Multispecific antibodies and uses thereof

Abstract

The present disclosure relates to multispecific antibodies (e.g., bispecific antibodies) or antigen binding fragments thereof. In one aspect, the multispecific antibody, or antigen-binding fragment thereof, binds to PD-L1 and / or VEGF, or a combination thereof. More specifically, the antibody or antigen-binding fragment comprises a sequence-specified heavy chain variable region and a sequence-specified light chain variable region, the sequence-specified heavy chain variable region and the sequence-specified light chain variable region further comprising sequence-specified CDR 1, 2 and 3, respectively.

Description

[0001] Cross-reference to related applications

[0002] This disclosure claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 63 / 401,086, filed on August 25, 2022, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically as an XML file named 52501-0007WO1.xml. The size of the XML file, created on August 22, 2023, is 194,051 bytes. The material in the XML file is hereby incorporated by reference in its entirety. Technical Field

[0005] The present disclosure relates to multispecific antibodies or antigen-binding fragments thereof. Background Art

[0006] Multispecific antibodies are artificial proteins that can bind to two or more different epitopes simultaneously. This opens up a wide range of applications, including redirecting T cells to tumor cells, blocking two different signaling pathways simultaneously, dual targeting of different disease mediators, and delivering payloads to targeted sites. The approval of catumaxomab (anti-EpCAM and anti-CD3) and blinatumomab (anti-CD19 and anti-CD3) has become a major milestone in the development of multispecific antibodies.

[0007] Since multispecific antibodies have various applications, there is a need to continue developing various therapeutic agents based on multispecific antibodies. Summary of the invention

[0008] The present disclosure relates to antibodies or antigen-binding fragments, wherein the antibodies or antigen-binding fragments specifically bind to PD-L1 and / or VEGF or a combination thereof. In some embodiments, the present disclosure relates to the development of PD-L1 / VEGF targeting bispecific antibodies.

[0009] In one aspect, the present disclosure relates to an antibody or an antigen-binding fragment thereof, which binds to programmed death-ligand 1 (PD-L1), the antibody or the antigen-binding fragment thereof comprising:

[0010] a heavy chain antibody variable domain (VHH), said VHH comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VHH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR1 amino acid sequence, the VHH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR2 amino acid sequence, and the VHH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR3 amino acid sequence;

[0011] wherein the selected VHH CDR 1, 2 and 3 amino acid sequences are as described in one of the following:

[0012] (1) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 96, 97 and 98, respectively;

[0013] (2) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 99, 100 and 101, respectively;

[0014] (3) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 102, 103 and 104, respectively;

[0015] (4) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 105, 106 and 107, respectively;

[0016] (5) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 108, 109 and 110, respectively;

[0017] (6) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 111, 112 and 113, respectively;

[0018] (7) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 114, 115 and 116, respectively;

[0019] (8) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 117, 118 and 119, respectively;

[0020] (9) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 120, 121, and 122, respectively;

[0021] (10) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 123, 124 and 125, respectively;

[0022] (11) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 126, 127, and 128, respectively;

[0023] (12) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 129, 130 and 131, respectively;

[0024] (13) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 132, 133, and 134, respectively;

[0025] (14) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 135, 136 and 137, respectively;

[0026] (15) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 138, 139 and 140, respectively;

[0027] (16) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 141, 142 and 143, respectively;

[0028] (17) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 144, 145 and 146, respectively;

[0029] (18) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 147, 148 and 149, respectively;

[0030] (19) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 150, 151 and 152, respectively;

[0031] (20) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 153, 154 and 155, respectively;

[0032] (21) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 156, 157 and 158, respectively;

[0033] (22) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 159, 160 and 161, respectively;

[0034] (23) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 162, 163 and 164, respectively;

[0035] (24) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 165, 166 and 167, respectively;

[0036] (25) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 168, 169 and 170, respectively;

[0037] (26) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 171, 172 and 173, respectively;

[0038] (27) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 174, 175 and 176, respectively;

[0039] (28) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 177, 178 and 179, respectively;

[0040] (29) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 180, 181 and 182, respectively; and

[0041] (30) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 183, 184 and 185, respectively.

[0042] In some embodiments, the VHH comprises CDRs 1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 183, 184, and 185, respectively.

[0043] In some embodiments, the VHH comprises CDRs 1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 171, 172, and 173, respectively.

[0044] In some embodiments, the VHH comprises CDRs 1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 177, 178, and 179, respectively.

[0045] In some embodiments, the VHH comprises CDRs 1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 180, 181, and 182, respectively.

[0046] In some embodiments, the VHH comprises CDRs 1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 132, 133, and 134, respectively.

[0047] In one aspect, the present disclosure relates to an antibody or an antigen-binding fragment thereof that binds to PD-L1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain antibody variable domain (VHH), wherein the VHH comprises an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is selected from the group consisting of SEQ ID NOs: 1-80.

[0048] In some embodiments, the VHH comprises the sequence of SEQ ID NO:79.

[0049] In some embodiments, the VHH comprises the sequence of SEQ ID NO:60.

[0050] In some embodiments, the VHH comprises the sequence of SEQ ID NO:68.

[0051] In some embodiments, the VHH comprises the sequence of SEQ ID NO:74.

[0052] In some embodiments, the VHH comprises the sequence of SEQ ID NO:78.

[0053] In some embodiments, wherein the VHH comprises the sequence of SEQ ID NO:33.

[0054] In some embodiments, the antibody or antigen-binding fragment specifically binds to PD-L1.

[0055] In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.

[0056] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof, comprising the VHH CDRs 1, 2, 3 of the antibody or antigen-binding fragment thereof described herein.

[0057] In some embodiments, the antibody or antigen-binding fragment comprises human IgG Fc.

[0058] In some embodiments, the antibody or antigen-binding fragment comprises two or more heavy chain antibody variable domains.

[0059] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that cross-competes with an antibody or antigen-binding fragment thereof described herein.

[0060] In one aspect, the present disclosure relates to an antibody or an antigen-binding fragment thereof, which binds to VEGF (vascular endothelial growth factor), the antibody or the antigen-binding fragment thereof comprising:

[0061] a heavy chain antibody variable domain (VHH), said VHH comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VHH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR1 amino acid sequence, the VHH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR2 amino acid sequence, and the VHH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR3 amino acid sequence;

[0062] wherein the selected VHH CDR 1, 2 and 3 amino acid sequences are as described in one of the following:

[0063] (1) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 186, 187 and 188, respectively;

[0064] (2) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 189, 190 and 191, respectively;

[0065] (3) the selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 192, 193 and 194, respectively; and

[0066] (4) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 195, 196 and 197, respectively.

[0067] In some embodiments, the VHH comprises CDRs 1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 186, 187, and 188, respectively.

[0068] In some embodiments, the VHH comprises CDRs 1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 189, 190, and 191, respectively.

[0069] In some embodiments, the VHH comprises CDRs 1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 192, 193, and 194, respectively.

[0070] In some embodiments, the VHH comprises CDRs 1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 195, 196, and 197, respectively.

[0071] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to VEGF, the antibody or antigen-binding fragment thereof comprising a heavy chain antibody variable domain (VHH), the VHH comprising an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is selected from the group consisting of SEQ ID NOs: 81-84.

[0072] In some embodiments, the VHH comprises the sequence of SEQ ID NO:81.

[0073] In some embodiments, the VHH comprises the sequence of SEQ ID NO:82.

[0074] In some embodiments, the VHH comprises the sequence of SEQ ID NO:83.

[0075] In some embodiments, the VHH comprises the sequence of SEQ ID NO:84.

[0076] In some embodiments, the antibody or antigen-binding fragment specifically binds to VEGF.

[0077] In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.

[0078] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof, comprising the VHH CDRs 1, 2, 3 of the antibody or antigen-binding fragment thereof described herein.

[0079] In some embodiments, the antibody or antigen-binding fragment comprises human IgG Fc.

[0080] In some embodiments, the antibody or antigen-binding fragment comprises two or more heavy chain antibody variable domains.

[0081] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that cross-competes with an antibody or antigen-binding fragment thereof described herein.

[0082] In one aspect, the present disclosure relates to a multispecific antibody or an antigen-binding fragment thereof, comprising a first VHH (VHH1) that specifically binds to VEGF and a second VHH (VHH2) that specifically binds to PD-L1.

[0083] In some embodiments, the multispecific antibody or antigen-binding fragment thereof further comprises a third VHH (VHH3) that specifically binds to VEGF and a fourth VHH (VHH4) that specifically binds to PD-L1.

[0084] In some embodiments, the VHH1 and / or the VHH3 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected CDR3 amino acid sequence; wherein the selected CDR 1, 2, and 3 amino acid sequences are in Fig.28 Listed in.

[0085] In some embodiments, said VHH1 and / or said VHH3 comprises an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein said selected VHH sequence is selected from the group consisting of SEQ ID NOs: 1-80.

