavy chain variable region CDRs selected from Figures 8, 9, 11, and 12. In some embodiments, an antibody, antigen-binding fragment thereof, or antigen-binding protein construct (e.g., a bispecific antibody) can comprise one, two, or three light chain variable region CDRs selected from Figures 8, 9, 11, and 12.

[0183] In some embodiments, the antibodies, antigen-binding fragments thereof, or antigen-binding protein constructs (e.g., bispecific antibodies) described herein can be conjugated to a therapeutic agent. The antibody drug conjugates comprising the antibodies or antigen-binding fragments thereof can be covalently or non-covalently bound to the therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., monomethylauristatin E, monomethylauristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, maytansinoids (such as DM-1 and DM-4), dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs).

[0184] Antibody multimerization can be achieved by natural aggregation of antibodies or by chemical or recombinant conjugation techniques well known in the art. For example, a percentage of purified antibody preparations (e.g., purified IgG1 molecules) naturally form protein aggregates containing antibody homodimers and other higher order antibody multimers.

[0185] In some embodiments, the multispecific antibody is a bispecific antibody. Bispecific antibodies can be produced by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface can contain at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is created in the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated by reference in its entirety.

[0186] Any of the antibodies, antigen-binding fragments thereof, or antigen-binding protein constructs (e.g., bispecific antibodies) described herein can be conjugated with a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of the antibody or antigen-binding fragment in vitro (e.g., when stored in tissue culture medium or as a pharmaceutical composition) or in vivo (e.g., in humans).

[0187] Antibodies, antigen-binding fragments thereof, or antigen-binding protein constructs (e.g., bispecific antibodies) can also take various forms. Many different formats of antigen-binding constructs are known in the art and are described, for example, in Suurs, et al. A review of bispecific antibodies and antibody constructs in oncology and clinical challenges, Pharmacology & therapeutics (2019), which is incorporated herein by reference in its entirety.

[0188] In some embodiments, the antigen binding protein construct is BiTe, (scFv)2, Nanobody, Nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiabody-HAS, or Tandem-scFv. In some embodiments, the antigen binding protein construct is selected from the group consisting of VHH-scAb, VHH-Fab, Dual scFab, F(ab')2, diabody, crossMab, DAF (2in1), DAF(4in1), DutaMab, DT-IgG, knobs-in-hole common light chain, knobs-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, orthogonal Fab, DVD-IgG, 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, DVI-IgG, diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, tandem scFv-Fc, Intrabody, Dock and Lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body, or scFv1-PEG-scFv2.

[0189] In some embodiments, the antigen binding protein construct can be a TrioMab, in which the two heavy chains are derived from different species and different sequences limit heavy-light chain pairing.

[0190] In some embodiments, the antigen binding protein construct has two different heavy chains and one common light chain. The heterodimerization of the heavy chains can be based on knobs-in-holes or some other heavy chain pairing technique.

[0191] In some embodiments, bispecific antibodies can be made using CrossMAb technology. CrossMAb technology can be used to enhance correct light chain association in bispecific heterodimeric IgG antibodies, allowing the generation of a variety of bispecific antibody formats, including bivalent (1+1), trivalent (2+1), and tetravalent (2+2) bispecific antibodies, as well as non-Fc tandem antigen-binding fragment (Fab)-based antibodies. These formats can be derived from any existing antibody pair using domain crossover without the need for identification of a common light chain, post-translational processing / in vitro chemical assembly, or the introduction of a series of mutations that enhance correct light chain association. This method is described in Klein et al., "The use of CrossMAb technology for the generation of bi-and multispecific antibodies." MAbs. Vol. 8. No. 6. Taylor & Francis, 2016, which is incorporated by reference in its entirety. In some embodiments, the CH1 in the heavy chain and the CL domain in the light chain are swapped.

[0192] The antigen-binding protein construct can be a Duobody. The Fab exchange mechanism naturally occurring in IgG4 antibodies is mimicked in a controlled manner in IgG1 antibodies, a mechanism called controlled Fab exchange. This format can ensure specific pairing between heavy and light chains.

[0193] In dual variable domain antibodies (DVD-Ig), an additional VH and variable light (VL) domain is added to each N-terminus for dual specific targeting. This format is similar to IgG-scFv, but the additional binding domains are individually attached to the corresponding N-terminus of each heavy chain instead of to the scFv.

[0194] In scFv-IgG, two scFvs are linked to the C-terminus of the heavy chain (CH3). The scFv-IgG format has two different bivalent binding sites, and is therefore also called tetravalent. In scFv-IgG, there is no problem with pairing of heavy and light chains.

[0195] In some embodiments, the antigen-binding protein construct can have an IgG-IgG format: two intact IgG antibodies are conjugated by chemically linking the C-termini of the heavy chains.

[0196] The antigen-binding protein construct can also have a Fab-scFv-Fc format, in which a light chain, a heavy chain and a third chain containing an Fc region and an scFv are assembled, which can ensure efficient production and purification.

[0197] In some embodiments, the antigen-binding protein construct can be TF. Three Fab fragments are linked by disulfide bridges. Two fragments target tumor-associated antigens (TAA) and one fragment targets a hapten. The TF format does not have an Fc region.

[0198] ADAPTIR has two scFvs attached on either side of an Fc region, which discards intact IgG as the basis for its construction but preserves the Fc region, extending half-life and facilitating purification.

[0199] A bispecific T cell engager ("BiTE") is composed of two scFvs, a VLA, a VHA, and a VHB VLB on a single peptide chain. BiTEs have a binding domain and no Fc region.

[0200] In BiTE-Fc, an Fc region is fused to the BiTE construct. Addition of the Fc region extends the half-life, provides a longer effective concentration, and avoids continuous IV.

[0201] Dual affinity retargeting (DART) has two peptide chains linking the opposite fragments (i.e., VLA to VHB, VLB to VHA) and a sulfur bond fusing them together at the C-terminus. In DART, the sulfur bond can improve stability over BiTE.

[0202] In DART-Fc, the Fc region is linked to the DART. It can be generated by assembling three chains (two via disulfide bonds, as in DART). One chain contains half of the Fc region, which dimerizes with the third chain, expressing only the Fc region. The addition of the Fc region extends the half-life, provides a longer effective concentration, and avoids continuous IV.

[0203] In tetravalent DART, four peptide chains are assembled. Essentially, two DART molecules are made with half of the Fc region and dimerize. This format has bivalent binding to both targets, therefore it is a tetravalent molecule.

[0204] Tandem diabodies (TandAb) contain two diabodies. Each diabody is composed of a VHA and a VLB fragment, and a VHA and a VLB fragment, covalently associated. The two diabodies are linked by a peptide chain. This can improve stability over diabodies composed of two scFvs. It has two bivalent binding sites.

[0205] The scFv-scFv-toxin comprises a toxin and two scFvs with stabilizing linkers, which can be used for specific delivery of a payload.

[0206] In the molecule scFv-scFv-scFv, an scFv directed against a TAA is tagged with a short recognizable peptide that is assembled against a bsAb composed of two scFvs, one directed against CD3 and one directed against a recognizable peptide.

[0207] In ImmTACs, a stabilized and soluble T cell receptor is fused to an scFv that recognizes CD3. Using the TCR, ImmTACs are directed against targets to be processed, such as intracellular proteins.

[0208] Trispecific nanobodies have two single variable domains (nanobodies) that contain an additional molecule for half-life extension. An additional module is added to extend the half-life.

[0209] In the Trispecific Killer Engager (TriKE), two scFvs are linked via a polypeptide linker that incorporates human IL-15. The linker for IL-15 is added to increase NK survival and proliferation.

[0210] In some embodiments, the antigen-binding protein construct is a bispecific antibody. In some embodiments, the bispecific antibody of the present disclosure is designed to be 1+1 (monovalent for each target) and has an IgG1 subtype structure. This can reduce the binding activity to cells with low expression levels of HER2 and TROP2, and can increase the binding activity to cells that co-express HER2 and TROP2, thereby achieving improved targeting function. Mutations S239D and / or I332E (SI mutations) can also be introduced into the antibody heavy chain to enhance the antibody affinity for FcγRIIIA.

[0211] In some embodiments, the antibody, antigen-binding fragment thereof, antigen-binding protein construct (e.g., an anti-TROP2 antibody, an anti-HER2 antibody, or a bispecific antibody), or related antibody drug conjugate (ADC) has a light chain constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:80, and a heavy chain constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:81 or SEQ ID NO:82.

[0212] In some embodiments, the bispecific antibodies or antigen-binding fragments thereof described herein share a common light chain.

[0213] Antibody Drug Conjugates (ADCs) The antibodies, antigen-binding fragments thereof, or antigen-binding protein constructs (e.g., bispecific antibodies) described herein can be conjugated with a therapeutic agent (drug). The therapeutic agent can be covalently or non-covalently attached to the antibody or antigen-binding fragment, or antigen-binding protein construct (e.g., bispecific antibody). In some embodiments, the bispecific antibody is an anti-HER2 / TROP2 bispecific antibody. In some embodiments, the bispecific antibody has a common light chain.

[0214] In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., monomethylauristatin E, monomethylauristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, maytansinoids (such as DM-1 and DM-4), dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, and analogs). Useful classes of cytotoxic, cytostatic, or immunoregulatory agents include, for example, antitubulin agents, DNA minor groove binding agents, DNA replication inhibitors, and alkylating agents.

[0215] In some embodiments, the therapeutic agent may include, but is not limited to, a cytotoxic agent (e.g., chemotherapeutic agent, immunotherapeutic agent, etc.), an antiviral agent, or an antibacterial agent. In some embodiments, the conjugable therapeutic agent may be selected from, but is not limited to, MMAE (monomethylauristatin E), MMAD (monomethylauristatin D), or MMAF (monomethylauristatin F).

[0216] In some embodiments, the therapeutic agent is an auristatin, such as auristatin E (also known in the art as a derivative of dolastatin-10), or a derivative thereof. The auristatin can be, for example, an ester formed with auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other exemplary auristatins include AFP, MMAF, and MMAE. The synthesis and structures of exemplary auristatins are described in U.S. Patent Publication No. 2003-0083263; International Patent Publication No. WO 04 / 010957; International Patent Publication No. WO 02 / 088172, as well as U.S. Patent Nos. 7,498,298; 6,884,869; 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,52 Nos. 1,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414, each of which is incorporated by reference herein in its entirety for all purposes.

[0217] Auristatins have been shown to interfere with microtubule dynamics, as well as nuclear and cell division, and have been shown to have anti-cancer activity. Auristatins can bind to tubulin and exert cytotoxic or cytostatic effects in cancer cells. There are many different assays known in the art that can be used to determine whether auristatins or the resulting antibody-drug conjugates exert cytostatic or cytotoxic effects in the desired cells.

[0218] In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; chlorambucil, chlornaphazine, colofosphamide, estram, and the like. Nitrogen mustards such as sucrine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, and chromomycin. Antibiotics such as mycobacterium, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, keramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; methotrexate and 5-fluorouracil (5- FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxiridine, 5-FU; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone;Antiadrenal agents such as aminoglutethimide, mitotein, and trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elfomitine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phena Met; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK7; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxanes, such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ), doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France), chlorambucil, gemcitabine, 6-thioguanine, platinum analogs such as cisplatin or carboplatin, vinblastine, platinum, etoposide (VP-16), ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, navelbine, novantrone, teniposide, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, the topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition includes, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)imidazoles, 4-hydroxytamoxifen, trioxyphene, ketoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;Also included are anti-hormonal agents that act to regulate or inhibit hormone action in tumors, such as anti-estrogens, including pharma- ceutically acceptable salts, acids, or derivatives of any of the above. A detailed description of chemotherapeutic agents can be found, for example, in US20180193477A1, which is incorporated by reference in its entirety.

[0219] In some embodiments, the antigen-binding construct is coupled to the drug via a cleavable linker, such as an SPBD linker or a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker. In some embodiments, the antigen-binding construct is coupled to the drug via a non-cleavable linker, such as an MCC linker formed with SMCC or sulfo-SMCC. The selection of an appropriate linker for a given ADC can be readily made by a person skilled in the art with knowledge of the art and taking into account relevant factors such as the site of attachment to the antigen-binding construct, any structural restrictions of the drug, and the hydrophobicity of the drug (see, for example, a review in Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer). A number of specific linker-toxin combinations have been described and can be used with the antigen-binding constructs described herein to prepare ADCs in certain embodiments. Examples include, but are not limited to, cleavable peptide-based linkers with auristatins such as MMAE and MMAF, camptothecins such as SN-38, duocarmycins, and PBD dimers; non-cleavable MC-based linkers with auristatins MMAF and MMAE; acid-labile hydrazone-based linkers with calicheamicin and doxorubicin; disulfide-based linkers with maytansinoids such as DM1 and DM4; and bismaleimide trioxyethylene glycol (BMPEO)-based linkers with the maytansinoid DM1. Some of these therapeutic agents and linkers are described, for example, in Peters & Brown, (2015) Biosci. Rep. e00225; Dosio et al., (2014) Recent Patents on Anti-Cancer Drug Discovery 9:35-65; U.S. Patent Publication No. U2015 / 0374847, and US20180193477A1, which are incorporated by reference in their entireties.

[0220] Depending on the desired drug and the selected linker, those skilled in the art can choose a suitable method for coupling them together. For example, some conventional coupling methods, such as amine coupling methods, can be used to form the desired drug-linker conjugate, which still contains a reactive group for covalently conjugating the antibody. In some embodiments, drug-maleimide conjugates (i.e., maleimide-linked drugs) can be used for the payloads with reactive groups in the present disclosure. The most common reactive group that can be linked to thiol groups in ADC preparation is maleimide. In addition, organic bromides and iodides are also frequently used.

[0221] ADCs can be prepared by one of several art-known routes using organic chemistry reactions, conditions, and reagents well known to those skilled in the art (see, for example, Bioconjugate Techniques (GT Hermanson, 2013, Academic Press)). For example, conjugation can be accomplished by (1) reacting a nucleophilic or electrophilic group of an antibody with a bivalent linker reagent to form an antibody-linker intermediate Ab-L, followed by reaction with an activated drug moiety D, via a covalent bond; or (2) reacting a nucleophilic or electrophilic group of a drug moiety with a linker reagent, followed by reaction with a nucleophilic or electrophilic group of an antibody, via a covalent bond, to form a drug-linker intermediate DL. Conjugation methods (1) and (2) can be used with a variety of antibodies, drug moieties, and linkers to prepare the ADCs described herein. The various linkers, linker components, and toxins prepared are either commercially available or can be prepared using standard synthetic organic chemistry techniques, as described, for example, in March's Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley); Toki et al., (2002) J. Org. Chem. 67:1866-1872; Frisch et al., (1997) Bioconj. Chem. 7:180-186; Bioconjugate Techniques (GTHermanson, 2013, Academic Press); US20210379193A1, and US20180193477A1, which are incorporated by reference in their entireties. Additionally, a number of preformed drug-linkers suitable for reaction with a selected antigen-binding construct are also commercially available, e.g., linker-toxins including DM1, DM4, MMAE, MMAF, or duocarmycin SA are available from Creative BioLabs (Shirley, NY).