[0086] In some embodiments, the VHH2 and / or the VHH4 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected CDR3 amino acid sequence; wherein the selected CDR 1, 2, and 3 amino acid sequences are in Fig.29 Listed in.

[0087] In some embodiments, said VHH3 and said VHH4 comprise an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein said selected VHH sequence is selected from the group consisting of SEQ ID NOs: 81-84.

[0088] In some embodiments, the multispecific antibody or antigen-binding fragment thereof comprises human IgG Fc.

[0089] In some embodiments, said VHH1 and said VHH3 are linked to the N-terminus or the C-terminus of said human IgG Fc.

[0090] In some embodiments, said VHH2 and said VHH4 are linked to the N-terminus or the C-terminus of said human IgG Fc.

[0091] In one aspect, the present disclosure relates to a polypeptide complex comprising

[0092] (a) a first polypeptide, which comprises, from N-terminus to C-terminus: a first heavy chain antibody variable domain (VHH1), a first hinge region, a first Fc region, and a second VHH (VHH2); and

[0093] (b) a second polypeptide, which comprises, from N-terminus to C-terminus: a third VHH (VHH3), a second hinge region, a second Fc region and a fourth VHH (VHH4),

[0094] wherein the VHH1 and the VHH3 specifically bind to PD-L1, and the VHH2 and the VHH4 specifically bind to VEGF.

[0095] In some embodiments, the first polypeptide comprises a sequence at least 80% identical to SEQ ID NO:85, 89, 90, 91, 92, 93 or 95; and / or wherein the second polypeptide comprises a sequence at least 80% identical to SEQ ID NO:85, 89, 90, 91, 92, 93 or 95.

[0096] In some embodiments, said VHH2 is linked to said C-terminus of said first Fc region via a first linker peptide sequence.

[0097] In some embodiments, said VHH4 is linked to said C-terminus of said second Fc region via a second linker peptide sequence.

[0098] In some embodiments, the first linker peptide sequence and / or the second linker peptide sequence is at least 80% identical to SEQ ID NO: 202 or 203.

[0099] In one aspect, the present disclosure relates to a polypeptide complex comprising

[0100] (a) a first polypeptide, which comprises, from N-terminus to C-terminus: VHH1, VHH2, a first hinge region, and a first Fc region; and

[0101] (b) a second polypeptide, which comprises, from N-terminus to C-terminus: VHH3, VHH4, a second hinge region, and a second Fc region,

[0102] wherein the VHH1 and the VHH3 specifically bind to PD-L1, and the VHH2 and the VHH4 specifically bind to VEGF.

[0103] In some embodiments, the VHH1 is connected to the N-terminus of the VHH2 via a first linker peptide sequence.

[0104] In some embodiments, the VHH3 is linked to the N-terminus of the VHH4 via a second linker peptide sequence.

[0105] In some embodiments, the first polypeptide comprises a sequence at least 80% identical to SEQ ID NO:86 or 94; and / or wherein the second polypeptide comprises a sequence at least 80% identical to SEQ ID NO:86 or 94.

[0106] In some embodiments, the first linker peptide sequence and / or the second linker peptide sequence is at least 80% identical to SEQ ID NO: 202 or 203.

[0107] In one aspect, the present disclosure relates to a polypeptide complex comprising

[0108] (a) a first polypeptide, which comprises, from N-terminus to C-terminus: VHH2, VHH1, a first hinge region, and a first Fc region; and

[0109] (b) a second polypeptide, which comprises, from N-terminus to C-terminus: VHH4, VHH3, a second hinge region, and a second Fc region,

[0110] wherein the VHH1 and the VHH3 specifically bind to PD-L1, and the VHH2 and the VHH4 specifically bind to VEGF.

[0111] In some embodiments, the VHH2 is linked to the N-terminus of the VHH1 via a first linker peptide sequence.

[0112] In some embodiments, the VHH4 is linked to the N-terminus of the VHH3 via a second linker peptide sequence.

[0113] In some embodiments, the first polypeptide comprises a sequence at least 80% identical to SEQ ID NO:87; and / or wherein the second polypeptide comprises a sequence at least 80% identical to SEQ ID NO:87.

[0114] In some embodiments, the first linker peptide sequence and / or the second linker peptide sequence is at least 80% identical to SEQ ID NO: 202 or 203.

[0115] In one aspect, the present disclosure relates to a polypeptide complex comprising

[0116] (a) a first polypeptide, which comprises, from N-terminus to C-terminus: VHH2, a first hinge region, a first Fc region and VHH1; and

[0117] (b) a second polypeptide, which comprises, from N-terminus to C-terminus: VHH4, a second hinge region, a second Fc region and VHH3,

[0118] wherein the VHH1 and the VHH3 specifically bind to PD-L1, and the VHH2 and the VHH4 specifically bind to VEGF.

[0119] In some embodiments, the first polypeptide comprises a sequence at least 80% identical to SEQ ID NO:88; and / or wherein the second polypeptide comprises a sequence at least 80% identical to SEQ ID NO:88.

[0120] In some embodiments, said VHH1 is linked to said C-terminus of said first Fc region via a first linker peptide sequence.

[0121] In some embodiments, said VHH3 is linked to said C-terminus of said second Fc region via a second linker peptide sequence.

[0122] In some embodiments, the first linker peptide sequence and / or the second linker peptide sequence is at least 80% identical to SEQ ID NO: 202 or 203.

[0123] In some embodiments, the VHH1 and / or the VHH3 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected CDR3 amino acid sequence; wherein the selected CDR 1, 2, and 3 amino acid sequences are in Fig.28 Listed in.

[0124] In some embodiments, said VHH1 and / or said VHH3 comprises an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein said selected VHH sequence is selected from the group consisting of SEQ ID NOs: 1-80.

[0125] In some embodiments, the VHH2 and / or the VHH4 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected CDR3 amino acid sequence; wherein the selected CDR 1, 2, and 3 amino acid sequences are in Fig.29 Listed in.

[0126] In some embodiments, said VHH2 and / or said VHH4 comprises an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein said selected VHH sequence is selected from the group consisting of SEQ ID NOs: 81-84.

[0127] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to programmed death-ligand 1 (PD-L1), the antibody or antigen-binding fragment thereof comprising: a heavy chain antibody variable domain (VHH), the VHH comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VHH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR1 amino acid sequence, the VHH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR2 amino acid sequence, and the VHH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR3 amino acid sequence, wherein the selected VHH CDR 1, 2, and 3 amino acid sequences are as described in one of the following:

[0128] (1) the selected VHH CDR3 is NARX1TTIY (SEQ ID NO: 198), wherein X1 = K, T or R; the selected VHH CDR1 is selected from the group consisting of: SEQ ID NO: 96, 99, 102, 105, 108, 111, 126 and 129; and the selected VHH CDR2 is selected from the group consisting of: SEQ ID NO: 97, 100, 103, 106, 109, 112, 127 and 130;

[0129] (2) the selected VHH CDR3 is NALVWX2GSSYNN (SEQ ID NO: 199), wherein X2 = Q, T, S or N; the selected VHH CDR1 is selected from the group consisting of: SEQ ID NO: 114, 117, 120, 123, 168, 171, 174, 177, 180 and 183; and the selected VHH CDR2 is selected from the group consisting of: SEQ ID NO: 115, 118, 121, 124, 169, 172, 175, 178, 181 and 184; and

[0130] (3) the selected VHH CDR3 is selected from the group consisting of SEQ ID NO: 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164 and 167; the selected VHH CDR1 is selected from the group consisting of SEQ ID NO: 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162 and 165; and the selected VHH CDR2 is selected from the group consisting of SEQ ID NO: 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163 and 166.

[0131] In some embodiments, the selected VHH CDR 1, 2, 3 amino acid sequence is as described in one of the following:

[0132] (1) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 96, 97 and 98, respectively;

[0133] (2) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 99, 100 and 101, respectively;

[0134] (3) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 102, 103 and 104, respectively;

[0135] (4) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 105, 106 and 107, respectively;

[0136] (5) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 108, 109 and 110, respectively;

[0137] (6) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 111, 112 and 113, respectively;

[0138] (7) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 114, 115 and 116, respectively;

[0139] (8) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 117, 118 and 119, respectively;

[0140] (9) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 120, 121, and 122, respectively;

[0141] (10) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 123, 124 and 125, respectively;

[0142] (11) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 126, 127, and 128, respectively;

[0143] (12) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 129, 130 and 131, respectively;

[0144] (13) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 132, 133, and 134, respectively;

[0145] (14) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 135, 136 and 137, respectively;

[0146] (15) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 138, 139 and 140, respectively;

[0147] (16) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 141, 142 and 143, respectively;

[0148] (17) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 144, 145 and 146, respectively;

[0149] (18) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 147, 148 and 149, respectively;

[0150] (19) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 150, 151 and 152, respectively;

[0151] (20) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 153, 154 and 155, respectively;

[0152] (21) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 156, 157 and 158, respectively;

[0153] (22) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 159, 160 and 161, respectively;

[0154] (23) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 162, 163 and 164, respectively;

[0155] (24) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 165, 166 and 167, respectively;

[0156] (25) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 168, 169 and 170, respectively;

[0157] (26) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 171, 172 and 173, respectively;

[0158] (27) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 174, 175 and 176, respectively;

[0159] (28) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 177, 178 and 179, respectively;

[0160] (29) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 180, 181 and 182, respectively; and

[0161] (30) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 183, 184 and 185, respectively.

[0162] In one aspect, the present disclosure relates to a nucleic acid comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof described herein, a multispecific antibody or antigen-binding fragment thereof described herein, or a polypeptide complex described herein.

[0163] In some embodiments, the nucleic acid is DNA (eg, cDNA) or RNA (eg, mRNA).

[0164] In one aspect, the disclosure relates to a vector comprising one or more of the nucleic acids described herein.

[0165] In one aspect, the disclosure relates to a cell comprising a vector described herein.

[0166] In some embodiments, the cell is a CHO cell.

[0167] In one aspect, the disclosure relates to a cell comprising one or more of the nucleic acids described herein.

[0168] In one aspect, the present disclosure relates to a method of producing an antibody or an antigen-binding fragment thereof, the method comprising:

[0169] (a) culturing the cells described herein under conditions sufficient for the cells to produce the antibody or the antigen-binding fragment; and

[0170] (b) collecting the antibody or the antigen-binding fragment produced by the cell.

[0171] In one aspect, the present disclosure relates to an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof described herein, a multispecific antibody or antigen-binding fragment thereof described herein, or a polypeptide complex described herein, wherein the antibody drug conjugate is covalently bound to a therapeutic agent.

[0172] In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

[0173] 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, a multispecific antibody or antigen-binding fragment thereof described herein, or a polypeptide complex described herein, or an antibody drug conjugate described herein.

[0174] In some embodiments, the subject has a cancer that expresses PD-L1.

[0175] In some embodiments, the subject has a cancer that expresses VEGF.

[0176] In some embodiments, the cancer is colon cancer, rectal cancer, lung cancer, breast cancer, kidney cancer, hepatocellular carcinoma, renal cancer, endometrial cancer, pancreatic cancer, head and neck cancer, or an advanced solid tumor.

[0177] In some embodiments, the cancer is non-small cell lung cancer (NSCLC).

[0178] In one aspect, the present disclosure relates to a method of reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein, a multispecific antibody or antigen-binding fragment thereof described herein, or a polypeptide complex described herein, or an antibody drug conjugate described herein.

[0179] In one aspect, the present disclosure relates to a method of killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein, a multispecific antibody or antigen-binding fragment thereof described herein, or a polypeptide complex described herein, or an antibody-drug conjugate described herein.

[0180] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein, a multispecific antibody or antigen-binding fragment thereof described herein, a polypeptide complex described herein, or an antibody-drug conjugate described herein, and a pharmaceutically acceptable carrier.

[0181] In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to PD-L1. In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to VEGF.

[0182] As used herein, the term "antibody" refers to any antigen binding molecule containing at least one (e.g., one, two, three, four, five or six) complementary determining regions (CDRs) (e.g., any CDR from the three CDRs of an immunoglobulin light chain or any CDR from the three CDRs of an immunoglobulin heavy chain) and capable of specifically binding to an epitope in an antigen. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, single variable domain (VHH) antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, the antibody may contain the Fc region of a human antibody. The term antibody also includes derivatives formed by these antibodies or antibody fragments, such as multispecific antibodies, bispecific antibodies, single-chain antibodies, bifunctional antibodies, and linear antibodies.

[0183] As used herein, the term "antigen binding fragment" refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of a light chain or VHH). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2 and Fv fragments, ScFv and VHH.

[0184] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification, and describe the animal, human or non-human being provided for treatment according to the method of the present invention. Veterinary and non-veterinary applications are contemplated in the present disclosure. Human patients can be adults or adolescents (e.g., people under the age of 18). In addition to people, patients include but are not limited to mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs and primates. Including, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, porcine animals (e.g., pigs, miniature pigs), horses, dogs, cats, cattle and other livestock animals, farm animals and zoo animals.

[0185] As used herein, the phrases "specifically binding" and "specifically binds" when referring to an antibody or antigen binding fragment refer to the interaction of an antibody or antigen binding fragment with its target molecule, preferably with other molecules, because the interaction depends on the presence of a specific structure (i.e., an antigenic determinant or epitope) on the target molecule; in other words, the agent generally recognizes and binds to molecules including a specific structure rather than all molecules. An antibody that specifically binds to a target molecule may be referred to as a target-specific antibody. For example, an antibody that specifically binds to VEGF may be referred to as a VEGF-specific antibody or an anti-VEGF antibody.

[0186] As used herein, the term "bispecific antibody" refers to an antibody that binds to two different epitopes. The epitopes can be on the same antigen or on different antigens.

[0187] As used herein, the term "trispecific antibody" refers to an antibody that binds to three different epitopes. The epitopes can be on the same antigen or on different antigens.

[0188] As used herein, the term "multispecific antibody" refers to an antibody that binds to two or more different epitopes. The epitopes can be on the same antigen or on different antigens. The multispecific antibody can be, for example, a bispecific antibody or a trispecific antibody. In some embodiments, the multispecific antibody binds to two, three, four, five or six different epitopes.

[0189] As used herein, "VHH" refers to the variable domain of the heavy chain of a heavy chain-only antibody and its variants. A heavy chain antibody is an antibody consisting of only two heavy chains but can still specifically bind to an antigen. In some embodiments, VHH is a humanized VHH.

[0190] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to amino acid polymers of any length of at least two amino acids.

[0191] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein to refer to nucleotide polymers of any length of at least two nucleotides, and include but are not limited to DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0192] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The 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 the event of a conflict, the present specification (including definitions) shall prevail.

[0193] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0194] Figure 1A Whole cell binding (WCB) of VHH bivalent antibodies to human PD-L1 (h-PD-L1) as determined by flow cytometry is shown. The X-axis is the value of antibody concentration in nanomolar units. The Y-axis is the value of median fluorescence intensity of AlexaFlour 488. Atezolizumab (Atezo) is a reference anti-h-PD-L1 antibody from Roche. 1F4, 5C12, 6D2, 4H8, 6A10, 1G4 and 6D1 are anti-human PD-L1 VHH-Fc clones.

[0195] Figure 1B The efficacy of the lead bivalent antibody blocking human PD-L1 activity as determined using a luciferase reporter gene assay is shown. In the presence of anti-h-PD-L1 VHH-Fc antibodies, CHO cells stably transfected with human PD-L1 were cultured with Jurkat cells stably transfected with PD1 and NFAT reporter genes. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the NFAT luciferase activity expressed in relative luminescence units. KN035 is a reference anti-h-PD-L1 antibody from AlphaMab. 1F4, 5C12, 6A10, 6D2, 4H8 and 6D1 are anti-h-PD-L1 VHH-Fc clones.

[0196] Figure 2Whole cell binding of mouse PD-L1 ELISA positive VHH-Fc antibodies to 293T cells stably transfected with mouse PD-L1 (m-PD-L1) as determined by flow cytometry is shown. The X-axis is the value of antibody concentration in nanomolar units. The Y-axis is the value of median fluorescence intensity of AlexaFlour 488. Atezolizumab (Atezo) is a reference anti-m-PD-L1 antibody from Roche. 5F8, 3H1, 2A11, 1C3, 6D2, and 4H8 are anti-PD-L1 VHH-Fc clones.

[0197] Figure 3A The efficacy of humanized variants hv7 and hv8 of 5F8 and hv7 of 3H1 in blocking human PD-L1 is shown. KN035 is a positive control. The efficacy (EC50 in nM) is also shown in the table below the accompanying drawings. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the NFAT luciferase activity expressed in relative luminescence units.

[0198] Figure 3B The whole cell binding activity of humanized variants and their parental clones to mouse PD-L1 is shown, as determined by flow cytometry using 293T cells stably transfected with mouse PD-L1 and anti-human IgG Fc-AlexaFlour 488 as secondary antibody. The X-axis is the value of antibody concentration in nanomolar. The Y-axis is the value of median fluorescence intensity. Atezolizumab (Atezo) is a reference control antibody from Roche.

[0199] Figure 3C The whole cell binding activity of 3H1-hv7 and 5F8-hv8 to mouse PD-L1 as determined in Hepa1-6 cells expressing endogenous m-PD-L1 with anti-human IgG Fc-AlexaFlour 488 as the secondary antibody is shown. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the median fluorescence intensity.