[0222] Some specific examples of methods for preparing ADCs are known in the art and are described in U.S. Patent No. 8,624,003 (Pot method), U.S. Patent No. 8,163,888 (one step), and U.S. Patent No. 5,208,020 (two step method), as well as US20180193477A1, which are incorporated by reference in their entireties. Other methods are known in the art and include those described in Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer.

[0223] Drug loading is expressed by the number of drug moieties per antibody in the ADC molecule. For some antibody drug conjugates, drug loading can be limited by the number of binding sites on the antibody. For example, when the linkage is a cysteine ​​thiol, as in certain exemplary embodiments described herein, drug loading can range from 0 to 8 drug moieties per antibody. In certain embodiments, high drug loading, e.g., p≧5, can cause aggregation, insolubility, toxicity, or cell permeability of certain antibody drug conjugates. In certain embodiments, the average drug loading for an antibody drug conjugate ranges from 1 to about 8, about 2 to about 6, or about 3 to about 5%. In fact, it has been shown that for certain antibody drug conjugates, the optimal ratio of drug moieties per antibody can be about 4. In some embodiments, the DAR is about or at least 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the average DAR in the composition is from about 1 to about 2, from about 2 to about 3, from about 3 to about 4, from about 4 to about 5, from about 5 to about 6, from about 6 to about 7, or from about 7 to about 8.

[0224] Antibody and ADC properties An anti-HER2 antigen binding protein construct (e.g., an antibody, bispecific antibody, or antibody fragment thereof), or an ADC derived therefrom, can comprise an antigen-binding region derived from any anti-HER2 antibody described herein, or any antigen-binding fragment thereof.

[0225] Common techniques that can be used to measure the affinity of an antibody to an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). Affinity can be estimated from the quotient of kinetic rate constants (KD=koff / ka). In some embodiments, an antibody, an antigen-binding fragment thereof, or an antigen-binding protein construct (e.g., a bispecific antibody) has a affinity of less than 0.1 s -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Less than or equal to 0.00001s -1 It can bind to ERBB2 (e.g., human HER2, canine ERBB2, monkey ERBB2, and / or mouse ERBB2) with a dissociation rate (koff) of less than 0.01 s. In some embodiments, the dissociation rate (koff) is less than 0.01 s. -1 Super, 0.001s -1 Super, 0.0001s -1 Super, 0.0001s -1 Greater than or equal to 0.00001s -1 In some embodiments, the dissociation rate (koff) is greater than 7×10 -4 s -1 is less than.

[0226] In some embodiments, the kinetic association rate (ka) is 1×10 2 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms or 1×10 6 In some embodiments, the kinetic association rate (ka) is greater than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 In some embodiments, the kinetic association rate (ka) is less than 1.3×10 5 / Over Ms.

[0227] In some embodiments, the antibody, antigen-binding fragment thereof, or antigen-binding protein construct (e.g., bispecific antibody) is administered to ERBB2 (e.g., human HER2, canine ERBB2, monkey ERBB2, and / or mouse ERBB2) at a concentration of 1×10 -6 Less than M, 1×10 -7 Less than M, 1×10 -8 Less than M, 1×10 -9 Less than M or 1 x 10 -10 In some embodiments, the KD is less than 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is less than 1×10 -7 Super M, 1×10 -8 Super M, 1×10 -9 More than M or 1×10 -10 In some embodiments, the antibody binds to human HER2 with a KD of about 5 nM, 4.5 nM, 3 nM, or 0.27 nM or less.

[0228] An anti-TROP2 antigen-binding protein construct (e.g., a bispecific antibody), or an ADC derived therefrom, can also comprise an antigen-binding region derived from any of the anti-TROP2 antibodies or antigen-binding fragments thereof described herein.

[0229] In some embodiments, the antibody, antigen-binding fragment thereof, or antigen-binding protein construct (e.g., bispecific antibody) is -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Less than or equal to 0.00001s -1 It can bind to TROP2 (e.g., human TROP2, canine TROP2, monkey TROP2, and / or mouse TROP2) with a k of less than 0.01 s. In some embodiments, the k is less than 0.01 s. -1 Super, 0.001s -1 Super, 0.0001s -1 Super, 0.0001s -1 Greater than or equal to 0.00001s -1 It's super.

[0230] In some embodiments, the kinetic association rate (ka) is 1×10 2 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms or 1×10 6 In some embodiments, the kinetic association rate (ka) is greater than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0231] Affinity can be estimated from the quotient of kinetic rate constants (KD=koff / ka). In some embodiments, KD is 1×10 -6 Less than M, 1×10 -7 Less than M, 1×10 -8 Less than M, 1×10 -9 Less than M or 1 x 10 -10 In some embodiments, the KD is less than 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is less than 1×10 -7 Super M, 1×10 -8 Super M, 1×10 -9 Super M, 1×10 -10 Super M, 1×10 -11 More than M or 1×10 -12 In some embodiments, the antibody binds to human TROP2 with a KD of about 15 nM or less than 10 nM.

[0232] Since the antigen-binding protein construct (e.g., bispecific antibody) binds to both TROP2 and HER2, for cells expressing both TROP2 and HER2, the antigen-binding protein construct has high binding affinity to these cells. The binding affinity of the antigen-binding protein construct to these cells can be measured using avidity. Avidity is the cumulative strength of the affinities of multiple individual non-covalent interactions.

[0233] Thermal stability can also be measured. The antibodies, antigen-binding fragments thereof, or antigen-binding protein constructs (e.g., bispecific antibodies), or ADCs derived therefrom, described herein can have a Tm of greater than 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, or 95°C. Since IgG can be described as a multidomain protein, the melting curve in some cases shows two transitions, the first denaturation temperature being Tm D1 and the second denaturation temperature being Tm D2. The presence of these two peaks often indicates the denaturation of the Fc domain (Tm D1) and the Fab domain (Tm D2), respectively. When there are two peaks, the Tm is usually referred to as Tm D2. Thus, in some embodiments, the antibodies or antigen-binding fragments described herein have a Tm D1 of greater than 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, or 95° C. In some embodiments, the antibodies or antigen-binding fragments described herein have a Tm D2 of greater than 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, or 95°C. In some embodiments, the Tm, Tm D1, Tm D2 are less than 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, or 95°C.

[0234] In some embodiments, the antibody, antigen-binding fragment thereof, antigen-binding protein construct (e.g., an anti-TROP2, HER2 antibody, or bispecific antibody), or ADC derived therefrom, has an endocytosis ratio within a cell (e.g., within NCI-N87 cells or NCI-H292 cells) that is at least 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the endocytosis ratio is less than 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0235] In some embodiments, the antibody, antigen-binding fragment thereof, antigen-binding protein construct (e.g., bispecific antibody), or ADC derived therefrom, can bind to canine ERRB2, monkey ERRB2, or mouse ERRB2. In some embodiments, binding is measured as the percentage of positive cells as measured by FACS. In some embodiments, the percentage of positive cells is greater than 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the percentage of positive cells is less than 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the antibody, antigen-binding fragment thereof, antigen-binding protein construct (e.g., bispecific antibody), or ADC derived therefrom is unable to bind to canine ERRB2, monkey ERRB2, or mouse ERRB2.

[0236] In some embodiments, the antibody, antigen-binding fragment thereof, antigen-binding protein construct (e.g., bispecific antibody), or ADC derived therefrom, can bind to canine TROP2, monkey TROP2, or mouse TROP2. In some embodiments, binding is measured as the percentage of positive cells as measured by FACS. In some embodiments, the percentage of positive cells is greater than 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the percentage of positive cells is less than 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the antibody, antigen-binding fragment thereof, antigen-binding protein construct (e.g., bispecific antibody), or ADC derived therefrom is unable to bind to canine TROP2, monkey TROP2, or mouse TROP2.

[0237] In some embodiments, the antibody, antigen-binding fragment thereof, antigen-binding protein construct (e.g., an anti-TROP2, HER2 antibody, or bispecific antibody), or ADC derived therefrom, has a purity of greater than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% as measured, for example, by HPLC. In some embodiments, the antibody has a purity of less than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% as measured, for example, by HPLC.

[0238] In some embodiments, the antibody, antigen-binding fragment thereof, antigen-binding protein construct (e.g., anti-TROP2, HER2 antibody, or bispecific antibody), or ADC derived therefrom has a yield of more than 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, or 700 (μg / mL). In some embodiments, the yield is less than 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, or 700 (μg / mL).

[0239] In some embodiments, the stability of an antibody, antigen-binding fragment thereof, antigen-binding protein construct (e.g., an anti-TROP2, HER2 antibody, or bispecific antibody), or an ADC derived therefrom, is measured by capillary isoelectric focusing (cIEF) (expressed as the percentage of major, acidic, and alkaline components). In some embodiments, the percentage of major components, e.g., as measured by cIEF, is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% after running through a variety of conditions. In some embodiments, the conditions are storing at 4° C. for at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, storing at 25° C. for at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or storing at 40° C. for at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, the conditions are freezing and thawing at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, or 50 times. In some embodiments, the conditions are storing the composition at pH 3.5 for about or at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In some embodiments, after treatment, the percent acidic components is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% as measured, for example, by cIEF. In some embodiments, after treatment, the percent alkaline content is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% as measured, for example, by cIEF.In some embodiments, after treatment, the percentage of major components is less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% as measured, for example, by cIEF. In some embodiments, the percentage of acidic components is less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, e.g., as measured by cIEF. In some embodiments, the percentage of alkaline components is less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, e.g., as measured by cIEF.

[0240] In some embodiments, the cell killing ability of an antibody, antigen-binding fragment thereof, antigen-binding protein construct (e.g., an anti-TROP2, HER2 antibody, or bispecific antibody), or an ADC derived therefrom is measured by IC50 (ng / mL) (e.g., in NCI-N87 or NCI-H292 cells). In some embodiments, the IC50 is greater than 2, 5, 10, 20, 30, 40, 50, 100, 200, 500, 1000, 2000, 50000, 100000, 200000, 50000, 100000, 200000, or 500000 ng / mL. In some embodiments, the IC50 is less than 2, 5, 10, 20, 30, 40, 50, 100, 200, 500, 1000, 2000, 5000, 10000, 20000, 50000, 100000, 200000, or 500000 ng / mL.

[0241] In some embodiments, the antibody, antigen-binding fragment thereof, antigen-binding protein construct (e.g., anti-TROP2, HER2 antibody, or bispecific antibody), or ADC derived therefrom has a tumor growth inhibition percentage (TGI%) of more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the antibody has a tumor growth inhibition percentage of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. TGI% can be measured, for example, 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, or 30 days after the start of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. As used herein, tumor growth inhibition percentage (TGI%) is calculated using the following formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)]×100

[0242] Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0.

[0243] Fc Region An antibody, antigen-binding fragment thereof, or antigen-binding protein construct (e.g., a bispecific antibody) has a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, one or both of the mutations S239D and / or I332E (SI mutations) are introduced into the antibody Fc region to enhance the antibody affinity to FcγRIIIA, thereby increasing the ADCC effect. A detailed description of SI mutations can be found in US7662925, which is incorporated by reference in its entirety.

[0244] Fc region antibodies, antigen-binding fragments thereof, or antigen-binding protein constructs (e.g., bispecific antibodies) that do not have a functional Fc region, e.g., antibodies or antigen-binding fragments are Fab, Fab', F(ab')2, and Fv fragments.

[0245] In some embodiments, the antibody, its antigen-binding fragment, or the antigen-binding protein construct (e.g., a bispecific antibody) is incorporated into an antibody drug conjugate.

[0246] Recombinant Vectors The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., such that the host cell contains the polynucleotides and / or vectors containing the polynucleotides), and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.

[0247] As used herein, a "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector is introduced. Thus, in an expression vector, a polynucleotide of interest is positioned for expression in the vector by being operably linked to control elements, such as promoters, enhancers, and / or polyA tails, either in the vector or in the genome of the host cell at, near, or adjacent to the integration site of the polynucleotide of interest, such that the polynucleotide of interest is translated in the host cell into which it is introduced with the expression vector.

[0248] Vectors can be introduced into host cells by methods well known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0249] In some embodiments, a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein) is introduced using a viral expression system (e.g., variola or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication-competent virus, or may use a replication-incompetent virus. In the latter case, viral propagation generally occurs only in complementary viral packaging cells. For example, Fisher-Hoch et al.,1989,Proc.Natl.Acad.Sci.USA 86:317-321, Flexner et al.,1989,Ann.NYAcad Sci.569:86-103, Flexner et al. al., 1990, Vaccine, 8:17-21, U.S. Patent No. 4,603,112, U.S. Patent No. 4,769,330, and U.S. Pat. 91 / 02805, Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al. Suitable systems are disclosed in: Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. The DNA can also be "naked," as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692. Uptake of naked DNA can be increased by coating the DNA onto biodegradable beads that are efficiently transported into cells.

[0250] For expression, a DNA insert comprising an antibody-encoding or polypeptide-encoding polynucleotide disclosed herein can be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters, and promoters of retroviral-type LTRs, to name a few. Other suitable promoters will be known to those skilled in the art. The expression construct can further contain sites for transcription initiation and termination, and, within the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct can include a translation initiation at the beginning and a termination codon (UAA, UGA or UAG) positioned approximately at the end of the polypeptide to be translated.

[0251] As indicated, the expression vector can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for E. coli and other bacterial culture. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium cells, fungal cells such as yeast cells, insect cells such as Drosophila melanogaster S2 and Spodoptera litura Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells, and plant cells. Appropriate culture media and conditions for the host cells described herein are well known in the art.

[0252] Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9 available from Qiagen, pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene, and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to those of skill in the art.

[0253] Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters, and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, promoters of retroviral-type LTRs such as those of Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0254] In the yeast Saccharomyces cerevisiae, several vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used. For reviews, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, and Grant et al., Methods Enzymol., 153:516-544 (1997).

[0255] Introduction of the construct into the host cell can be accomplished by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, or other methods, which are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which are incorporated herein by reference in their entirety.

[0256] Transcription of DNA encoding the antibody of the present disclosure in more eukaryotic organisms can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 bp, that serve to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer, located at base pairs 100-270 on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0257] To allow secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals can be incorporated into the expressed polypeptide. The signals can be endogenous to the polypeptide or the signals can be heterologous signals.