[0200] Figure 4 Whole cell binding of selected high affinity PD-L1 clones to endogenously expressed human PD-L1 in T24 cells is shown, as determined by flow cytometry using anti-human IgG Fc-AlexaFlour 488 as the secondary antibody. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the median fluorescence intensity of AlexaFlour 488. Atezolizumab (Atezo) is a reference anti-h-PD-L1 antibody from Roche. KN035 is also a reference anti-h-PD-L1 antibody from Corning Jeol. 5C12, 1F4, 4D2, 4E2, and 5F8 are lead anti-PD-L1 VHH-Fc clones.

[0201] Figures 5A-5D SEC-HPLC data obtained using an Agilent AdvanceBio SEC 300A 2.7um column and an Agilent 1200 HPLC are shown. SEC was performed in 150 mM sodium phosphate buffer with 10 ul of protein injected at 25 ml / min at a concentration of approximately 1-2 mg / ml. Figure 5A Data for the 5C12-parent are shown. Figure 5B Data for 5C12-NS are shown. Figure 5C Data for 5C12-NT are shown. Figure 5D Data for 5C12-NQ are shown.

[0202] Fig. 6A Shown is the whole cell binding of 5C12 hotspot correction variants to human PD-L1 in h-PD-L1 stably transfected cells, as determined by flow cytometry using anti-human IgG Fc-AlexaFlour 488 as the secondary antibody. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the median fluorescence intensity of AlexaFlour 488.

[0203] Figure 6B The efficacy of 5C12 hotspot correction variants in blocking human PD-L1 activity as determined using a luciferase reporter assay is shown. The X-axis is the value of antibody concentration in nanomolar units. The Y-axis is the value of NFAT luciferase activity expressed in relative luminescence units. Atezolizumab (Atezo) is a reference anti-h-PD-L1 antibody from Roche.

[0204] Fig. 7A Shown is the whole cell binding of 5C12 humanized variants to human PD-L1 in cells stably transfected with h-PD-L1, as determined by flow cytometry using anti-human IgG Fc-AlexaFlour 488 as the secondary antibody (data for selected humanized variants are shown). The X-axis is the value of antibody concentration in nanomolar units. The Y-axis is the value of median fluorescence intensity of AlexaFlour 488.

[0205] Figure 7BWhole cell binding of 5C12 CDR3 hotspot correction variants (N to T) and humanized variants to human PD-L1 in cells stably transfected with h-PD-L1 is shown, as determined by flow cytometry using anti-human IgG Fc-AlexaFlour 488 as the secondary antibody, with data for selected variants shown. The X-axis is the value of antibody concentration in nanomolar units. The Y-axis is the value of median fluorescence intensity of AlexaFlour 488. Avelumab is a reference anti-PD-L1 antibody from Pfizer.

[0206] Figure 7C Whole cell binding of 5C12 CDR3 hotspot corrected variants (N to T) and humanized variants to NCI-H441 lung tumor cells is shown, as determined by flow cytometry using anti-human IgG Fc-AlexaFlour 488 as the secondary antibody, with data for selected variants shown. The X-axis is the value of antibody concentration in nanomolar. The Y-axis is the value of median fluorescence intensity of AlexaFlour 488. Avelumab is a reference anti-PD-L1 antibody from Pfizer.

[0207] Fig.7D The efficacy of selected 5C12 CDR3 hotspot correction variants and humanized variants in blocking human PD-L1 activity using a luciferase reporter assay is shown. The X-axis is the value of antibody concentration in nanomolar units. The Y-axis is the value of NFAT luciferase activity expressed in relative luminescence units. Atezolizumab (Atezo) is a reference anti-h-PD-L1 antibody from Roche.

[0208] Fig. 8A Whole cell binding of 1F4 CDR3 hotspot corrected variants and humanized variants to human PD-L1 in h-PD-L1 stably transfected cells is shown, as determined by flow cytometry using anti-human IgG Fc-AlexaFlour 488 as the secondary antibody (data for selected humanized variants are shown). The X-axis is the value of antibody concentration in nanomolar units. The Y-axis is the value of median fluorescence intensity of AlexaFlour 488. Avelumab is a reference anti-PD-L1 antibody from Pfizer.

[0209] Figure 8B The efficacy of selected 1F4 CDR3 hotspot correction variants and humanized variants in blocking human PD-L1 activity as determined using a luciferase reporter assay is shown. The X-axis is the value of antibody concentration in nanomolar units. The Y-axis is the value of NFAT luciferase activity expressed in relative luminescence units.

[0210] Fig.9AWhole cell binding of 4D2 and 4E2 CDR3 hotspot corrected variants and humanized variants to human PD-L1 in cells stably transfected with h-PD-L1 is shown, as determined by flow cytometry using anti-human IgG Fc-AlexaFlour488 as the secondary antibody (data for selected variants are shown). The X-axis is the value of antibody concentration in nanomolar units. The Y-axis is the value of median fluorescence intensity of AlexaFlour 488. Avelumab is a reference anti-PD-L1 antibody from Pfizer.

[0211] Fig. 9B Whole cell binding of 4D2 and 4E2 CDR3 hotspot corrected variants and humanized variants to NCI-H441 lung cancer cells is shown, as determined by flow cytometry using anti-human IgG Fc-AlexaFlour 488 as the secondary antibody (data for selected variants are shown). The X-axis is the value of antibody concentration in nanomolar units. The Y-axis is the value of median fluorescence intensity of AlexaFlour 488. Avelumab is a reference anti-PD-L1 antibody from Pfizer.

[0212] Fig.10 The efficacy of 4D2 and 4E2 CDR3 hotspot correction variants and humanized variants in blocking human PD-L1 activity as determined using a luciferase reporter assay is shown. The X-axis is the value of antibody concentration in nanomolar units. The Y-axis is the value of NFAT luciferase activity expressed in relative luminescence units.

[0213] Fig.11 The effectiveness of the NFAT luciferase activity of the lead anti-VEGF clone blocking human VEGFA mediation is shown, as determined by the luciferase reporter gene assay using cells stably transfected with KDR (VEGFR2) and NFAT luciferase reporter genes. In the presence of 50ng / ml of human VEGFA, the stable cells in the suspension were incubated for 4-5 hours with the serial dilutions of the anti-VEGF antibody, and the luciferase activity was determined by adding the Bright-Glo luciferase assay buffer with substrate, and read by a plate reader. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the NFAT luciferase activity represented by relative luminous units.

[0214] Fig.12The effectiveness of the lead anti-VEGF clone blocking the NFAT luciferase activity mediated by mouse VEGFA is shown, as determined by luciferase reporter assay using cells stably transfected with KDR (VEGFR2) and NFAT luciferase reporter genes. In the presence of 50ng / ml mouse VEGFA, the stable cells in the suspension were incubated for 4-5 hours with the serial dilutions of anti-VEGF antibodies, and the luciferase activity was determined by adding Bright-Glo luciferase assay buffer with substrate, and read by a plate reader. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the NFAT luciferase activity represented by relative luminous units.

[0215] Figures 13A-13D Schematic diagrams showing different formats of BsAb constructs.

[0216] Fig.14A The efficacy of different forms of BsAb blocking human PD-L1 activity as determined using a luciferase reporter gene assay is shown. The X-axis is the value of antibody concentration in nanomolar units. The Y-axis is the value of NFAT luciferase activity expressed in relative luminescence units.

[0217] Fig. 14B The efficacy of different forms of BsAb in blocking human VEGFA (VEGF165) is shown. The X-axis is the value of antibody concentration in nanomolar units. The Y-axis is the value of NFAT luciferase activity expressed in relative luminescence units.

[0218] Figures 15A-15F The dual binding capacity of BsAb or monospecific Ab to h-PD-L1 ECD or h-VEGFA as determined by BLI binding assay is shown. Briefly, the sensor is loaded with 300nM of antibody and then immersed in a solution containing 200nM h-PD-L1 ECD recombinant protein (his-tagged). After 130 seconds of association, the sensor is moved and immersed in another solution containing 200nM h-VEGFA (VEGF165, his-tagged). The Y axis is the displacement in nm and the X axis is the time in seconds.

[0219] Figures 16A-16E Schematic diagram of BsAb constructs with different lead anti-PD-L1 clones is shown.

[0220] Fig.17A The binding ability of the lead BsAb and its parental bivalent antibody to h-PD-L1 endogenously expressed in T24 tumor cells in whole cell binding is shown.

[0221] Fig. 17BThe efficacy of the lead BsAb and its parent bivalent antibody in blocking human PD-L1 activity as determined using a luciferase reporter gene assay is shown. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of NFAT luciferase activity expressed in relative luminescence units.

[0222] Fig. 17C The efficacy of the lead BsAb blocking human VEGFA (VEGF165) as determined using a luciferase reporter gene assay is shown. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the NFAT luciferase activity expressed in relative luminescence units.

[0223] Figures 18A-18B Schematic representation of two lead alternative BsAb constructs is shown.

[0224] Fig.19A The effectiveness of two different forms of alternative BsAb blocking mouse VEGFA (VEGF164) as determined using luciferase reporter gene assay is shown. The X-axis is the value of antibody concentration in nanomolar units. The Y-axis is the value of luciferase activity expressed in relative luminescent units. The EC50 value is shown below each figure.

[0225] Fig.19B The efficacy of two different forms of alternative BsAbs blocking mouse PD-L1 as determined using a luciferase reporter gene assay is shown. The X-axis is the value of antibody concentration in nanomolar units. The Y-axis is the value of luciferase activity expressed in relative luminescence units. The EC50 values ​​are shown below each figure.

[0226] Fig. 20A The efficacy of the lead surrogate BsAb 3B9_Fc_7H3 in blocking mouse PD-L1 as determined using a luciferase reporter assay for samples treated with mouse serum is shown. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the luciferase activity expressed in relative luminescence units. The EC50 values ​​are shown below each figure.

[0227] Fig. 20B The effectiveness of the lead replacement BsAb 3B9-Fc-7H3 blocking mouse VEGFA as determined by using a luciferase reporter gene assay for samples treated with mouse serum is shown. The X-axis is the value of the antibody concentration in nanomoles. The Y-axis is the value of the luciferase activity represented by relative luminescent units. The EC50 value is shown below each accompanying drawing.

[0228] Fig.21A Shown are tumor volume data from an in vivo efficacy study in the MC38 syngeneic model. The X-axis is the number of days treated. The Y-axis is tumor volume.

[0229] Fig. 21B Body weight data from an in vivo efficacy study in the MC38 syngeneic model are shown. The X-axis is the number of days of treatment. The Y-axis is the % change in body weight.

[0230] Fig. 22 The individual tumor volumes of each mouse are shown. The X-axis is the number of days after treatment. The Y-axis is the tumor volume.

[0231] Fig.23 The comparison of lead BsAb and IMM2510 in whole cell binding to CHO cells stably transfected with human PD-L1 is shown. The X-axis is the value of antibody concentration in nanomolar units. The Y-axis is the value of median fluorescence intensity of AlexaFlour 488.

[0232] Fig.24 The comparison of the lead BsAb and the reference BsAb in blocking human PD-L1 activity is shown. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the NFAT luciferase activity expressed in relative luminescence units.

[0233] Fig.25 A comparison of the lead BsAb and the reference BsAb in blocking human VEGFA is shown. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the NFAT luciferase activity expressed in relative luminescence units.

[0234] Fig.26A Tumor volume data from an in vivo efficacy study in a MC38-PD-L1 syngeneic mouse model is shown. The X-axis is treatment days. The Y-axis is tumor volume.

[0235] Fig.26B Body weight data from an in vivo efficacy study in a MC38-PD-L1 syngeneic mouse model are shown. The X-axis is the number of days of treatment. The Y-axis is the % change in body weight.

[0236] Fig. 27 The individual tumor volumes for each mouse are shown. The X-axis is the number of days treated. The Y-axis is the tumor volume.

[0237] Fig.28 The CDR sequences of the PD-L1 antibodies described in this disclosure are listed.

[0238] Fig.29 The CDR sequences of the VEGF antibodies described in this disclosure are listed.

[0239] Fig.30 The amino acid sequences of VHHs as described in the present disclosure are listed. DETAILED DESCRIPTION

[0240] Cancer cells have developed several mechanisms to evade host immune surveillance. For example, many tumors or cancer cells express high levels of PD-L1 and PD-L2 on their surface, which bind to PD-1 on the surface of T cells, thereby inducing T cell apoptosis.

[0241] In addition, the growth of cancer cells depends on adequate nutrient supply. Cancer cells themselves can secrete factors that promote blood vessel growth, such as vascular epithelial growth factor (VEGF). Inhibiting the activity of VEGF or its receptors will stop the blood supply to solid tumors, thereby inhibiting their growth.

[0242] The present disclosure relates to antibodies or antigen-binding fragments, wherein the antibodies or antigen-binding fragments specifically bind to PD-L1 and / or VEGF or a combination thereof. In some embodiments, the present disclosure relates to the development of PD-L1 / VEGF targeting bispecific antibodies.

[0243] PD-L1

[0244] Programmed death ligand 1 (PD-L1 or PDL1) is the major ligand for programmed death 1 (PD-1), a co-inhibitory receptor that can be constitutively expressed or induced in myeloid, lymphoid, normal epithelial cells, and cancer. Under physiological conditions, the PD-1 / PD-L1 interaction is crucial in the development of immune tolerance that prevents excessive immune cell activity that can lead to tissue destruction and autoimmunity. PD-L1 expression is an immune evasion mechanism exploited by various malignancies and is often associated with a poor prognosis. PD-L1 expression has also been considered a predictive biomarker for response to anti-PD-1 / PD-L1 therapy; however, there is conflicting evidence regarding its role in tissue typing. Over the years, anti-PD-1 / PD-L1 agents have gained momentum as novel anticancer therapeutics by inducing durable tumor regression in many malignancies, including metastatic lung cancer, melanoma, and others.

[0245] PD-L1 expression can be constitutive or inducible. Constitutive low-level PD-L1 expression can be found in resting lymphocytes, antigen presenting cells (APCs), as well as corneal cells, syncytiotrophoblasts, and Langerhans' islet cells, where it contributes to tissue homeostasis in proinflammatory responses. PD-L1 confers an 'immune privileged' state to certain tissues (such as the placenta, testis, and anterior chamber of the eye), in which vaccination with exogenous antigens is tolerated without inducing an inflammatory / immune response.

[0246] In carcinogenesis, PD-L1 may be overexpressed as a driver of oncogenic events. Epidermal growth factor receptor (EGFR) mutations, for example, are positively correlated with PD-L1 expression in lung cancer, where EGFR inhibitors act as repressors of PD-L1 transcription. In phosphatase and tensin homolog (PTEN) mutant tumors, PD-L1 overexpression is maintained by unrestrained activation of the PI3K / AKT pathway. In T-cell lymphomas, the nucleophosmin (NPM) / anaplastic lymphoma kinase (ALK) fusion gene upregulates PD-L1 through constitutive STAT3 activation.

[0247] The biological function of PD-L1 depends on binding to PD-1 (CD279), a 288 amino acid long type 1 transmembrane receptor encoded by the PDCD1 gene and physiologically expressed on lymphocytes and myeloid cells. PD-1 consists of an extracellular IgV-like domain and a transmembrane region. Its intracellular tail consists of a tyrosine-based switch motif (ITSM) and an immunoreceptor tyrosine-based inhibitory motif sequence. Upon ligation with PD-L1, recruitment of the Src homology 2 domain containing phosphatases 1 and 2 (SHP-1 / SHP-2) to the ITSM leads to dephosphorylation of signaling kinases such as CD3ζ, PKCθ, and ZAP70, resulting in an overall inhibition of T cell expansion. Such an inhibitory response is secondary to the inactivation of the PI3K-Akt and Ras-MEK-ERK cascades. Casein kinase 2 is a target of SHP-2. Dephosphorylation of casein kinase 2 (CK-2) leads to unrestrained activation of PTEN, a physiological PI3K-Akt signaling antagonist. The inhibitory effect of PD-1 on the Ras-MEK-ERK cascade depends primarily on direct inhibition of Ras and dephosphorylation of phospholipase Cγ.

[0248] A detailed review of PD-L1 and its functions can be found in the following references: (1) Kythreotou, Anthousa et al. "PD-L1." Journal of Clinical Pathology 71.3 (2018): 189-194; and (2) Gong, Jun et al. "Development of PD-1 and PD-L1 inhibitors as a form of cancer immunotherapy: a comprehensive review of registration trials and future considerations." Journal for immunotherapy of cancer 6.1 (2018): 1-18, each of which is incorporated by reference in its entirety.

[0249] VEGF

[0250] VEGF is a key pleiotropic growth factor controlling tissue / wound repair processes and is classified as VEGF-A, VEGF-B, VEGF-C, VEGF-D and PIGF. During the tissue healing process, VEGF simultaneously drives the formation of new blood vessels (angiogenesis) while downregulating immunity (Canic M et al. The role of vascular endothelial growth factor in wound healing. J Surg Res. 2009, 153: 347-358; Leung DW et al. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science. 1989, 246: 1306-1309; Voron T et al. Control of the immune response by pro-angiogenic factors. Front Oncol. 2014, 4: 70). These two properties of VEGF are crucial to the tumorigenic process because they can promote the development of tumor blood vessels and suppress anti-cancer immunity (Manahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011, 144: 646-674). Specifically, VEGF is believed to exert its immunosuppressive effects through three key mechanisms: inhibiting DC maturation, reducing T cell tumor infiltration, and increasing suppressor cells in the tumor microenvironment. Abnormal VEGF expression can also lead to other diseases, including rheumatoid arthritis, diabetic retinopathy, wet age-related macular degeneration and glomerular hypertrophy. The FDA-approved anti-VEGF monoclonal antibody drug Avastin treats cancer (colon cancer, lung cancer) by inhibiting the biological activity of VEGF. Another protein drug that targets VEGF, aflibercept, is approved in the United States and Europe as Eylea for the treatment of wet macular degeneration and as Zaltrap for metastatic colorectal cancer.