[0258] Polypeptides (e.g., antibodies) can be expressed in modified forms, such as fusion proteins (e.g., GST fusions) or with histidine tags, and can contain not only secretion signals, but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of a polypeptide to improve stability and durability in the host cell during purification or during subsequent handling and storage. Peptide moieties can also be added to the polypeptide to facilitate purification. Such regions can be removed before final preparation of the polypeptide. It is well known and routine in the art to add peptide moieties to polypeptides to, among other things, effect secretion or excretion, improve stability, and facilitate purification.

[0259] The present disclosure also provides nucleic acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the nucleotide sequences described herein, and Also provided are amino acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any amino acid sequence described herein.

[0260] The present disclosure also provides nucleic acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology to any of the nucleotide sequences described herein; Also provided are amino acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology to any of the amino acid sequences described herein.

[0261] In some embodiments, the disclosure relates to a nucleotide sequence encoding any of the peptides described herein or any amino acid sequence encoded by any of the nucleotide sequences described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

[0262] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences described herein.

[0263] In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences described herein.

[0264] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment for comparison, and non-homologous sequences may be ignored). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are considered to be identical at that position (as used herein, amino acid or nucleic acid "identity" corresponds to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences. For example, sequence comparison and percent identity determination between two sequences can be performed using the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0265] Percentages of sequence homology (e.g., amino acid sequence homology or nucleic acid homology) can also be measured. Methods for measuring percent sequence homology are well known in the art. In some embodiments, conserved amino acid residues with similar physicochemical properties (% homology), e.g., leucine and isoleucine, can be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include, for example, amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the percentage of homology will be higher than the percentage of identity.

[0266] The present disclosure provides one or more nucleic acids encoding any of the polypeptides described herein. In some embodiments, the nucleic acid (e.g., cDNA) comprises a polynucleotide encoding a heavy chain polypeptide described herein. In some embodiments, the nucleic acid comprises a polynucleotide encoding a light chain polypeptide described herein. In some embodiments, the nucleic acid comprises a polynucleotide encoding a scFv polypeptide described herein.

[0267] In some embodiments, the vector can have two of the nucleic acids described herein, the vectors encoding VL and VH regions that both bind to HER2. In some embodiments, a pair of vectors is provided, each vector comprising one of the nucleic acids described herein, the pair of vectors together encoding VL and VH regions that both bind to HER2. In some embodiments, the vector comprises two of the nucleic acids described herein, the vectors together encoding VL and VH regions that both bind to TROP2. In some embodiments, a pair of vectors is provided, each vector comprising one of the nucleic acids described herein, the pair of vectors together encoding VL and VH regions that both bind to TROP2. In some embodiments, the VL regions are identical.

[0268] Vectors can also be constructed to express specific antibodies or polypeptides. In some embodiments, vectors can be constructed to co-express the light chain (HER2-K) and the heavy chain (HER2-H) of an anti-HER2 antibody. In some embodiments, the vector can contain, from the 5' to the 3' end, the sequences of a cytomegalovirus promoter (CMV), HER2-K, polyadenylation (polyA), CMV, HER2-H, simian vacuolar virus 40 terminator (SV40), and glutamine synthetase marker (GS). In some embodiments, vectors can be constructed to co-express the light chain (TROP2-K) and the heavy chain (TROP2-H) of an anti-TROP2 antibody. In some embodiments, the vector can contain, from the 5' to the 3' end, the sequences of CMV, HER2-K, polyA, HER2-H, SV40, and GS. In some embodiments, vectors can be constructed to express an anti-TROP2 antibody scFv polypeptide chain.

[0269] Methods for producing antibodies, antigen-binding fragments, and antigen-binding protein constructs Isolated fragments of human proteins can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of the antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species being immunized. Animals can be injected with the antigenic peptide or protein more than once (e.g., 2, 3, or 4 times).

[0270] The full-length polypeptide or protein can be used, or alternatively, an antigenic peptide fragment thereof can be used as the immunogen. An antigenic peptide of a protein contains at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of the protein and encompasses an epitope of the protein such that antibodies raised against the peptide form specific immune complexes with the protein.

[0271] An immunogen is typically used for the preparation of antibodies by immunizing a suitable subject (e.g., a human or a transgenic animal expressing at least one human immunoglobulin locus). An appropriate immunogenic preparation can contain, for example, a recombinantly expressed or chemically synthesized polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulant.

[0272] Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with the polypeptide, or an antigenic peptide thereof (e.g., a portion of the protein) as an immunogen. Antibody titers in the immunized subject can be monitored over time by standard techniques, such as enzyme-linked immunosorbent assay (ELISA), using immobilized polypeptide or peptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A on protein G chromatography, to obtain an IgG fraction. At an appropriate time after immunization, e.g., when the specific antibody titer is at its highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256:495-497, 1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96, 1985), or the trioma technique. Techniques for producing hybridomas are well known (see generally, Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY)). For example, hybridoma cells producing monoclonal antibodies are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest using a standard ELISA assay.

[0273] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the human, humanized, or chimeric antibodies, or antibodies or antigen-binding fragments thereof described herein, or by peptide synthesis. Such variants include, for example, deletion, insertion, or substitution of residues within the amino acids of the sequence that make up the antigen-binding site or domain of the antibody. In such a population of variants, some of the antibodies or antigen-binding fragments have increased affinity for the target protein. Any combination of deletion, insertion, and / or combination can be achieved in an antibody or antigen-binding fragment thereof with increased binding affinity for the target. Antibodies or antigen-binding fragments can be altered or new post-translational modifications can be introduced into the antibody or antigen-binding fragment by changing the number (e.g., increasing or decreasing) of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence so that a different sugar is attached by an enzyme present in the cell), or introducing a new glycosylation site.

[0274] The antibodies disclosed herein can be derived from any species of animal, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates, such as monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents that have been genetically engineered to produce human antibodies.

[0275] Phage display (panning) can be used to optimize antibody sequences with desired binding affinities. In this technique, a gene encoding a single chain Fv (including VH or VL) can be inserted into a phage coat protein gene, allowing the phage to "display" the scFv on its outside while containing the gene for the protein on its inside, resulting in a link between genotype and phenotype. Displaying phage can then be screened against a target antigen to detect the interaction of the displayed antigen-binding site with the target antigen. Thus, large libraries of proteins can be screened and amplified in a process called in vitro selection to obtain antibody sequences with desired binding affinities.

[0276] Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequences derived from) human germline immunoglobulin sequences. Human antibodies can include, for example, amino acid residues within the CDRs that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0277] In some embodiments, covalent modifications can be added to antibodies, antigen-binding fragments thereof, or antigen-binding protein constructs (e.g., bispecific antibodies). These covalent modifications can be added by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with organic derivatizing agents capable of reacting with selected side chains or with the N- or C-terminal residues.

[0278] In some embodiments, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be between 1% and 80%, between 1% and 65%, between 5% and 65%, or between 20% and 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at position 297 (position 314 in the Eu numbering of Fc region residues, or Kabat numbering) in the Fc region, however, Asn297 may also be located about ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, the Fc region of the antibody is further engineered to replace the asparagine at position 297 with alanine (N297A) to reduce glycan heterogeneity.

[0279] In some embodiments, the Fc region of the antibody is further engineered to replace the serine at position 228 (EU numbering) of IgG4 with a proline (S228P) to promote production efficiency by avoiding Fab-arm exchange. A detailed description of the S228 mutation is provided, for example, in Silva et al., "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation." Journal of Biological Chemistry 290.9(2015):5462-5469, which is incorporated by reference in its entirety.

[0280] In some embodiments, the methods described herein are designed to generate bispecific antibodies. Bispecific antibodies can be produced by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface can contain at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is created in the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers over other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated by reference in its entirety.

[0281] In some embodiments, knob-into-hole (KIH) technology can be used, which involves recombining CH3 domains to create either "knobs" or "holes" in each heavy chain to promote heterodimerization. KIH technology is described, for example, in Xu, Yiren, et al. Production of bispecific antibodies in 'knobs-into-holes' using a cell-free expression system. MAbs. Vol. 7. No. 1. Taylor & Francis, 2015, which is incorporated by reference in its entirety. In some embodiments, one heavy chain has T366W and / or S354C (knobs) substitutions (EU numbering) and the other heavy chain has Y349C, T366S, L368A, and / or Y407V (holes) substitutions (EU numbering). In some embodiments, one heavy chain has one or more of the substitutions Y349C and T366W (EU numbering). The other heavy chain may have one or more of the substitutions E356C, T366S, L368A, and Y407V (EU numbering). Furthermore, it is also possible to introduce substitutions (-ppcpScp-->-ppcpPcp-) in the hinge region of both substituted IgGs.

[0282] Furthermore, anion exchange chromatography can be used to purify bispecific antibodies. Anion exchange chromatography is a process that uses an ion exchange resin containing a positively charged group, such as a diethylaminoethyl group (DEAE), to separate substances based on their charges. In solution, the resin is coated with a positively charged counterion (cation). The anion exchange resin binds to negatively charged molecules and replaces the counterion. Anion exchange chromatography can be used to purify proteins based on their isoelectric point (pI). The isoelectric point is defined as the pH at which a protein has no net charge. When pH > pI, the protein has a negative net charge, and when pH < pI, the protein has a positive net charge. Thus, in some embodiments, different amino acid substitutions can be introduced into the two heavy chains such that the pI for the homodimer containing the two Arm A's, and the pI for the homodimer containing the two Arm B's, are different. The pI for the bispecific antibody having Arm A and Arm B is somewhere between the two pI's of the homodimers. Thus, the two homodimers, and the bispecific antibody, may be eluted under different pH conditions. The present disclosure shows that the pI can be adjusted by introducing some amino acid residue substitutions into the heavy chain.

[0283] Bispecific antibodies can also include, for example, cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies within the heteroconjugate can be bound to avidin and the other to biotin. Heteroconjugate antibodies can also be produced using any convenient cross-linking method. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is hereby incorporated by reference in its entirety.

[0284] Treatment methods The methods described herein include methods for treating cancer and related disorders. In general, the methods include administering a therapeutically effective amount of a recombinant antibody, antigen-binding fragment thereof, antigen-binding protein construct (e.g., bispecific antibody), or antibody-drug conjugate described herein to a subject in need of such treatment or determined to be in need of such treatment.

[0285] As used in this context, "treatment" means alleviating at least one symptom of a disorder associated with cancer. In many cases, cancer results in death. Thus, treatment can result in an increase in life expectancy (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years). Administering a therapeutically effective amount of an agent described herein for treating a condition associated with cancer results in a reduction in the number of cancer cells and / or alleviation of symptoms.

[0286] As used herein, the term "cancer" refers to an abnormal situation or condition characterized by cells with autonomous growth potential, i.e., rapidly proliferating cell proliferation. The term is meant to include any kind of cancerous growth or oncogenic process, metastatic tissue or malignantly transformed cells, tissues, or organs, regardless of the type of tissue change or stage of invasiveness. As used herein, the term "tumor" refers to a cancerous cell, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of various organ systems, such as those affecting the lung, breast, thyroid, lymphatic system, gastrointestinal, and genitourinary tract, as well as adenocarcinomas, including malignancies such as most colon cancers, renal cell carcinoma, prostate cancer, and / or testicular cancer, non-small cell carcinoma of the lung, small intestine cancer, and esophageal cancer. In some embodiments, the agents described herein are designed to treat or diagnose carcinoma in a subject. The term "carcinoma" is art-recognized and refers to malignant tumors of epithelial or endocrine tissue, including respiratory, digestive, genitourinary, testicular, breast, prostate, endocrine, and melanoma. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the esophagus, cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcomas, which include, for example, malignant tumors composed of carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to carcinomas derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art-recognized and refers to malignant tumors of mesenchymal origin. In some embodiments, the cancer is a chemotherapy-resistant cancer.

[0287] In one aspect, the present disclosure also provides a method of treating cancer in a subject, reducing the rate of increase in tumor volume in a subject over time, reducing the risk of developing metastasis, or reducing the risk of developing further metastasis in a subject. In some embodiments, the treatment can halt, slow, prevent, or inhibit the progression of cancer. In some embodiments, the treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0288] In one aspect, the disclosure relates to a method comprising administering a therapeutically effective amount of an antibody, antigen-binding fragment thereof, antigen-binding protein construct (e.g., bispecific antibody), or antibody drug conjugate described herein to a subject in need thereof, e.g., a subject having, or identified or diagnosed as having, cancer, e.g., breast cancer, carcinoid, cervical cancer, colorectal cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, gastric cancer, esophageal carcinoma, testicular cancer, thyroid cancer, or urothelial cancer.

[0289] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and refer to an animal, human, or non-human to which treatment according to the methods of the invention is provided. Veterinary and non-veterinary uses are contemplated by the present invention. A human patient can be an adult human or a juvenile human (e.g., a human under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. For example, patients include non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, porcines (e.g., pigs, minipigs), equines, canines, felines, bovines, and other domestic, livestock, and zoo animals. In some embodiments, the subject is a human. In some embodiments, the subject is a dog.

[0290] In some embodiments, the cancer is thyroid cancer, urothelial cancer, breast cancer, colorectal cancer, renal cancer, cervical cancer, ovarian cancer, lung cancer, uterine cancer, skin cancer, gastric cancer, esophageal carcinoma, pancreatic cancer, prostate cancer, liver cancer, lymphoma, or glioma.

[0291] In some embodiments, the cancer is cervical cancer, prostate cancer, thyroid cancer, urothelial cancer, head and neck cancer, uterine cancer, ovarian cancer, lung cancer, breast cancer, carcinoid, skin cancer, liver cancer, or testicular cancer.

[0292] In some embodiments, the cancer is pancreatic cancer, lung cancer, gastric cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, skin cancer, or brain cancer.

[0293] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for cancer. Patients suffering from cancer can be identified in a variety of ways well known in the art.

[0294] As used herein, an "effective amount" refers to an amount or dosage sufficient to bring about a beneficial or desired result, including halting, slowing, preventing, or inhibiting the progression of a disease, e.g., cancer. The effective amount varies depending on, for example, the age and weight of the subject to which the antibody, antigen-binding fragment, antibody drug conjugate, polynucleotide encoding the antibody, vector comprising the polynucleotide, and / or composition is administered, the severity of the symptoms, and the route of administration, and thus dosing can be determined on an individual basis.

[0295] An effective amount can be administered in one or more administrations. For example, an effective amount of an antibody, antigen-binding fragment, or antibody drug conjugate is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or an amount sufficient to ameliorate, stop, stabilize, reverse, slow, and / or delay the proliferation of a cell (e.g., a biopsy cell, any of the cancer cells described herein, or a cell line (e.g., a cancer cell line)) in vitro. As is understood in the art, an effective amount of an antibody, antigen-binding fragment, or antibody drug conjugate may vary depending on other factors, such as, inter alia, the medical history of the patient, as well as the type (and / or dose) of the composition used.