[0251] The discovery that anti-VEGF antibodies reduce the growth of tumor cells implanted in immunodeficient mice has opened up translational possibilities for targeting VEGF-VEGFR signaling. Additionally, it has been demonstrated that inactivation of a single allele of the vegfa gene in mice results in vascular developmental defects and early embryonic lethality, thus emphasizing the importance of VEGF during embryonic development. Inactivation of both copies of vegfr2 phenotypically mimics vegfa monoallelic deletion to a large extent. With the advent of the cre-lox system, the ability to delete VEGF in target tissues has created the possibility to assess the role of VEGF in individual tissues / cells. Many studies using this approach have demonstrated the important role of VEGF in angiogenesis and homeostasis in various pathophysiological situations.

[0252] VEGF secreted by tumor cells and surrounding stroma stimulates the proliferation and survival of endothelial cells, resulting in the formation of new blood vessels, which may be abnormal and leaky in structure. VEGF mRNA is overexpressed in most human tumors and is associated with invasiveness, vascular density, metastasis, recurrence and prognosis. Several strategies have been designed to inhibit the VEGF-VEGFR signal transduction pathway to treat cancer.

[0253] Detailed reviews of VEGF and its functions can be found in the following references: (1) Apte et al. "VEGF in signaling and disease: beyond discovery and development". Cell 176.6 (2019): 1248-1264; and (2) Yang et al. "Targeting VEGF / VEGFR to modulate antitumor immunity". Frontiers in Immunology 9 (2018): 978, each of which is incorporated by reference in its entirety.

[0254] Heavy chain antibody variable domain (VHH)

[0255] Monoclonal antibodies and recombinant antibodies are important tools in medicine and biotechnology. Like all mammals, Camelidae (e.g., llamas and alpacas) can produce conventional antibodies consisting of two heavy chains and two light chains that are bound together with a disulfide bond in a Y shape (e.g., IgG1). However, Camelidae also produces two unique IgG subclasses: IgG2 and IgG3, also referred to as heavy chain antibodies. These antibodies consist of only two heavy chains that lack the CH1 region but still carry the antigen-binding domain known as VHH (or nanobody) at its N-terminus. Conventional Ig requires the association of the variable regions from both heavy and light chains to allow the high diversity of antigen-antibody interactions. Although the heavy and light chains of separation still show this ability, when compared with paired heavy and light chains, it shows very low affinity. The unique feature of heavy chain antibodies is that their monomeric antigen-binding regions are capable of binding antigens with specificity, affinity, and diversity in particular, which is comparable to conventional antibodies that do not need to be paired with another and individual regions. This feature is mainly due to some major variations in the amino acid sequence of the variable regions of the two heavy chains, which induce profound conformational changes compared to conventional Ig. The major substitutions in the variable regions prevent the light chain from binding to the heavy chain, but also prevent the unbound heavy chain from being retrieved by the immunoglobulin binding protein.

[0256] The single variable domain (named as VHH, sdAb, nano antibody or heavy chain antibody variable domain) of these antibodies is the minimum antigen binding domain produced by the adaptive immune system. It is often found that the third complementary determining region (CDR3) of the variable region of these antibodies is twice as long as conventional antibodies. This increases the interaction surface with the antigen and the diversity of antigen-antibody interactions, which compensates for the lack of light chain. In the case of long complementary determining region 3 (CDR3), VHH can extend to protein clefts that conventional antibodies cannot reach, including functional sites of concern, such as the active site of an enzyme or receptor binding canyons on a virus surface. In addition, other cysteine ​​residues make the structure more stable, thereby increasing the intensity of interaction.

[0257] Compared with conventional antibodies carrying VH and VL domains, VHH provides many other advantages, including higher stability, solubility, expression yield and refolding ability and better in vivo tissue penetration. In addition, compared with the VH domain of conventional antibodies, VHH does not show an inherent tendency to bind to the light chain. This is conducive to inducing heavy chain antibodies in the presence of functional light chain loci. Further, since VHH does not bind to the VL domain, it is much easier to reformat VHH into a multispecific antibody construct than to reformat it into a construct containing a conventional VH-VL pair or a single domain based on the VH domain.

[0258] Anti-PD-L1 antibodies and antigen-binding fragments

[0259] The present disclosure provides, for example, anti-PD-L1 antibodies, modified antibodies thereof, chimeric antibodies thereof, and humanized antibodies thereof. The present disclosure also provides variable domains (e.g., VHH) of these antibodies. These VHHs can be used in various multispecific antibody constructs as described herein.

[0260] In some embodiments, the CDR sequences of R1-1C3 and R1-1C3-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domains as shown in SEQ ID NOs: 96, 97, 98, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 1.

[0261] In some embodiments, the CDR sequences of R2-1G4 and R2-1G4-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domains as shown in SEQ ID NOs: 99, 100, 101, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 2.

[0262] In some embodiments, the CDR sequences of R2-1A3 and R2-1A3-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 102, 103, 104, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:3.

[0263] In some embodiments, the CDR sequences of R2-3A5 and R2-3A5-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 99, 100, 101, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:4.

[0264] In some embodiments, the CDR sequences of R2-1G10 and R2-1G10-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domains as shown in SEQ ID NOs: 96, 97, 98, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:5.

[0265] In some embodiments, the CDR sequences of R2-6D2 and R2-6D2-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 105, 106, 107, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:6.

[0266] In some embodiments, the CDR sequences of R2-4H8 and R2-4H8-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 108, 109, 110, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:7.

[0267] In some embodiments, the CDR sequences of R2-6A10 and R2-6A10-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 111, 112, 113, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:8.

[0268] In some embodiments, the CDR sequences of R2-5C12 and R2-5C12-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 114, 115, 116, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:9.

[0269] In some embodiments, the CDR sequences of R2-1F4 and R2-1F4-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 117, 118, 119, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 10.

[0270] In some embodiments, the CDR sequences of R2-4D2 and R2-4D2-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 120, 121, 122, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 11.

[0271] In some embodiments, the CDR sequences of R2-4E2 and R2-4E2-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 123, 124, 125, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 12.

[0272] In some embodiments, the CDR sequences of R3-3H1 and R3-3H1-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 126, 127, 128, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 13.

[0273] In some embodiments, the CDR sequences of R3-5F8 and R3-5F8-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 129, 130, 131, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 14.

[0274] In some embodiments, the CDR sequences of R3-2A11 and R3-2A11-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 99, 100, 101, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 15.

[0275] In some embodiments, the CDR sequences of 5F8-alpaca and 5F8-alpaca derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 129, 130, 131, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 16.

[0276] In some embodiments, the CDR sequences of 5F8-hv1 and 5F8-hv1-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 129, 130, 131, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:17.

[0277] In some embodiments, the CDR sequences of 5F8-hv2 and 5F8-hv2-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 129, 130, 131, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 18.

[0278] In some embodiments, the CDR sequences of 5F8-hv3 and 5F8-hv3-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 129, 130, 131, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 19.

[0279] In some embodiments, the CDR sequences of 5F8-hv4 and 5F8-hv4-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 129, 130, 131, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 20.

[0280] In some embodiments, the CDR sequences of 5F8-hv5 and 5F8-hv5-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 129, 130, 131, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 21.

[0281] In some embodiments, the CDR sequences of 5F8-hv6 and 5F8-hv6-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 129, 130, 131, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 22.

[0282] In some embodiments, the CDR sequences of 5F8-hv7 and 5F8-hv7-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 129, 130, 131, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:23.

[0283] In some embodiments, the CDR sequences of 5F8-hv8 and 5F8-hv8-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 129, 130, 131, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 24.

[0284] In some embodiments, the CDR sequences of 3H1-alpaca and 3H1-alpaca-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 126, 127, 128, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 25.

[0285] In some embodiments, the CDR sequences of 3H1-hv1 and 3H1-hv1-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 126, 127, 128, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:26.

[0286] In some embodiments, the CDR sequences of 3H1-hv2 and 3H1-hv2-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 126, 127, 128, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 27.

[0287] In some embodiments, the CDR sequences of 3H1-hv3 and 3H1-hv3-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 126, 127, 128, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 28.

[0288] In some embodiments, the CDR sequences of 3H1-hv4 and 3H1-hv4-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 126, 127, 128, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 29.

[0289] In some embodiments, the CDR sequences of 3H1-hv5 and 3H1-hv5-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 126, 127, 128, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 30.

[0290] In some embodiments, the CDR sequences of 3H1-hv6 and 3H1-hv6-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 126, 127, 128, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 31.

[0291] In some embodiments, the CDR sequences of 3H1-hv7 and 3H1-hv7-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 126, 127, 128, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 32.

[0292] In some embodiments, the CDR sequences of 3H1-3B9 and 3H1-3B9-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 132, 133, 134, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 33.

[0293] In some embodiments, the CDR sequences of 3H1-2A2 and 3H1-2A2-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 111, 112, 113, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:34.

[0294] In some embodiments, the CDR sequences of 3H1-2G3 and 3H1-2G3-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 135, 136, 137, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 35.

[0295] In some embodiments, the CDR sequences of 3H1-1G9 and 3H1-1G9-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 138, 139, 140, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 36.

[0296] In some embodiments, the CDR sequences of 3H1-6D2 and 3H1-6D2-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 141, 142, 143, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 37.

[0297] In some embodiments, the CDR sequences of 3H1-2F4 and 3H1-2F4-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 144, 145, 146, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 38.

[0298] In some embodiments, the CDR sequences of 5F8-3A3 and 5F8-3A3-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 147, 148, 149, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 39.

[0299] In some embodiments, the CDR sequences of 5F8-8E3 and 5F8-8E3-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 150, 151, 152, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 40.

[0300] In some embodiments, the CDR sequences of 5F8-8D4 and 5F8-8D4-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 153, 154, 155, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 41.

[0301] In some embodiments, the CDR sequences of 5F8-4B12 and 5F8-4B12-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 156, 157, 158, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:42.

[0302] In some embodiments, the CDR sequences of 5F8-6D6 and 5F8-6D6-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 159, 160, 161, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:43.

[0303] In some embodiments, the CDR sequences of 5F8-8H3 and 5F8-8H3-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 162, 163, 164, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 44.

[0304] In some embodiments, the CDR sequences of 5F8-7D5 and 5F8-7D5-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 165, 166, 167, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 45.

[0305] In some embodiments, the CDR sequences of 5C12-NQ and 5C12-NQ-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 168, 169, 170, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 46.

[0306] In some embodiments, the CDR sequences of 5C12-NT and 5C12-NT-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 171, 172, 173, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 47.

[0307] In some embodiments, the CDR sequences of 5C12-NS and 5C12-NS-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 174, 175, 176, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 48.

[0308] In some embodiments, the CDR sequences of 5C12-hv1 and 5C12-hv1-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 114, 115, 116, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:49.

[0309] In some embodiments, the CDR sequences of 5C12-hv1-NQ and 5C12-hv1-NQ-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 168, 169, 170, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 50.

[0310] In some embodiments, the CDR sequences of 5C12-hv1-NT and 5C12-hv1-NT-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 171, 172, 173, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 51.

[0311] In some embodiments, the CDR sequences of 5C12-hv1-NS and 5C12-hv1-NS-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 174, 175, 176, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 52.

[0312] In some embodiments, the CDR sequences of 5C12-hv2 and 5C12-hv2-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 114, 115, 116, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:53.

[0313] In some embodiments, the CDR sequences of 5C12-hv3 and 5C12-hv3-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 114, 115, 116, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:54.

[0314] In some embodiments, the CDR sequences of 5C12-hv4 and 5C12-hv4-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 114, 115, 116, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:55.

[0315] In some embodiments, the CDR sequences of 5C12-hv5 and 5C12-hv5-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 114, 115, 116, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:56.

[0316] In some embodiments, the CDR sequences of 5C12-hv6 and 5C12-hv6-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 114, 115, 116, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:57.

[0317] In some embodiments, the CDR sequences of 5C12-hv7 and 5C12-hv7-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 114, 115, 116, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:58.

[0318] In some embodiments, the CDR sequences of 5C12-hv8 and 5C12-hv8-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 114, 115, 116, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:59.

[0319] In some embodiments, the CDR sequences of 5C12-hv7-NT and 5C12-hv7-NT-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 171, 172, 173, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 60.

[0320] In some embodiments, the CDR sequences of 5C12-hv9-NT and 5C12-hv9-NT-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 171, 172, 173, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 61.

[0321] In some embodiments, the CDR sequences of 1F4-hv1-NT and 1F4-hv1-NT-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 177, 178, 179, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 62.

[0322] In some embodiments, the CDR sequences of 1F4-hv2-NT and 1F4-hv2-NT-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 177, 178, 179, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 63.

[0323] In some embodiments, the CDR sequences of 1F4-hv3-NT and 1F4-hv3-NT-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 177, 178, 179, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 64.

[0324] In some embodiments, the CDR sequences of 1F4-hv4-NT and 1F4-hv4-NT-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 177, 178, 179, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 65.

[0325] In some embodiments, the CDR sequences of 1F4-hv5-NT and 1F4-hv5-NT derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 177, 178, 179, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 66.

[0326] In some embodiments, the CDR sequences of 1F4-hv6-NT and 1F4-hv6-NT-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 177, 178, 179, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 67.

[0327] In some embodiments, the CDR sequences of 1F4-hv7-NT and 1F4-hv7-NT-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 177, 178, 179, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 68.

[0328] In some embodiments, the CDR sequences of 1F4-hv8-NT and 1F4-hv8-NT-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 177, 178, 179, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 69.

[0329] In some embodiments, the CDR sequences of 1F4-hv9-NT and 1F4-hv9-NT-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 177, 178, 179, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 70.

[0330] In some embodiments, the CDR sequences of 1F4-hv10-NT and 1F4-hv10-NT-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 177, 178, 179, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:71.

[0331] In some embodiments, the CDR sequences of 1F4-hv11-NT and 1F4-hv11-NT-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 177, 178, 179, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 72.

[0332] In some embodiments, the CDR sequences of 1F4-hv12-NT and 1F4-hv12-NT-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 177, 178, 179, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:73.

[0333] In some embodiments, the CDR sequences of 4D2-hv1 and 4D2-hv1-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 180, 181, 182, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:74.

[0334] In some embodiments, the CDR sequences of 4D2-hv2 and 4D2-hv2-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 180, 181, 182, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:75.

[0335] In some embodiments, the CDR sequences of 4D2-hv3 and 4D2-hv3-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 180, 181, 182, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:76.

[0336] In some embodiments, the CDR sequences of 4D2-hv4 and 4D2-hv4-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 180, 181, 182, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:77.

[0337] In some embodiments, the CDR sequences of 4E2_Alpaca and 4E2_Alpaca-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 183, 184, 185, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 201.

[0338] In some embodiments, the CDR sequences of 4E2-hv1 and 4E2-hv1-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 183, 184, 185, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:78.

[0339] In some embodiments, the CDR sequences of 4E2-hv2 and 4E2-hv2-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 183, 184, 185, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO:79.

[0340] In some embodiments, the CDR sequences of 4E2-hv3 and 4E2-hv3-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 183, 184, 185, respectively. In some embodiments, the amino acid sequence of the VHH domain is shown in SEQ ID NO: 80.

[0341] Also provided are the amino acid sequences of various modified or humanized VHHs. Because there are different ways to modify or humanize Ilama antibodies (e.g., different amino acids can be substituted for the modified sequence), the heavy and light chains of the antibodies can have more than one version of the humanized sequence. In some embodiments, the humanized VHH domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of the sequences of SEQ ID NO: 1-80.

[0342] Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may further contain one, two or three VHH domain CDRs, wherein the VHH domain CDRs are selected from the group consisting of SEQ ID NOs: 96-185.

[0343] In some embodiments, the antibody may have a heavy chain antibody variable domain (VHH) comprising complementarity determining regions (CDR) 1, 2, and 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 VHH 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 VHH 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 VHH CDR3 amino acid sequence. Fig.28 Selected VHH CDR 1, 2, 3 amino acid sequences are shown in .

[0344] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain antibody variable domain (VHH) containing one, two or three of the following: a VHH CDR1 having zero, one or two amino acid insertions, deletions or substitutions; a VHH CDR2 having zero, one or two amino acid insertions, deletions or substitutions; a VHH CDR3 having zero, one or two amino acid insertions, deletions or substitutions, wherein VHH CDR1, VHH CDR2 and VHH CDR3 are selected from Fig.28 CDRs in .

[0345] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain antibody variable domain (VHH) containing one, two, or three of the CDRs of SEQ ID NOs: 96-185 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0346] The insertions, deletions and substitutions may be within the CDR sequence, or at one or both ends of the CDR sequence. In some embodiments, the CDRs are determined based on the Kabat numbering scheme. In some embodiments, the CDRs are determined based on the Chothia numbering scheme. In some embodiments, the CDRs are determined based on the IMGT numbering scheme.

[0347] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to PD-L1. The antibody or antigen-binding fragment thereof contains a heavy chain antibody variable domain (VHH), and the VHH comprises an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VHH sequence or consists of the amino acid sequence. In some embodiments, the selected VHH sequence is selected from SEQ ID NO: 1-80.