[0296] Effective amounts and schedules for administering the antibodies, polynucleotides encoding the antibodies, antibody drug conjugates, and / or compositions disclosed herein can be determined by experimentation, and making such determinations is within the skill of one in the art. One of skill in the art will understand that the dosage required to be administered will vary depending on, for example, the mammal receiving the antibodies, polynucleotides encoding the antibodies, antibody drug conjugates, and / or compositions disclosed herein, the route of administration, the particular type of antibody, polynucleotide encoding the antibody, antigen-binding fragment, antibody drug conjugate, and / or compositions disclosed herein that are used, and other agents administered to the mammal.

[0297] A typical daily dose of an effective amount of an antibody, antigen-binding fragment thereof, antigen-binding protein construct (e.g., bispecific antibody), or antibody-drug conjugate is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose can be less than 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dose can be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or 0.01 mg / kg. In some embodiments, the dose is about, or at least, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0298] In any of the methods described herein, at least one antibody, antigen-binding fragment thereof, or antigen-binding protein construct (e.g., bispecific antibody), antibody drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, antibody drug conjugates, or pharmaceutical compositions described herein), and optionally at least one additional therapeutic agent, can be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered in the same composition (e.g., liquid composition). In some embodiments, at least one antibody, antigen-binding fragment, antigen-binding protein construct (e.g., bispecific antibody), or antibody drug conjugate, and at least one additional therapeutic agent are administered in the same composition (e.g., liquid composition). In some embodiments, the at least one antibody or antigen-binding fragment and the at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing the at least one antibody or antigen-binding fragment and a solid oral composition containing the at least one additional therapeutic agent). In some embodiments, the at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained release oral formulation.

[0299] In some embodiments, one or more additional therapeutic agents can be administered to the subject before or after administration of at least one antibody, antigen-binding antibody fragment, antibody drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, the one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, antibody drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, antibody drug conjugates, or pharmaceutical compositions described herein) are administered to the subject such that there is an overlap in the period of biological activity of the one or more additional therapeutic agents with the period of biological activity of the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) or antibody drug conjugate in the subject.

[0300] In some embodiments, at least one antibody, antigen-binding antibody fragment, antibody drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) can be administered to a subject over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional can determine the length of the treatment period using any of the methods described herein to diagnose or track the effectiveness of the treatment (e.g., to observe at least one symptom of cancer). As described herein, a skilled medical professional can also vary (e.g., increase or decrease) the identity and number of antibodies or antigen-binding antibody fragments, antibody drug conjugates (and / or one or more additional therapeutic agents) administered to a subject, and can adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) to a subject based on an evaluation of the effectiveness of the treatment (e.g., using any of the methods described herein and well known in the art).

[0301] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agents can include one or more inhibitors selected from the group consisting of inhibitors of B-Raf, EGFR inhibitors, inhibitors of MEK, inhibitors of ERK, inhibitors of K-Ras, inhibitors of c-Met, inhibitors of anaplastic lymphoma kinase (ALK), inhibitors of phosphatidylinositol 3-kinase (PI3K), inhibitors of Akt, inhibitors of mTOR, dual PI3K / mTOR inhibitors, inhibitors of Bruton's tyrosine kinase (BTK), and inhibitors of isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-1 (IDO1) (e.g., epacadostat).

[0302] In some embodiments, the additional therapeutic agent can include one or more inhibitors selected from the group consisting of inhibitors of HER3, inhibitors of LSD1, inhibitors of MDM2, inhibitors of BCL2, inhibitors of CHK1, inhibitors of the activated hedgehog signaling pathway, and agents that selectively degrade the estrogen receptor.

[0303] In some embodiments, the additional therapeutic agent is trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, leolysin, Alimta, Dicaida, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, pazopanib, IMA-901, AGS-00 3, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0304] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, a tumor necrosis factor (TNF), IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a CX3CL1 targeted therapy, a CXCL9 targeted therapy, a CXCL10 targeted therapy, a CCL5 targeted therapy, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0305] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

[0306] In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, or an anti-GITR antibody.

[0307] Pharmaceutical Compositions and Routes of Administration Also provided herein is a pharmaceutical composition containing at least one (e.g., 1, 2, 3, or 4) of the antigen-binding protein constructs, antibodies (e.g., bispecific antibodies), antigen-binding fragments, or antibody-drug conjugates described herein. Two or more (e.g., 2, 3, or 4) of any of the antigen-binding protein constructs, antibodies, antigen-binding fragments, or antibody-drug conjugates described herein can be present in the pharmaceutical composition in any combination. The pharmaceutical composition can be formulated in any manner known in the art.

[0308] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The compositions may contain a sterile diluent (e.g., sterile water or saline), fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial or antifungal agents (e.g., benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetates, citrates, or phosphates), and isotonic agents (e.g., sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride)), or any combination thereof. Liposomal suspensions may also be used as pharma- ceutically acceptable carriers (see, e.g., U.S. Pat. No. 4,522,811). The preparation of the composition can be formulated and enclosed in ampoules, disposable syringes, or multiple dose vials. Where necessary (e.g., in injectable formulations), proper fluidity can be maintained, for example, by the use of a coating such as lecithin, or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, sustained release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0309] Compositions containing any one or more of the antigen binding protein constructs, antibodies, antigen binding fragments, antibody drug conjugates described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in unit dosage form (i.e., physically discrete units containing a predetermined amount of active compound for ease of administration and uniformity of dosage).

[0310] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). The LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population) can be determined, and the therapeutic index is the ratio of LD50:ED50. Drugs that exhibit a high therapeutic index are preferred. If a drug exhibits undesirable side effects, care should be taken to minimize the potential for harm (i.e., to reduce the undesirable side effects). The toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0311] Exemplary doses include amounts (in milligrams or micrograms) of any of the antigen-binding protein constructs, antibodies, antigen-binding fragments, or antibody drug conjugates described herein per kilogram of subject body weight (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; or about 0.1 mg / kg to about 0.5 mg / kg).

[0312] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.The disclosure also provides methods of producing the antibodies or antigen-binding fragments thereof, or antibody-drug conjugates for the various uses described herein. EXAMPLES

[0313] Working Example The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0314] Example 1. Detection of HER2 and TROP2 Expression Functional genomic RNA-seq was performed to measure the expression levels of HER2 and TROP2 in various human tumor cell lines. The results are shown in the table below.

[0315] [Table 1]

[0316] In addition, flow cytometry (FACS) was used to detect the expression of HER2 and TROP2 proteins in the following human tumor cells: conjugated antibodies including anti-hTROP2-PE (R&D Systems, Inc., Catalog No. FAB650P) and PE anti-human CD340 (erbB2 / HER-2) (Biolegend Inc., Catalog No. 324406) were used. The geometric mean fluorescence intensity (MFI) results are shown in the table below.

[0317] [Table 2]

[0318] The data showed that HER2 and TROP2 were highly expressed in various tumor cell lines.

[0319] Example 2. Anti-HER2 antibodies Generation of anti-HER2 antibodies Human HER2 protein (ACRO Biosystems Inc., Catalog Number: HE2-H5253, comprising positions 23-652 of SEQ ID NO: 64 of human HER2 protein), or DNA encoding this protein, was emulsified with adjuvant and used to immunize RenLite™ mice (Biocytogen, fully human heavy chain variable domains combined with common light chain substitutions in situ). Mice are described, for example, in PCT / CN2021 / 097652, which is incorporated herein by reference in its entirety. Retro-orbital bleeds were taken prior to immunization as a negative control.

[0320] The first immunization was with Freund's complete adjuvant CFA, and the second, third, and fourth immunizations were with Freund's incomplete adjuvant IFA. A total of four immunizations were performed. The first and second immunizations were spaced two weeks apart, and the remaining immunizations were spaced one week apart. One week after the fourth immunization, retro-orbital blood was collected and serum antibody titers were detected by ELISA. One week later, mice with high titers were further injected with human HER2 protein via the tail vein for booster immunization.

[0321] Antigen-specific immune cells were isolated from the immunized mice, and anti-HER2 antibodies were obtained, or light and heavy chain variable region sequences of anti-HER2 antibodies were obtained. For example, single cell technology (e.g., using Beacon® Optofluidic System, Berkeley Lights Inc.) was used to screen and find plasma cells secreting antigen-specific monoclonal antibodies, and antibody variable region sequences were obtained using reverse transcription and PCR sequencing. The obtained variable region sequences were used for antibody expression, and FACS was used to verify binding affinity to HER2. Exemplary antibodies obtained by this method included H-2A10, H-1H2, H-2B2, H-3E5, H-3C6, and H-3C8. These antibodies contain substantially the same light chain, and their VL and VL CDR 1-3 sequences are shown in Figures 8 and 9. The VH and VL regions of H-1H2, H-2B2, H-3E5, H-3C6, and H-3C8 are shown in FIG.

[0322] Various IgG1, IgG2, and IgG4 antibodies were generated. Regarding the names of the antibodies, when the antibody VH\VL are linked to various isotypes, the isotype is added to the name. For example, when the VH and VL of H-1H2 are linked to the IgG1 constant region, the antibody is named H-1H2-IgG1. Examples of other isotypes are: H-1H2-IgG2, H-1H2-IgG4.

[0323] Anti-HER2 antibody endocytosis test Sample anti-HER2 antibody (2.5 μg / mL) and pHAb goat anti-human IgG secondary antibody (Promega (Beijing) Biotech Co., Ltd., Catalog No.: G9845) were added to BT-474 cells with high HER2 expression. After 1 hour of incubation, the cells were centrifuged and washed with FACS buffer. The MFI value was measured by flow cytometer, and the endocytosis ratio of anti-HER2 antibody was calculated. The results are shown in the table below (Table 3). For isotype control, an antibody targeting an irrelevant target protein was used.

[0324] [Table 3]

[0325] Trastuzumab is a HER2-targeting humanized monoclonal antibody, and its heavy and light chain sequences are shown in SEQ ID NOs:65-66.

[0326] Pertuzumab is also a HER2-targeting antibody, and its heavy and light chain sequences are shown in SEQ ID NOs: 67-68.

[0327] The data showed that H-1H2-IgG1, H-2B2-IgG1, H-3E5-IgG1, H-3C6-IgG1, and H-3C8-IgG1 had better cell endocytosis rates compared to the isotype control (ISO), positive controls trastuzumab and pertuzumab.

[0328] Cross-species binding analysis of anti-HER2 antibodies BT-474 cells expressing human HER2 (hHER2), CHO-S cells expressing monkey HER2 (fasHER2) (CHO-fasHER2), and NIH3T3 cells expressing Canis lupus familiaris HER2 (dHER2) (NIH3T3-dHER2) were cultured at 5 × 10 4 The cells were transferred to a 96-well plate at a density of 1000 cells / well. Serially diluted sample anti-HER2 antibodies were transferred to the 96-well plate and incubated at 4° C. for 30 minutes. The cells were then incubated with secondary antibody anti-hIgG-Fc-Alex Flour 647 (RL1-H) (Jackson ImmunoResearch Laboratories, Inc., 109-606-170) for 15 minutes in the dark at 4° C., followed by flow cytometry analysis.

[0329] CHO-fasHER2 cells were obtained by transferring the amino acid sequence of Macaca fascicularis (cynomolgus monkey) HER2 (SEQ ID NO: 74) to positions 174-804 in CHO-S cells. NIH3T3-dHER2 cells were obtained by transfecting NIH3T3 (ATCC, Catalog No.: CRL1658) cells with a construct containing the amino acid sequence of Canis lupus familiaris (dog) HER2 (SEQ ID NO: 75) (the construct contains CAG promoter-dog HER2 CDS-P2A-EGFP-WPRE-PA).

[0330] The test results are shown in the table below. All five anti-HER2 antibodies can bind to hHER2 and fasHER2. Among these five antibodies, H-1H2-IgG1, H-2B2-IgG1, and H-3C8-IgG1 also showed cross-species binding to hHER2, fasHER2, and dHER2.

[0331] [Table 4]

[0332] Anti-HER2 antibody affinity testing The affinity of anti-HER2 antibodies to His-tagged HER2 proteins from humans (hHER2, ACROBiosystems Inc., Catalog No. HE2-H5225) (positions 23-652 of SEQ ID NO: 64), mice (mHER2, ACROBiosystems Inc., Catalog No. ER2-M5220) (SEQ ID NO: 76), dogs (dHER2, Sino Biological Inc., Catalog No. 70024-D08H) (positions 1-652 of SEQ ID NO: 75), or monkeys (fasHER2, ACROBiosystems Inc., Catalog No. HE2-C52Hb) (SEQ ID NO: 74) was measured by biolayer interferometry (BLI) at 30°C using a ForteBio Octet system. A total of five monoclonal antibodies were tested: H-1H2-IgG1, H-2B2-IgG1, H-3E5-IgG1, H-3C6-IgG1, and H-3C8-IgG1.

[0333] Anti-HER2 antibodies were loaded onto an AHC biosensor (ForteBio Inc., Cat. No.: 18-5060) at 10 μg / mL, giving a response of 1.0 nm. Kinetic measurements were performed with recombinant His-tagged HER2 protein at concentrations of 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM. The association phase lasted 180 s, the dissociation phase lasted 300 s, followed by a regeneration step with 10 mM glycine-HCl (pH 1.7). Data analysis was performed using the Octet data analysis program (DataAnalysis11) using a standard 1:1 binding model. Affinity values ​​were estimated from the quotient of the kinetic rate constants (KD=koff / kon).

[0334] The same method, with appropriate adjustments of parameters (e.g., antibody concentration), as would be understood by one of skill in the art, was performed for each of the anti-HER2 antibodies tested. The results for the tested antibodies are summarized in the table below.

[0335] [Table 5]

[0336] The results showed that all five anti-HER2 antibodies showed good binding affinity to human HER2 and monkey HER2. Among these five antibodies, H-1H2-IgG1, H-2B2-IgG1, and H-3C8-IgG1 also showed good binding affinity to canine HER2.

[0337] Example 3. Preparation and analysis of anti-TROP2 antibodies Generation of anti-TROP2 antibodies RenLite™ mice were cross-immunized with hTrop2-Fc (ACROBiosystems Inc., Catalog No. TR2-H5253, containing amino acids 31-274 of human TROP2 protein SEQ ID NO: 69) and mTrop2-his (ACROBiosystems Inc., Catalog No. TR2-M52H6, containing amino acids 25-270 of mouse TROP2 protein SEQ ID NO: 79). Prior to immunization, retro-orbital blood was collected from mice as a negative control. Freund's complete adjuvant CFA was used for the first immunization, and Freund's incomplete adjuvant IFA was used for the second, third, and fourth immunizations. A total of four immunizations were performed. One week later, the third immunization was performed, and retro-orbital blood was collected to detect serum titers of anti-human TROP2 and anti-mouse TROP2 antibodies by ELISA. One week later, high titer mice were selected for a fourth immunization with mTrop2-his by subcutaneous injection. After the fourth immunization, CHO-S cells expressing human TROP2 antigen were selected for prime immunization by tail vein injection, and hTROP2-Fc was selected for prime immunization by intraperitoneal injection.

[0338] Antigen-specific immune cells were isolated (from immune organs of immunized mice) and anti-TROP2 antibodies were obtained, or anti-TROP2 antibody light and heavy chain V-region sequences were obtained. Plasma cells secreting antigen-specific monoclonal antibodies were screened and found using single-cell technology (such as Beacon® Optofluidic System, Berkeley Lights Inc.), and then antibody variable region sequences were obtained using reverse transcription and PCR sequencing. The variable region sequences were cloned into a backbone vector containing human IgG constant regions for antibody expression, and the binding of the expressed antibodies to TROP2 was verified by FACS. Exemplary fully human antibodies obtained by this method included T-3A4, T-4B9, T-4C12, T-5C8, and T-6F7. These antibodies have the same light chain, and the sequence matches the common light chain sequence of anti-HER2 antibodies. The CDR sequences, and VH and VL sequences of T-3A4, T-4B9, and T-6F7 are shown in Figures 11 to 13.

[0339] Various IgG1, IgG2 and IgG4 antibodies were produced. The constant region can also contain some mutations. For example, SI mutations (EU numbering: S239D and I332E mutations) are introduced into the Fc region of T-6F7-IgG1, and the resulting antibody is named T-6F7-IgG1-SI.

[0340] Endocytosis detection of anti-TROP2 antibody Anti-TROP2 antibody (1.25 μg / mL) and pHAb goat anti-human IgG secondary antibody were added to CHO cells (CHO-hTROP2) highly expressing human TROP2 (SEQ ID NO: 69). After 1 hour of incubation, the cells were centrifuged and washed with FACS buffer. The MFI was detected by a flow cytometer and the endocytosis rate of the anti-TROP2 antibody was calculated. The results are shown in the table below.

[0341] [Table 6]

[0342] Sacituzumab govitecan (Trodelvy™) is a humanized anti-TROP2 monoclonal antibody drug conjugate. The heavy and light chain sequences of sacituzumab govitecan analogs are shown as SEQ ID NOs: 70-71.

[0343] DS-1062 is a TROP2-directed antibody-drug conjugate that is in Phase III clinical trials in patients with advanced or metastatic non-small cell lung cancer (NSCLC). The heavy and light chain sequences of DS-1062 are shown as SEQ ID NOs: 72-73.

[0344] For comparison, the antibody moieties of sacituzumab govitecan and DS-1062 were used.The data showed that T-3A4-IgG1, T-4B9-IgG1, T-4C12-IgG1, T-5C8-IgG1, and T-6F7-IgG1 all had good endocytosis rates compared to the controls (sacituzumab govitecan antibody analog and DS-1062 antibody analog).

[0345] Cross-species binding analysis of anti-TROP2 CHO-hTROP, CHO-S cells expressing Macaca fascicularis (cynomolgus monkey) TROP2 (fasTROP2, SEQ ID NO: 77) (CHO-fasTROP2), and CHO-S cells expressing Canis lupus familiaris (dog) TROP2 (dTROP2, SEQ ID NO: 78) (CHO-dTROP2) were cultured at 5 × 10 4 The cells were transferred to a 96-well plate at a density of 1000 cells / well. Gradient dilutions of sample antibodies were added to the 96-well plate and incubated at 4°C for 30 minutes. The cells were then incubated with anti-hIgG-Fc-Alex Flour 647 (RL1-H) (Jackson ImmunoResearch Laboratories, Inc., Catalog No.: 109-606-170) for 15 minutes in the dark at 4°C, followed by flow cytometry analysis. The results are shown in the table below:

[0346] [Table 7]

[0347] The results showed that T-6F7-IgG1-SI, T-4C12-IgG1-SI, T-3A4-IgG1-SI, T-5C8-IgG1-SI, and T-4B9-IgG1-SI were all capable of binding to human TROP2, monkey TROP2, and canine TROP2.

[0348] Anti-TROP2 antibody affinity test The affinity of anti-TROP2 antibodies to His-tagged TROP2 proteins from human (hTROP2, ACROBiosystems Inc., Catalog No.: TR2-H5223), dog (dTROP2, ACROBiosystems Inc., Catalog No.: TR2-C52H4), and rhesus / cynomolgus monkeys (fasTROP2, ACROBiosystems Inc., Catalog No.: R52H3) was measured by surface plasmon resonance (SPR) using Biacore (Biacore, INC, Piscataway NJ) 8K biosensors equipped with pre-immobilized Protein A sensor chips.

[0349] Purified anti-TROP2 antibodies were diluted to 0.5 μg / mL and then injected onto a Biacore 8K biosensor at 10 μL / min for approximately 50 seconds to achieve the desired protein density (e.g., approximately 120 response units (RU)). His-tagged TROP2 protein at a concentration of 200 nM was then injected at 30 μL / min for 180 seconds. Dissociation was monitored for 600 seconds. After the last injection of each titration, the chip was regenerated with glycine (pH 2.0, 30 μL / min for 30 seconds).

[0350] Kinetic association rates (k) and dissociation rates (k) were obtained simultaneously by fitting the entire data to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) using the Biacore 8K Evaluation software 3.0. The affinity was estimated from the quotient of the kinetic rate constants (K = k / k).

[0351] The same method was performed for each antibody tested, with appropriate adjustments to parameters (e.g., antibody concentration), as would be understood by one of skill in the art. The results for the antibodies tested are summarized in the table below.

[0352] [Table 8]

[0353] The results show that all tested antibodies have good binding affinity to human TROP2. Among the tested antibodies, T-3A4-IgG1-SI, T-4B9-IgG1-SI, and T-6F7-IgG1-SI also have cross-species binding affinity to monkey and canine TROP2.

[0354] Example 4. Anti-HER2 / TROP2 bispecific antibodies Preparation of anti-HER2 / TROP2 bispecific antibodies

[0355] Anti-HER2 antibodies (H-1H2, H-2B2, H-3E5, H-3C6, and H-3C8) and anti-TROP2 antibodies (T-3A4, T-4B9, T-4C12, T-5C8, and T-6F7) can be paired to form various bispecific antibodies. Vectors for the light and heavy chains of the antibodies were generated. The three vectors were co-transfected into CHO-S cells. After culturing for 14 days, the cell supernatants were collected and purified by protein A affinity chromatography.

[0356] Various methods can be used to reduce the possibility of incorrect pairing between the two heavy chains. In the Fc region, knob-into-hole mutations were introduced into the anti-TROP2 arm heavy chain and the anti-HER2 arm heavy chain. The resulting exemplary bispecific antibodies included H-1H2-T-6F7, H-2B2-T-6F7, H-3C8-T-6F7, and T-6F7-H-1H2. To verify the binding affinity of the bispecific antibodies, anti-HER2 or anti-TROP control bispecific antibodies were also constructed, where one arm of the control bispecific antibody recognizes HER2 or TROP and the other arm recognizes CD28 (CD28RenLite co-light chain antibody). These control bispecific antibodies were named H-2B2-CD28, H-3C8-CD28, CD28-H-1H2, and CD28-T-6F7.

[0357] All of these antibodies have knob-into-hole mutations. In H-1H2-T-6F7, the heavy chain constant region of H-1H2 has a knob mutation and the constant region of T-6F7 has a hole mutation. In T-6F7-H-1H2, the heavy chain constant region of T-6F7 has a knob mutation and the heavy chain constant region of H-1H2 has a hole mutation. Exemplary antibody structures are shown in FIG. 1, where target 1 and target 2 can be HER2 and TROP2, respectively, or TROP2 and HER2, respectively, or HER2 and CD28, respectively, or CD28 and HER2, respectively, or CD28 and TROP2, respectively.

[0358] The sequences of the light chain constant region, the heavy chain constant region with the knob mutation, and the heavy chain constant region with the hole mutation are shown in FIG.

[0359] The purified anti-HER2 / TROP2 bispecific antibody was analyzed by non-reducing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and SEC-HPLC (size exclusion chromatography-high performance liquid chromatography).

[0360] Non-reducing SDS-PAGE was performed using 4–12% acrylamide gels. Protein samples were prepared as follows: First, 2.4 μL of protein sample was mixed with 6 μL of Tris-glycine SDS sample buffer (2×) (Invitrogen LC2676) and 3.6 μL of distilled water. Then, the mixture was boiled for 2 min and immediately centrifuged before loading. 4 μg of each sample was loaded onto the gel.

[0361] In the SEC-HPLC method, the antibody sample was diluted to 1 mg / mL with purified water and an Agilent 1290 chromatographic system was used, connected with an XBridge™ Protein BEH SEC column (200 Å, Waters Corporation). The following parameters were used: mobile phase: 25 mmol / L phosphate buffer (PB) + 300 mmol / L NaCl, pH 6.8; flow rate: 1.8 mL / min; column temperature: 25° C.; detection wavelength: 280 nm; injection volume: 10 μL; sample tray temperature: about 4° C.; and run time: 7 min. The results are summarized in the table below.

[0362] [Table 9]

[0363] Verification of the binding activity of anti-HER2 / TROP2 bispecific antibodies The binding activity of anti-HER2 / TROP2 antibodies to NCI-H292 cells and NCI-N87 cells was verified by flow cytometry. The secondary antibody used in the experiment was AF647-conjugated goat anti-human IgG (Jackson ImmunoResearch Laboratories, Inc. Catalog No.: 109-606-170). The results are summarized in the table below.

[0364] [Table 10]

[0365] The results show that H-2B2-T-6F7 and T-6F7-H-1H2 can bind to NCI-H292 cells and NCI-N87 cells, which can simultaneously express HER2 and TROP2.

[0366] Validation of antibody binding to canine tumor cell lines The binding activity of HER2 antibody, anti-TROP antibody, and anti-HER2 / TROP2 antibody to the canine breast cancer cell line CMT-U27 (ATCC, Catalog No.: CRL-3456) was measured by flow cytometry. The secondary antibody used in the experiment was FITC-conjugated goat anti-human IgG (Jackson ImmunoResearch Laboratories, Inc. Catalog No.: 109-096-170). The results are summarized in the table below.

[0367] [Table 11]

[0368] The results showed that the anti-HER2 monoclonal antibodies H-1H2-IgG1, H-2B2-IgG1, and H-3C8-IgG1, and the anti-HER2 / TROP2 bispecific antibodies T-6F7-H-1H2, H-2B2-T-6F7, and H-3C8-T-6F7 were all capable of binding to the canine cell line CMT-U27.

[0369] Anti-HER2 / TROP2 bispecific antibody endocytosis assay Anti-HER2 antibody, anti-TROP2 antibody, or anti-HER2 / TROP2 bispecific antibody, and / or goat anti-human IgG secondary antibody were added to NCI-N87 cells, which highly express human HER2 and TROP2, respectively, and incubated for 1 hour. Cells were centrifuged and washed with FACS buffer. MFI was measured by flow cytometer. Endocytosis ratio of antibodies against NCI-N87 was calculated. Results are shown in the following table (Table 12). For isotype control, antibodies targeting irrelevant target proteins were used.

[0370] For direct detection, the primary antibody (e.g., anti-HER2 antibody, anti-TROP2 antibody, or anti-HER2 / TROP2 bispecific antibody) was directly labeled with a pH-sensitive marker and their endocytosis rate was detected. For indirect detection, a goat anti-human IgG secondary antibody labeled with a pH-sensitive marker was used to detect endocytosis of the primary antibody (e.g., anti-HER2 antibody, anti-TROP2 antibody, or anti-HER2 / TROP2 bispecific antibody).

[0371] [Table 12]

[0372] The results showed the following results regarding endocytosis rate: H-3C8-T-6F7>H-2B2-T-6F7>T-6F7-H-1H2 for bispecific antibodies. The bispecific antibodies showed similar or higher endocytosis rate than the corresponding monoclonal antibodies. The single-arm control bispecific antibodies (H-2B2-CD28, H-3C8-CD28, CD28-H-1H2, and CD28-T-6F7) showed significantly lower endocytosis rate than the corresponding bispecific antibodies or monoclonal antibodies.

[0373] For indirect detection, a marker-labeled goat anti-human secondary antibody may cause cross-linking of the primary antibody (e.g., an anti-HER2 antibody, an anti-TROP2 antibody, or an anti-HER2 / TROP2 bispecific antibody), resulting in enhanced endocytosis.

[0374] Furthermore, purified antibodies H-1H2-T-6F7, H-2B2-T-6F7, H-3C8-T-6F7, and T-6F7-H-1H2 were incubated in cell culture with NCI-N87 cells or NCI-H292 cells, respectively. IncuCyte (Sartorius AG, IncuCyte® S3) was incubated for 24 hours to detect endocytosis of the antibodies. The endocytosis results of NCI-N87 and NCI-H292 cells are shown in Figures 2 to 5.

[0375] The endocytosis results in NCI-N87 cells are shown in FIG. 2 and FIG.

[0376] In terms of endocytosis efficiency over 24 hours in the NCI-N87 cell line, the bispecific antibodies showed similar or higher endocytosis than the corresponding monoclonal antibodies, while the control bispecific antibody showed significantly reduced endocytosis activity. The endocytosis ratios of the bispecific antibodies are as follows: H-3C8-T-6F7>H-2B2-T-6F7>T-6F7-H-1H2.

[0377] The endocytosis results in NCI-H292 cells are shown in FIG.

[0378] In the NCI-H292 cell line, the endocytosis rates within 24 hours are as follows: the endocytosis efficiency of the bispecific antibody is weaker than that of the T-6F7-IgG1-SI monoclonal antibody, but stronger than that of the anti-HER2 monoclonal antibody and the control bispecific antibody.

[0379] Stability of anti-HER2 / TROP2 bispecific antibodies Four anti-HER2 / TROP2 antibodies, H-1H2-T-6F7, H-2B2-T-6F7, T-6F7-H-1H2, and H-3C8-T-6F7, were diluted to 5 mg / mL with a pH 6.0 buffer (3 mg / mL histidine, 80 mg / mL sucrose, and 0.2 mg / mL Tween® 80). The diluted antibodies were kept in a sealed Eppendorf tube at 4±3°C (hereinafter referred to as 4°C) for 7 days or at 40±3°C (hereinafter referred to as 40°C) for 7 days to evaluate thermal stability.

[0380] Specifically, the following tests were performed: (1) observing the appearance of the solution and the presence of visible non-soluble objects; (2) detecting changes in the purity of the antibody by size-exclusion high-performance liquid chromatography (SEC-HPLC), shown as the percentage of the main peak area relative to the sum of all peak areas (purity, %); (3) detecting changes in the nominal hydrophobicity of the antibody using a hydrophobic interaction chromatography-high-performance liquid chromatography (HIC-HPLC) method, shown as the retention time of the main peak (HIC, minutes); (4) detecting the percentage purity of the antibody by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) under reducing (CE-SDS(R) and non-reducing (CE-SDS(NR)) conditions, shown as the percentage of the main peak area relative to the sum of all peak areas (purity, %); (5) detecting charge variants in the antibody by capillary isoelectric focusing (cIEF) method, shown as the percentage (%) of the main, acidic, and alkaline components.

[0381] In the SEC-HPLC experiments, antibody samples were diluted to 1 mg / mL with purified water and an Agilent 1290 chromatographic system was used, connected to an XBridge™ Protein BEH SEC column (200 Å, Waters Corporation). The following parameters were used: mobile phase: 25 mmol / L phosphate buffer (PB) + 300 mmol / L NaCl, pH 6.8; flow rate: 1.8 mL / min; column temperature: 25° C.; detection wavelength: 280 nm; injection volume: 10 μL; sample tray temperature: approx. 4° C.; and run time: 7 min.

[0382] For HIC-HPLC experiments, an Agilent 1260 chromatographic system (connected with a ProPasc™ HIC-10 column (4.6x250mm, Thermo Scientific)) was used, and samples were diluted to 0.5mg / mL with mobile phase A. The following parameters were used: mobile phase A: 1.0M ammonium sulfate, 20mM sodium acetate, 10% acetonitrile (pH 6.5); mobile phase B: 20mM sodium acetate, 10% acetonitrile (pH 6.5); flow rate: 0.8mL / min; gradient: 0min 100% A, 2min 100% A, 32min 100% B, 34min 100% B, 35min 100% A, and 45min 100% A; column temperature: 30°C; detection wavelength: 280nm; injection volume: 10μL; sample tray temperature: about 10°C; and run time: 30min.

[0383] For cIEF experiments, a Maurice cIEF Method Development Kit (Protein Simple, Catalog Number: PS-MDK01-C) was used for sample preparation. Specifically, 8 μL of protein sample was mixed with the following reagents in the kit: 1 μL Maurice cIEF pI Marker-4.05, 1 μL Maurice cIEF pI Marker-9.99, 35 μL 1% methylcellulose solution, 2 μL Maurice cIEF 500 mM arginine, 4 μL ampholytes (Pharmalyte pH range 3-10), and water (add to a final volume of 100 μL). Imaging capillary isoelectric focusing spectra were generated using a Maurice cIEF cartridge (PS-MC02-C) on a Maurice analyzer (Protein Simple, Santa Clara, CA). Samples were focused for a total of 10 min. Protein absorbance focused at 280 nm was integrated using analysis software installed on the instrument.

[0384] For CE-SDS experiments, Maurice (Protein simple, Maurice™) and Maurice CE-SDS size application kit (Protein simple, Catalogue number: PS-MAK02-S) were used.

[0385] In CE-SDS (NR), 54 μL sample buffer, 6 μL antibody sample, 2.4 μL 25x internal standard, 3 μL 250 nM iodoacetamide (SIGMA, Catalog No.: 16125) were added to a microcentrifuge tube, followed by centrifugation at 3000 rpm for 1 minute and heating in a 70°C water bath for 10 minutes. The samples were then cooled to room temperature, followed by centrifugation at 10000 rpm for 3 minutes. The supernatant sample preparations were then transferred to a 96-well plate and tested on the Maurice. The following parameters were used: injection voltage 4.6 kV, injection time 20 seconds, separation voltage 5.75 kV, and separation time 40 minutes.

[0386] In CE-SDS®, 54 μL of sample buffer, 6 μL of antibody sample, 2.4 μL of 25x internal standard, 3 μL of 2-mercaptoethanol (SIGMA, Cat. No.: M6250) were added to a microcentrifuge tube, followed by centrifugation at 3000 rpm for 1 minute and heating in a 70°C water bath for 10 minutes. The sample was then cooled to room temperature, followed by centrifugation at 10000 rpm for 3 minutes. 50 μL of the supernatant sample preparation was then transferred to a 96-well plate and tested on the Maurice. The injection voltage was 4.6 kV, the injection time was 20 seconds, the separation voltage was 5.75 kV, and the separation time was 30 minutes.

[0387] Detailed results of H-1H2-T-6F7, H-2B2-T-6F7, T-6F7-H-1H2, and H-3C8-T-6F are shown in Figure 6.

[0388] The results showed that H-3C8-T-6F7-IgGI and H-2B2-T-6F7-IgG1 have better stability, and physical and chemical properties.

[0389] Example 5. Antibody Drug Conjugates After Protein A purification, H-2B2-IgG1, H-3C8-IgG1, H-1H2-IgG1, T-6F7-IgG1-SI, H-2B2-CD28, H-3C8-CD28, CD28-T-6F7, CD28-H-1H2, H-2B2-T-6F7, H-3C8-T-6F7, H-1H2-T-6F7, and T-6F7-H-1H2 were dialyzed and concentrated by ultrafiltration into PBS buffer. Concentrations were measured by UV absorption. These antibodies were used in the subsequent antibody-drug coupling reaction.

[0390] Coupling of antibodies with drug molecules The purified antibodies H-2B2-IgG1, H-3C8-IgG1, H-1H2-IgG1, T-6F7-IgG1-SI, H-2B2-CD28, H-3C8-CD28, CD28-T-6F7, CD28-H-1H2, H-2B2-T-6F7, H-3C8-T-6F7, H-1H2-T-6F7, and T-6F7-H-1H2 were coupled to MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F) by a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker. The drug-antibody ratio (DAR) of the resulting ADCs was 3-4.

[0391] For the name of the antibody drug conjugate, "ADC" is added immediately after the antibody name. For example, H-2B2-IgG1 coupled to MMAE would be named H-2B2-IgG1-ADC.

[0392] HPLC and HIC-HPLC were used to detect the coupling of the antibody to the drug molecule. The results of the SEC-HPLC test are shown in the table below (Table 13). For the isotype control ADC, a human IgG1 isotype control was coupled to MMAE to form an isotype control ADC.

[0393] [Table 13]

[0394] The detection results of HIC-HPLC are shown in Table 14. The results showed that the DAR of the ADC was about 3, where the average DAR is measured by multiplying the corresponding drug loading of 0, 2, 4, 6, or 8 by the PA% (PA% is the percentage of the peak area as measured by the original area of ​​the 280 nm peak) divided by 100. For example, the average DAR of H-2B2-IgG1-ADC is [(7.81×0)+(36.65×2)+(39.81×4)+(12.01×6)+(3.71×8)] / 100=3.34.

[0395] [Table 14]

[0396] in vitro killing activity Various concentrations of purified ADC (10000ng / mL, 2000ng / mL, 400ng / mL, 80ng / mL, 16ng / mL, 3.2ng / mL, 0.64ng / mL, 0.13ng / mL) were used to treat human gastric cancer cell line NCI-N87 or human lung cancer cell line NCI-H292 cultured in cell culture plates, and the killing activity was detected after 72 hours of incubation with IncuCyte (Sartorius AG, IncuCyte® S3). The results are shown in the table below.

[0397] [Table 15]

[0398] In the NCI-N87 cell line, the HER2 / TROP2 bispecific antibody ADC showed a stronger killing ability than the control bispecific antibody ADC (IC50>10-fold). In the NCI-H292 cell line, the HER2 / TROP2 bispecific antibody ADC killing was lower than the TROP2 monoclonal antibody ADC, but more potent than the HER2 monoclonal antibody ADC and the control bispecific antibody ADC. The data showed that the HER2 / TROP2 bispecific antibody ADC selectively killed the HER2 / TROP2 dual target expressing cell line. In addition, the in vitro cell killing ability of H-2B2-T-6F7-ADC was similar to that of the parent T-6F7-IgG1-SI-ADC, suggesting that H-2B2-T-6F7-ADC has potential efficacy against TROP2 high / HER2 low tumors.

[0399] Example 6. Antitumor activity in the NCI-H1975 xenograft model The ADC was tested for its effect on tumor growth in vivo in a model of lung adenocarcinoma. 6 NCI-H1975 (lung adenocarcinoma cells) were subcutaneously injected into B-NDG mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., Beijing, China; Catalog No. B-CM-002). The tumor volume in the mice was approximately 100 mm 3 Once tumor volume reached 100 μg / kg, mice were randomly divided into different groups based on tumor volume. Mice were then injected intravenously (iv) with phosphate buffered saline (PBS) or ADC.

[0400] The injection volume was calculated based on the mouse weight and the desired dose of 3 mg / kg. The long and short axes of the tumor were measured and the tumor volume was calculated as 0.5 x (long axis) x (shorter axis). 2 The body weight of the mice was also measured twice a week.

[0401] Tumor growth inhibition percentage (TGI%) is calculated using the following formula: [1-(Ti-T0) / (Vi-V0)]×100. Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0.

[0402] T-test was performed for statistical analysis. A TGI% of more than 60% indicates a clear inhibition of tumor growth. P<0.05 was the threshold for indicating significant differences.

[0403] [Table 16]

[0404] The weights of the mice in the different groups all increased. On the day of group allocation (day 0), the mean weight of each group ranged from 22.0 g to 22.6 g. At the end of the experiment (day 28), the mean weight of each group ranged from 21.3 g to 24.1 g, and the mean weight of each group ranged from 94.1% to 112.2%. The results showed that all the tested ADCs were well tolerated and had no obvious toxicity to the mice.

[0405] Tumor sizes in the ADC treated groups are shown in Figure 7. The table below lists the results for this experiment, including tumor volumes on the day of grouping (day 0), 11 days after grouping (day 11), 21 days after grouping (day 21), and at the end of the experiment (day 28); survival rate of mice; tumor growth inhibition values ​​(TGI); and statistical differences (P values) of tumor volumes and body weights between treated and control groups.

[0406] [Table 17]

[0407] The tumor volumes in all treatment groups (G3-G15) were smaller than those in the control groups (G1 and G2). The treatment groups had different tumor-inhibitory effects. Three bispecific antibody ADCs, including T-6F7-H-1H2-ADC (G5), H-3C8-T-6F7-ADC (G6), and H-2B2-T-6F7-ADC (G7), showed sustained and potent tumor-inhibitory effects.

[0408] The corresponding monoclonal antibody ADCs (G8-G11) and control ADCs (G12-G14) except for G15 also had potent tumor suppressor effects. The isotype control (G2) also showed potent tumor inhibitory effects, which may be due to an unstable linker or nonspecific binding of the isotype control, resulting in vc-MMAE release in mice, leading to nonspecific killing.

[0409] In another experiment, about 5 × 10 6 NCI-H1975 cells were injected subcutaneously into B-NDG mice, and tumor volumes were approximately 300 mm 3 Once grown to 100%, cells were divided into a control group and various treatment groups (5 mice per group) based on tumor size. Treatment groups were randomly selected for H-2B2-T-6F7 treatment (G2 (3 mg / kg) and G3 (10 mg / kg)), H-2B2-T-6F7-ADC treatment (G4, 3 mg / kg), or MMAE treatment (G5, 0.06 mg / kg, equimolar dose to G4). Mice in the control group were injected with PBS (G1). The dosing frequency was once a week (total, 2 doses). Tumor volumes were measured twice a week, as were the weights of the mice.

[0410] The tumor sizes of the antibody-treated groups are shown in Figure 15 and Table 18. This shows that compared with the control group, the tumor growth in the treatment groups was inhibited to different degrees, and the anti-HER2 / TROP2 bispecific antibody ADC H-2B2-T-6F7-ADC at a dose of 3 mg / kg obtained a better tumor inhibition effect compared with the anti-HER2 / TROP2 bispecific antibody H-2B2-T-6F7 at a dose of 10 mg / kg. In the MMAE-treated group, limited tumor growth inhibition was obtained, suggesting that the in vivo efficacy of H-2B2-T-6F7-ADC was dependent on the binding of HER2 and TROP2 antigens.

[0411] [Table 18]

[0412] In another experiment, approximately 1 × 10 6 NCI-H1975 cells were injected subcutaneously into B-NDG mice, and tumor volumes were approximately 300 mm 3 Once grown to 100%, cells were divided into different treatment groups based on tumor size. The dosing scheme is detailed in the table below.

[0413] [Table 19]

[0414] Dicitamab vedotin (RC48, RemeGen Co., Ltd., SEQ ID NO: 83; SEQ ID NO: 84) is an ADC developed by RemeGen that contains a recombinant humanized monoclonal antibody against HER2 and an MMAE payload. In 2021, the product was conditionally approved in China for the treatment of HER2-expressing locally advanced / metastatic gastric cancer (gastroesophageal junction carcinoma).

[0415] Trastuzumab deruxtecan (Enhertu®, Daiichi Sankyo Company, Limited., SEQ ID NO: 85; SEQ ID NO: 86) is an ADC comprising a humanized anti-HER2 antibody linked to a topoisomerase I inhibitor payload by a tetrapeptide linker. The product was initially launched in the United States in 2020 for the treatment of adult patients with unresectable or metastatic HER2-positive breast cancer who have received two or more prior anti-HER2-based regimens in the metastatic setting.

[0416] Tumor volumes were measured twice weekly, and the results are shown in Figure 16 and Table 20. These show that at the same dose and dosing frequency, the anti-HER2 / TROP2 bispecific antibody ADC H-2B2-T-6F7-ADC inhibited tumor growth with a higher TGI% than that of the sacituzumab govitecan analogs, dicitamab vedotin, or trastuzumab deruxtecan. In addition, the tumor inhibition of H-2B2-T-6F7-ADC showed a correlation trend with increasing dose.

[0417] [Table 20]

[0418] Example 7. Antitumor activity in SK-OV-3 xenograft model The ADC was tested for its effect on tumor growth in vivo in a model of ovarian cancer. 6 SK-OV-3 cells were subcutaneously injected into B-NDG mice. The tumor volume in the mice was approximately 200–250 mm. 3 Once tumor volume reached 100 mg / kg, the mice were randomly divided into different groups based on tumor volume. The mice were then injected with PBS or ADC by intravenous (iv) administration. The administration frequency was once a week (total, 3 administrations). The doses of the first and second administrations were both 3 mg / kg, and the final administration was 5 mg / kg. The details of the administration scheme are shown in the table below.

[0419] [Table 21]

[0420] Tumor volumes were measured twice weekly and the results are shown in Figure 17 and Table 22. These show that the anti-HER2 / TROP2 bispecific antibody ADC H-2B2-T-6F7-ADC had the greatest tumor growth inhibition at 3 mg / kg in the ovarian cancer model. Furthermore, the anti-HER2 monoclonal antibody ADC H-2B2-IgG1-ADC provided better efficacy than the positive control trastuzumab deruxtecan analog, and the anti-TROP2 monoclonal antibody ADC H-2B2-T-6F7-ADC provided better efficacy than the positive control DS-1062 analog.

[0421] [Table 22]

[0422] In another experiment, about 5 × 10 6 SK-OV-3 cells were subcutaneously injected into B-NDG mice, and tumor volumes were approximately 200–250 mm 3 Once the tumors had grown to 100%, the cells were divided into a control group and four treatment groups (5 mice per group) based on tumor size. Treatment group mice were randomly selected for intravenous (iv) administration of H-2B2-T-6F7-ADC at 0.3 mg / kg (G2), 1 mg / kg (G3), 3 mg / kg (G4), or 10 mg / kg (G5). Control group mice were injected with an equal volume of PBS (G1). The administration frequency was once a week (total of 2 administrations). Tumor volumes were measured twice a week, and the results are shown in Table 23. This indicates that H-2B2-T-6F7-ADC exhibits sustained tumor inhibitory activity in a dose-dependent manner.

[0423] [Table 23]

[0424] Example 8. Antitumor activity in the NCI-N87 model The ADC was tested for its effect on tumor growth in vivo in a model of gastric cancer. 6 NCI-N87 cells were injected subcutaneously into B-NDG mice. The tumor volume in the mice was approximately 150 mm. 3 Once tumor volume reached 100 mg / kg, mice were randomly divided into different groups based on tumor volume. Mice were then injected intravenously (iv) with PBS or ADC. The administration scheme is detailed in the table below.

[0425] [Table 24]

[0426] Tumor volumes were measured twice weekly, and the results are shown in Table 25. This shows that the anti-HER2 / TROP2 bispecific antibody ADC H-2B2-T-6F7-ADC had the greatest tumor growth inhibition at 3 mg / kg in the NCI-N87 gastric cancer model. Among the anti-TROP2 monoclonal antibody ADCs, T-6F7-IgG1-SI-ADC showed better efficacy than the positive control sacituzumab govitecan analog at both 1 mg / kg and 3 mg / kg doses. The anti-HER2 monoclonal antibody ADC H-2B2-IgG1-ADC also showed better efficacy than the positive controls dicitamab vedotin and trastuzumab deruxtecan at both 1 mg / kg and 3 mg / kg doses.

[0427] [Table 25]

[0428] Example 9. Antitumor activity in the BT474 model The ADCs were tested for their effect on tumor growth in vivo in a model of breast cancer. 7 BT-474 cells were injected subcutaneously into B-NDG mice. The tumor volume in the mice was approximately 200 mm. 3Once tumor volume reached 100 mg / kg, mice were randomly divided into different groups based on tumor volume. Mice were then injected intravenously (iv) with PBS or ADC. The administration scheme is detailed in the table below.

[0429] [Table 26]

[0430] Tumor volumes were measured twice weekly, and the results are shown in Figure 18. It shows that the anti-HER2 / TROP2 bispecific antibody ADC H-2B2-T-6F7-ADC had the greatest tumor growth inhibition at 3 mg / kg in the HER2-highly expressing breast cancer BT-474 model (TGI%=122.0% at day 23). Among the anti-TROP2 monoclonal antibody ADCs, T-6F7-IgG1-SI-ADC showed better efficacy than the positive control sacituzumab govitecan analog (TGI%=68.7% and 51.4%, respectively). The anti-HER2 monoclonal antibody ADC H-2B2-IgG1-ADC also showed better efficacy than the positive controls dicitamab vedotin and trastuzumab deruxtecan (TGI%=121.9%, 94.4%, and 92.9%, respectively).

[0431] Example 10. Antitumor activity in the NCI-H292 model The effect of the ADC on tumor growth in the NCI-H292 lung cancer model was examined. 6 NCI-H292 cells were injected subcutaneously into B-NDG mice. The tumor volume in the mice was approximately 300 mm. 3 Once tumor volume reached 100 mg / kg, mice were randomly divided into different groups based on tumor volume. Mice were then injected intravenously (iv) with PBS or ADC. The administration scheme is detailed in the table below.

[0432] [Table 27]

[0433] Tumor volumes were measured twice weekly and the results are shown in Table 28, which shows that anti-HER2 / TROP2 bispecific antibody ADC H-2B2-T-6F7-ADC inhibited tumor growth in a dose-dependent manner, with maximum TGI% in the treatment groups at 1 mg / kg, 3 mg / kg, and 10 mg / kg, respectively. The results indicate that anti-HER2 / TROP2 bispecific antibody ADC H-2B2-T-6F7-ADC has therapeutic potential for the treatment of HER2 low-expressing tumors.

[0434] [Table 28]

[0435] Example 11. In vivo efficacy in a human pancreatic patient-derived xenograft (PDX) model

[0436] The efficacy of ADC was tested in two human pancreatic PDX models (PDX001 and PDX002). Immunofluorescence staining of patient-derived pancreatic tumor sections was performed and images were analyzed with HALO 3.2 version. The results showed that in PDX001, HER2-positive cells and TROP2-positive cells were 84.72% and 89.09%, respectively. In PDX002, HER2-positive cells and TROP2-positive cells were 86.34% and 89.17%, respectively.

[0437] In the PDX001 model, patient-derived pancreatic tumor fragments (2 mm × 2 mm × 2 mm) were implanted into the right flank of B-NDG mice. Tumor volumes in mice were approximately 300–400 mm. 3 Once tumor volume reached 100 mg / kg, mice were randomly divided into different groups based on tumor volume. Mice were then injected intravenously (iv) with PBS or ADC. The administration scheme is detailed in the table below.

[0438] [Table 29]

[0439] Tumor volumes were measured twice weekly and the results are shown in Figure 19, which shows that treatment with H-2B2-T-6F7-ADC (G2), H-2B2-IgG1-ADC (G3), and T-6F7-IgG1-SI-ADC (G4) resulted in significant tumor growth inhibition in the HER2 / TROP2 co-expressing human pancreatic PDX models, with TGI% of 100.5%, 88.2%, and 66.4%, respectively, at day 38 (38 days after group placement).

[0440] In the PDX002 model, tumors in mice grew to approximately 250–300 mm 3 When tumors reached a volume of 1000 μg / ml, the cells were divided into a control group and various treatment groups (5 mice per group) based on tumor size. Treatment groups were randomly selected for 3 mg / kg H-2B2-T-6F7-ADC treatment (G2), H-2B2-IgG1-ADC treatment (G3), T-6F7-IgG1-SI-ADC treatment (G4), sacituzumab govitecan analog treatment (G5), dicitamab vedotin treatment (G6), or trastuzumab deruxtecan treatment (G7). Mice in the control group were injected with PBS (G1). The dosing frequency was once a week (total, 1 dose).

[0441] Tumor sizes in the ADC-treated groups are shown in Figure 20. Consistent with the in vivo findings above, H-2B2-T-6F7-ADC (G2), H-2B2-IgG1-ADC (G3), and T-6F7-IgG1-SI-ADC (G4) provided substantial antitumor activity with TGI% of 107.5%, 91.9%, and 88.2%, respectively, at day 21 (21 days after group placement).

[0442] Example 12. Toxicological studies Toxicological study in B-NDG mice The toxicity of anti-HER2 / TROP2 bispecific antibody ADC was measured in B-NDG mice. Specifically, mice were placed into six groups (12 mice per group) and administered saline (G1) or H-2B2-T-6F7-ADC at 10 mg / kg (G2), 30 mg / kg (G3), 50 mg / kg (G4), 70 mg / kg (G5), or 100 mg / kg (G6) by intravenous injection. The dosing frequency was once a week (total, one dose). The details of the dosing scheme are shown in the table below.

[0443] [Table 30]

[0444] Body weight was measured daily for the first week and then twice weekly until the end of the experiment after 4 weeks. As shown in Figures 21A-21B, mice in groups G5 and G6 showed significant weight loss, while mice in other treatment groups showed no significant difference in body weight compared to control mice. One mouse died in group G6 receiving 100 mg / kg H-2B2-T-6F7-ADC.

[0445] Toxicological studies in C57BL / 6 and hHER2 / TROP2 mice As in the previous experiment, the toxicity of the anti-HER2 / TROP2 bispecific antibody ADC was measured in C57BL / 6 mice and hHER2 / TROP2 mice.

[0446] HER2 humanized mice were crossed with TROP2 humanized mice to generate hHER2 / TROP2 mice. HER2 humanized mice were engineered to express chimeric HER2 protein (SEQ ID NO: 87), in which the extracellular and transmembrane domains of the mouse HER2 protein were replaced with the corresponding human TNFR2 extracellular and transmembrane domains. TROP2 humanized mice were engineered to express human TROP2 protein (SEQ ID NO: 88), in which the coding region of the mouse TROP2 gene was replaced with the corresponding human TROP2 coding region. Detailed descriptions of HER2 humanized mice and TROP2 humanized mice can be found in CN202110959814.8 and CN202111119814.3, each of which is incorporated by reference in its entirety herein.

[0447] hHER2 / TROP2 mice and C57BL / 6 mice were divided into different groups (6 mice per group) based on body weight and administered saline, H-2B2-T-6F7-ADC (10 mg / kg, 30 mg / kg, 90 mg / kg), or MMAE (0.19 mg / kg, 0.57 mg / kg, 1.14 mg / kg, and 1.71 mg / kg, equivalent to H-2B2-T-6F7-ADC at 10 mg / kg, 30 mg / kg, 60 mg / kg, and 90 mg / kg, respectively) by intravenous injection. The administration frequency was once a week (total, one administration). The administration scheme and details of survival at day 7 are shown in the table below.

[0448] [Table 31]

[0449] Body weight was measured daily until the end of the experiment one week later. The results showed that all mice survived in groups G1-G6 and G9-G10. However, treatment groups G7, G8, and G11 showed some toxicity.

[0450] Example 13. In vivo efficacy in human colorectal cancer PDX models The efficacy of ADC was tested in a xenograft model derived from a human colorectal cancer patient. Immunofluorescence staining of patient-derived colon tumor fragments was performed, and images were analyzed with HALO 3.2 version. The results showed that the percentage of HER2-positive cells and TROP2-positive cells in human colon tumor tissues was 13.28% and 15.30%, respectively. Patient-derived colon tumor fragments (2 mm x 2 mm x 2 mm) were implanted into the right flank of B-NDG mice. The tumor volume in the mice was approximately 250-300 mm. 3 Once tumor volume reached 100 mg / kg, mice were randomly divided into different groups based on tumor volume. Mice were then injected intravenously (iv) with PBS or ADC. The administration scheme is detailed in the table below.

[0451] [Table 32]

[0452] Tumor volumes were measured twice weekly, and the results are shown in Table 33 and Figure 22. Compared with PBS (G1) and sacituzumab govitecan (G3), H-2B2-T-6F7-ADC (G2), dicitamab vedotin (G4), and trastuzumab deruxtecan (G5) showed remarkable antitumor effects, with H-2B2-T-6F7-ADC inhibiting tumor growth with a high TGI% of 96.4% on day 39. In addition, the H-2B2-T-6F7-ADC and trastuzumab deruxtecan treatment groups were followed for 49 days after grouping (day 49), and H-2B2-T-6F7-ADC showed better tumor inhibition than the trastuzumab deruxtecan treatment group in the HER2-low-expressing human colon PDX model.

[0453] [Table 33]

[0454] Example 14. In vivo efficacy in human lung cancer PDX models The efficacy of ADC was tested in a xenograft model derived from a human lung cancer patient. Immunofluorescence staining of the patient-derived lung tumor fragments was performed, and the images were analyzed with HALO 3.2 version. The results showed that the percentage of HER2-positive cells and TROP2-positive cells in human lung tumor tissues was 67.05% and 72.04%, respectively. Patient-derived lung tumor fragments (2 mm x 2 mm x 2 mm) were transplanted into the right flank of B-NDG mice. The tumor volume in the mice was approximately 250-300 mm. 3 Once tumor volume reached 100 mg / kg, mice were randomly divided into different groups based on tumor volume. Mice were then injected intravenously (iv) with PBS or ADC. The administration scheme is detailed in the table below.

[0455] [Table 34]

[0456] Tumor volumes were measured twice weekly, and the results are shown in Table 35 and Figure 23. H-2B2-T-6F7-ADC (G2) at 3 mg / kg provided better antitumor activity than parental monoclonal antibodies H-2B2-IgG1-ADC (G4) and T-6F7-IgG1-SI-ADC (G5). In addition, H-2B2-IgG1-ADC provided more significant tumor growth inhibition, even at a dose of 3 mg / kg, than the positive controls dicatomab vedotin and trastuzumab deruxtecan at a dose of 6 mg / kg in the HER2-low-expressing human lung PDX model.

[0457] [Table 35]

[0458] Mouse tumor volume and survival were continuously monitored after day 28. At the end of the experiment (day 63), all mice in groups G1-G8 (except G3) were euthanized due to excessive tumor volume, and in group G3, all mice had tumors of 514±172 mm. 3 100% CI, 0.01 to 0.25, respectively, indicating that H-2B2-T-6F7-ADC has potent therapeutic potential.

[0459] Example 15. In vivo efficacy in human gastric cancer PDX models The efficacy of ADC was tested in a xenograft model derived from a human gastric cancer patient. Immunofluorescence staining of patient-derived gastric tumor fragments was performed, and the results showed that the percentage of HER2-positive cells and TROP2-positive cells was 0.08% and 0.34%, respectively. Patient-derived gastric cancer tumor fragments (2 mm x 2 mm x 2 mm) were implanted into the right flank of B-NDG mice. The tumor volume in the mice was approximately 150-200 mm. 3 Once tumor volume reached 100 mg / kg, mice were randomly divided into different groups based on tumor volume. Mice were then injected intravenously (iv) with PBS or ADC. The administration scheme is detailed in the table below.

[0460] [Table 36]

[0461] Tumor volumes were measured twice weekly, and the results are shown in Table 37 and Figure 24. Experimental results show that H-2B2-T-6F7-ADC exhibited better antitumor activity than the positive controls dicitamab vedotin and trastuzumab deruxtecan at a dose of 6 mg / kg in a HER2-low-expressing human gastric PDX model.

[0462] [Table 37]

[0463] Example 16. In vitro plasma stability study of anti-HER2 / TROP2 ADCs H-2B2-T-6F7-ADC, human plasma, monkey plasma, and 0.5% BSA PBS solution were each filtered through a 0.22 μm filter for sterilization. H-2B2-T-6F7-ADC was added to the sterilized plasma or 0.5% BSA solution in PBS at a final concentration of 0.1 mg / mL, and the reaction solution was incubated in an incubator at 37° C. The incubation day was recorded as day 0, and samples were taken on days 1, 2, 6, 8, 11, and 14, respectively, for detection of free MMAE by LC-MS (liquid chromatography-mass spectrometry). The ratio of free MMAE to the total antibody MMAE (hereinafter referred to as MMAE release rate (%)) was calculated.

[0464] The results are shown in Figure 25, which indicates that H-2B2-T-6F7-ADC is fairly stable in both human and monkey plasma, as well as in a solution of 0.5% BSA in PBS, with a maximum release rate of less than 1.0% free MMAE.

[0465] Other embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the above description is for illustrative purposes only and is not intended to limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; a light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence; The selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are as follows: (1) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 49 to 51, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1 to 3, respectively; (2) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 58-60, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively; (3) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 43 to 45, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1 to 3, respectively; (4) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 46-48, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; (5) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 52-54, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively; (6) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 55 to 57, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 4 to 6, respectively; An antibody or antigen-binding fragment thereof that binds to TROP2 (trophoblast surface antigen 2), one of the antigens listed above.

2. wherein the VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 49 to 51, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 1 to 3, respectively, according to the Kabat numbering scheme; or According to the Chothia numbering scheme, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 58-60, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 4-6, respectively; or the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOS: 43-45, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOS: 1-3, respectively, according to the Kabat numbering scheme; or According to the Kabat numbering scheme, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 46-48, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 1-3, respectively; or the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOS: 52-54, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOS: 4-6, respectively, according to the Chothia numbering scheme; or 2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 55 to 57, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 4 to 6, respectively, according to the Chothia numbering scheme.

3. a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90% identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are: (1) the selected VH sequence is SEQ ID NO: 63 and the selected VL sequence is SEQ ID NO: 37; (2) the selected VH sequence is SEQ ID NO: 61 and the selected VL sequence is SEQ ID NO: 37; (3) the selected VH sequence is SEQ ID NO: 62 and the selected VL sequence is SEQ ID NO: 37; An antibody or antigen-binding fragment thereof that binds to TROP2, which is one of the antibodies listed above. Claim 4: The VH comprises the sequence of SEQ ID NO: 63 and the VL comprises the sequence of SEQ ID NO: 37; or said VH comprising the sequence of SEQ ID NO: 61 and said VL comprising the sequence of SEQ ID NO: 37; or The antibody or antigen-binding fragment thereof of claim 3, wherein the VH comprises the sequence of SEQ ID NO: 62 and the VL comprises the sequence of SEQ ID NO:

37.

5. (1) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 49 to 51, wherein the VH binds to TROP2 when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 37; or (2) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 58 to 60, wherein the VH binds to TROP2 when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 37; or (3) An immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 1 to 3, wherein the VL binds to TROP2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 63; or (4) An immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 4 to 6, wherein the VL binds to TROP2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 63; or (5) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 43 to 45, wherein the VH binds to TROP2 when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 37; (6) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 46 to 48, wherein the VH binds to TROP2 when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 37; (7) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 52 to 54, wherein the VH binds to TROP2 when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 37; (8) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 55 to 57, wherein the VH binds to TROP2 when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 37; (9) An immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 1 to 3, wherein the VL binds to TROP2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 61; (10) An immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 1 to 3, wherein the VL binds to TROP2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 62; (11) An immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 4 to 6, wherein the VL binds to TROP2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 61; (12) An immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 4 to 6, wherein the VL binds to TROP2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 62; A nucleic acid comprising a polynucleotide encoding a polypeptide comprising:

6. An antigen-binding protein construct comprising a first antigen-binding domain that specifically binds to HER2 and a second antigen-binding domain that specifically binds to TROP2, wherein the second antigen-binding domain comprises the antigen-binding fragment of any one of claims 1 to 4.

7. The first antigen-binding domain comprises a first heavy chain variable region (VH1) comprising CDR1, 2, and 3, and a first light chain variable region (VL1) comprising CDR1, 2, and 3; the second antigen-binding domain comprises a second heavy chain variable region (VH2) comprising CDR1, 2, and 3, and a second light chain variable region (VL2) comprising CDR1, 2, and 3; (1) The VH1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 10 to 12, respectively, the VL1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively, the VH2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 49 to 51, respectively, and the VL2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively; (2) The VH1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 19 to 21, respectively, the VL1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively, the VH2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 49 to 51, respectively, and the VL2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively; (3) The VH1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 7 to 9, respectively, the VL1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively, the VH2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 49 to 51, respectively, and the VL2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively; (4) The VH1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 7 to 9, respectively, the VL1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively, the VH2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 46 to 48, respectively, and the VL2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively; (5) The VH1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 10 to 12, respectively, the VL1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively, the VH2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 46 to 48, respectively, and the VL2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively; (6) The VH1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 19 to 21, respectively, the VL1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively, the VH2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 46 to 48, respectively, and the VL2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively; (7) The VH1 CDR1, 2, and 3 amino acid sequences are respectively represented by SEQ ID NOs: 7 to 9, the VL1 CDR1, 2, and 3 amino acid sequences are respectively represented by SEQ ID NOs: 1 to 3, the VH2 CDR1, 2, and 3 amino acid sequences are respectively represented by SEQ ID NOs: 43 to 45, and the VL2 CDR1, 2, and 3 amino acid sequences are respectively represented by SEQ ID NOs: 1 to 3; (8) The VH1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 10 to 12, respectively, the VL1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively, the VH2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 43 to 45, respectively, and the VL2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively; (9) The VH1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 19 to 21, respectively, the VL1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively, the VH2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 43 to 45, respectively, and the VL2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively; (10) The VH1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 13 to 15, respectively, the VL1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively, the VH2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 49 to 51, respectively, and the VL2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively; (11) The VH1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 13 to 15, respectively; the VL1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively; the VH2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 46 to 48, respectively; and the VL2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively; (12) The VH1 CDR1, 2, and 3 amino acid sequences are respectively represented by SEQ ID NOs: 13 to 15, the VL1 CDR1, 2, and 3 amino acid sequences are respectively represented by SEQ ID NOs: 1 to 3, the VH2 CDR1, 2, and 3 amino acid sequences are respectively represented by SEQ ID NOs: 43 to 45, and the VL2 CDR1, 2, and 3 amino acid sequences are respectively represented by SEQ ID NOs: 1 to 3; (13) The VH1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 16 to 18, respectively; the VL1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively; the VH2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 49 to 51, respectively; and the VL2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively; (14) The VH1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 16 to 18, respectively; the VL1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively; the VH2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 46 to 48, respectively; and the VL2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively; (15) The VH1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 16 to 18, respectively; the VL1 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively; the VH2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 43 to 45, respectively; and the VL2 CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 1 to 3, respectively; 7. The antigen-binding protein construct of claim 6.

8. The first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2), and the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:39, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:63, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37; or the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:42, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:63, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37; or the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:38, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:63, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37; or the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:38, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:62, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37; or the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:39, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:62, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37; or the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:42, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:62, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37; or the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:38, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:61, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37; or the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:39, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:61, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37; or the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:42, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:61, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37; or the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:40, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:63, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37; or the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:41, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:63, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37; or the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:40, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:62, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37; or the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:41, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:62, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37; or the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:40, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:61, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37; or 7. The antigen-binding protein construct of claim 6, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:41, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:37, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:61, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:

37.

9. An antibody or antigen-binding fragment thereof that binds to HER2 (human epidermal growth factor receptor 2), a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; a light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence; wherein the selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are as follows: (1) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 10 to 12, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1 to 3, respectively; (2) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 25 to 27, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 4 to 6, respectively; (3) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 7 to 9, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1 to 3, respectively; (4) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 13 to 15, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1 to 3, respectively; (5) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 16 to 18, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1 to 3, respectively; (6) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 19 to 21, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1 to 3, respectively; (7) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 22 to 24, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 4 to 6, respectively; (8) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 28 to 30, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 4 to 6, respectively; (9) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 31 to 33, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 4 to 6, respectively; (10) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 34 to 36, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 4 to 6, respectively; It is one of the Optionally, where: (i) the antibody or antigen-binding fragment specifically binds to human HER2 or canine HER2; (ii) the antibody or antigen-binding fragment is a human or humanized antibody or antigen-binding fragment thereof; and / or (iii) the antibody or antigen-binding fragment is a single-chain variable fragment (scFv); An antibody or antigen-binding fragment thereof that binds to HER2 (human epidermal growth factor receptor 2).

10. The VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 10-12, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 1-3, respectively, according to the Kabat numbering scheme; or According to the Chothia numbering scheme, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs:25-27, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs:4-6, respectively; or According to the Kabat numbering scheme, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOS: 7-9, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOS: 1-3, respectively; or According to the Kabat numbering scheme, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 13-15, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 1-3, respectively; or According to the Kabat numbering scheme, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOS: 16-18, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOS: 1-3, respectively; or According to the Kabat numbering scheme, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 19-21, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 1-3, respectively; or According to the Chothia numbering scheme, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOS: 22-24, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOS: 4-6, respectively; or According to the Chothia numbering scheme, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOS: 28-30, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOS: 4-6, respectively; or According to the Chothia numbering scheme, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 31-33, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 4-6, respectively; or 10. The antibody or antigen-binding fragment thereof according to claim 9, wherein the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 34 to 36, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 4 to 6, respectively, according to the Chothia numbering scheme.

11. An antibody or antigen-binding fragment thereof that binds to HER2, comprising: a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90% identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are: (1) the selected VH sequence is SEQ ID NO: 39 and the selected VL sequence is SEQ ID NO: 37; (2) the selected VH sequence is SEQ ID NO: 38 and the selected VL sequence is SEQ ID NO: 37; (3) the selected VH sequence is SEQ ID NO: 40 and the selected VL sequence is SEQ ID NO: 37; (4) the selected VH sequence is SEQ ID NO: 41 and the selected VL sequence is SEQ ID NO: 37; (5) the selected VH sequence is SEQ ID NO: 42 and the selected VL sequence is SEQ ID NO: 37; It is one of the Optionally here (i) the antibody or antigen-binding fragment specifically binds to human HER2 or canine HER2; (ii) the antibody or antigen-binding fragment is a human or humanized antibody or antigen-binding fragment thereof; and / or (iii) the antibody or antigen-binding fragment is a single-chain variable fragment (scFv); An antibody or antigen-binding fragment thereof that binds to HER2. Claim 12: The VH comprises the sequence of SEQ ID NO: 39 and the VL comprises the sequence of SEQ ID NO: 37; or the VH comprises the sequence of SEQ ID NO: 38 and the VL comprises the sequence of SEQ ID NO: 37; or the VH comprises the sequence of SEQ ID NO: 40 and the VL comprises the sequence of SEQ ID NO: 37; or the VH comprises the sequence of SEQ ID NO: 41 and the VL comprises the sequence of SEQ ID NO: 37; or The antibody or antigen-binding fragment thereof of claim 11, wherein the VH comprises the sequence of SEQ ID NO: 42 and the VL comprises the sequence of SEQ ID NO:

37.

13. The antibody or antigen-binding fragment thereof of claim 9, wherein the antibody or antigen-binding fragment thereof is a bispecific or multispecific antibody or antigen-binding fragment thereof; optionally, wherein the antibody or antigen-binding fragment thereof further specifically binds to TROP2. (1) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 10-12, wherein the VH binds to HER2 when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 37; (2) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 25 to 27, wherein the VH binds to HER2 when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 37; (3) An immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 1 to 3, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 39; (4) An immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 4 to 6, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 39; (5) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 7 to 9, wherein the VH binds to HER2 when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 37; (6) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 13 to 15, wherein the VH binds to HER2 when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 37; (7) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 16 to 18, wherein the VH binds to HER2 when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 37; (8) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 19 to 21, wherein the VH binds to HER2 when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 37; (9) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 22 to 24, wherein the VH binds to HER2 when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 37; (10) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 28 to 30, wherein the VH binds to HER2 when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 37; (11) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 31 to 33, wherein the VH binds to HER2 when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 37; (12) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 34 to 36, wherein the VH binds to HER2 when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 37; (13) An immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 1 to 3, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 38; (14) An immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 1 to 3, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 40; (15) An immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 1 to 3, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 41; (16) An immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 1 to 3, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 42; (17) An immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 4 to 6, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 38; (18) An immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 4 to 6, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 40; (19) An immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 4 to 6, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 41; or (20) An immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 4 to 6, wherein the VL binds to HER2 when paired with a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 42; A nucleic acid comprising a polynucleotide encoding a polypeptide comprising:

15. A vector comprising one or more of the nucleic acids according to claim 5 or 14, or a nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and 9 to 13, or a nucleic acid encoding an antigen-binding protein construct comprising a first antigen-binding domain that specifically binds to HER2 and a second antigen-binding domain that specifically binds to TROP2, wherein the second antigen-binding domain comprises the antigen-binding fragment of any one of claims 1 to 4.

16. A cell comprising the vector of claim 15.

17. (a) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and 9 to 13; or (b) An antigen-binding protein construct comprising a first antigen-binding domain that specifically binds to HER2 and a second antigen-binding domain that specifically binds to TROP2, wherein the second antigen-binding domain comprises the antigen-binding fragment of any one of claims 1 to 4.

1. An antibody drug conjugate comprising a therapeutic agent covalently attached to Optionally, the therapeutic agent is a cytotoxic or cytostatic agent; The antibody drug conjugate, optionally wherein the therapeutic agent is MMAE or MMAF.

18. A composition for treating cancer in a subject, the composition comprising: (i) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and 9 to 13; (ii) an antigen-binding protein construct comprising a first antigen-binding domain that specifically binds to HER2 and a second antigen-binding domain that specifically binds to TROP2, wherein the second antigen-binding domain comprises the antigen-binding fragment of any one of claims 1 to 4; or (iii) an antibody drug conjugate comprising a therapeutic agent covalently bonded to the antibody or antigen-binding fragment thereof of (i) or the antigen-binding protein construct of (ii); Optionally, the subject has a solid tumor; Optionally, the cancer is thyroid cancer, urothelial cancer, breast cancer, colorectal cancer, renal cancer, cervical cancer, ovarian cancer, lung cancer, endometrial cancer, skin cancer, gastric cancer, esophageal carcinoma, pancreatic cancer, prostate cancer, liver cancer, lymphoma, or glioma; Optionally, the cancer is cervical cancer, prostate cancer, thyroid cancer, urothelial cancer, head and neck cancer, endometrial cancer, ovarian cancer, lung cancer, breast cancer, carcinoid, skin cancer, liver cancer, or testicular cancer; Optionally, the cancer is multiple myeloma or renal carcinoma; Optionally, the subject is a human; Optionally, the subject is a non-human animal.

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