[0348] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, for the purpose of optimal comparison, the sequences are compared (for example, a gap can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal comparison, and non-homologous sequences can be ignored for comparison purposes). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When the position in the first sequence is occupied by the amino acid residue or nucleotide identical to the corresponding position in the second sequence, the molecules are identical at the position. The number of gaps and the length of each gap (which needs to be introduced to achieve the optimal comparison of the two sequences) are taken into account, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences. For example, the comparison of the sequence and the determination of the percent identity between the two sequences can be completed, for example, using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0349] The present disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides comprising immunoglobulin heavy chain antibody variable domains (VHH). VHH comprises Fig.28 The CDRs shown, or having Fig.30 sequence shown.

[0350] The antibodies and antigen-binding fragments can also be antibody or antibody fragments and multispecific (e.g., bispecific) antibodies or antibody variants (including derivatives and conjugates) of antibody or antibody fragments. Other antibodies provided herein are polyclonal, monoclonal, multispecific (multimeric, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, antibodies produced in cells (i.e., in vivo antibodies) and antigen-binding fragments thereof.

[0351] In some embodiments, the antibody or antigen-binding fragment thereof comprises an Fc domain, which may be derived from various types (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), classes (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclasses. In some embodiments, the Fc domain is derived from an IgG antibody or antigen-binding fragment thereof. In some embodiments, the Fc domain comprises one, two, three, four, or more heavy chain constant regions.

[0352] Anti-VEGF Antibodies and Antigen-binding Fragments

[0353] The present disclosure provides, for example, anti-VEGF antibodies, modified antibodies thereof, chimeric antibodies thereof, and humanized antibodies thereof. The present disclosure also provides VHHs of these antibodies. These VHHs can be used in various multispecific antibody constructs as described herein.

[0354] The CDR sequences of 7H3 and 7H3-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 186, 187, and 188, respectively. The amino acid sequence of the VHH domain of the 7H3 antibody is shown in SEQ ID NO:81.

[0355] The CDR sequences of 1C8 and 1C8-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 189, 190, and 191, respectively. The amino acid sequence of the VHH domain of the 1C8 antibody is shown in SEQ ID NO:82.

[0356] The CDR sequences of 4B11 and 4B11-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 192, 193, and 194, respectively. The amino acid sequence of the VHH domain of the 4B11 antibody is shown in SEQ ID NO:83.

[0357] The CDR sequences of 1G1 and 1G1-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 195, 196, and 197, respectively. The amino acid sequence of the VHH domain of the 1G1 antibody is shown in SEQ ID NO:84.

[0358] Also provided are the amino acid sequences of various modified or humanized VHHs. Because there are different ways to modify or humanize Ilama antibodies (e.g., different amino acids can be substituted for the modified sequence), the heavy and light chains of the antibodies can have more than one version of the humanized sequence. In some embodiments, the humanized VHH domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of the sequences of SEQ ID NOs: 81-84.

[0359] Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may further contain one, two or three VHH domain CDRs, wherein the VHH domain CDRs are selected from the group consisting of SEQ ID NOs: 186-197.

[0360] In some embodiments, the antibody may have a heavy chain antibody variable domain (VHH) comprising complementarity determining regions (CDR) 1, 2, and 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 VHH 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 VHH 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 VHH CDR3 amino acid sequence. Fig.29 Selected VHH CDR 1, 2, 3 amino acid sequences are shown in .

[0361] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain antibody variable domain (VHH) containing one, two or three of the following: a VHH CDR1 having zero, one or two amino acid insertions, deletions or substitutions; a VHH CDR2 having zero, one or two amino acid insertions, deletions or substitutions; a VHH CDR3 having zero, one or two amino acid insertions, deletions or substitutions, wherein VHH CDR1, VHH CDR2 and VHH CDR3 are selected from Fig.29 CDRs in .

[0362] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain antibody variable domain (VHH) containing one, two, or three of the CDRs of SEQ ID NOs: 186-197 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0363] The insertions, deletions and substitutions may be within the CDR sequence, or at one or both ends of the CDR sequence. In some embodiments, the CDRs are determined based on the Kabat numbering scheme. In some embodiments, the CDRs are determined based on the Chothia numbering scheme. In some embodiments, the CDRs are determined based on the IMGT numbering scheme.

[0364] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to VEGF. The antibody or antigen-binding fragment thereof contains a heavy chain antibody variable domain (VHH), and the VHH comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VHH sequence. In some embodiments, the selected VHH sequence is selected from SEQ ID NO: 81-84.

[0365] The present disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides comprising immunoglobulin heavy chain antibody variable domains (VHH). VHH comprises Fig.29 The CDRs shown, or having Fig.30 sequence shown.

[0366] The antibodies and antigen-binding fragments can also be antibody or antibody fragments and multispecific (e.g., bispecific) antibodies or antibody variants (including derivatives and conjugates) of antibody or antibody fragments. Other antibodies provided herein are polyclonal, monoclonal, multispecific (multimeric, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, antibodies produced in cells (i.e., in vivo antibodies) and antigen-binding fragments thereof.

[0367] In some embodiments, the antibody or antigen-binding fragment thereof comprises an Fc domain, which may be derived from various types (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), classes (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclasses. In some embodiments, the Fc domain is derived from an IgG antibody or antigen-binding fragment thereof. In some embodiments, the Fc domain comprises one, two, three, four, or more heavy chain constant regions.

[0368] Structure of PD-L1 / VEGF bispecific antibody

[0369] In some embodiments, the bispecific antibody is designed to include a VHH targeting PD-L1 and a VHH targeting VEGF. In some embodiments, the present disclosure provides bispecific antibodies that bind to both PD-L1 and VEGF. The bispecific antibody can be used to treat a subject's PD-L1 or VEGF-positive cancer (e.g., non-small cell lung cancer).

[0370] PD-L1 / VEGF bispecific antibodies with specific structures are described below.

[0371] Bispecific-V1 structure

[0372] like Fig.13A As shown, a PD-L1 / VEGF bispecific antibody having a bispecific-V1 structure can be prepared. Specifically, the PD-L1 / VEGF bispecific antibody comprises (a) a first polypeptide, which comprises from N-terminus to C-terminus: a first heavy chain antibody variable domain (VHH1), a first hinge region, a first Fc region, and a third heavy chain antibody variable domain (VHH3); and (b) a second polypeptide, which comprises from N-terminus to C-terminus: a second heavy chain antibody variable region (VHH2), a second hinge region, a second Fc region, and a fourth heavy chain antibody variable region (VHH4).

[0373] In some embodiments, VHH1 and VHH2 specifically bind to PD-L1. In some embodiments, VHH3 and VHH4 specifically bind to VEGF. In some embodiments, the sequences of VHH1 and VHH2 are identical. In some embodiments, the sequences of VHH3 and VHH4 are identical.

[0374] In some embodiments, the PD-L1 / VEGF bispecific antibody comprises a knob-in-hole mutation. In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the first polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 85, 89, 90, 91, 92, 93, or 95. In some embodiments, the second polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 85, 89, 90, 91, 92, 93, or 95.

[0375] In some embodiments, the first Fc region and / or the second Fc region comprises aspartic acid (Asp) at position 239 according to EU numbering. In some embodiments, the first Fc region and / or the second Fc region comprises glutamic acid (Glu) at position 332 according to EU numbering.

[0376] In some embodiments, the VHH3 is connected to the C-terminus of the first Fc region via a first linker peptide sequence. In some embodiments, the VHH4 is connected to the C-terminus of the second Fc region via a second linker peptide sequence.

[0377] In some embodiments, the first joint peptide sequence and / or the second joint peptide sequence comprise a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) repeats of GGGGS. In some embodiments, the first joint peptide sequence and / or the second joint peptide sequence comprise a sequence selected from GSGGSGGSGGSG (SEQ ID NO: 202) and GSGGSGGSGGSGGSG (SEQ ID NO: 203).

[0378] Bispecific-V2 structure

[0379] like Fig. 13B As shown, a PD-L1 / VEGF bispecific antibody having a bispecific-V2 structure can be prepared. Specifically, the PD-L1 / VEGF bispecific antibody comprises (a) a first polypeptide, which comprises, from N-terminus to C-terminus: VHH1, VHH3, a first hinge region, and a first Fc region; and (b) a second polypeptide, which comprises, from N-terminus to C-terminus: VHH2, VHH4, a second hinge region, and a second Fc region.

[0380] In some embodiments, VHH1 and VHH2 specifically bind to PD-L1. In some embodiments, VHH3 and VHH4 specifically bind to VEGF. In some embodiments, the sequences of VHH1 and VHH2 are identical. In some embodiments, the sequences of VHH3 and VHH4 are identical.

[0381] In some embodiments, the PD-L1 / VEGF bispecific antibody comprises a knob-in-hole mutation. In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the first polypeptide comprises a sequence at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 86 or 94. In some embodiments, the second polypeptide comprises a sequence at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 86 or 94.

[0382] In some embodiments, the first Fc region and / or the second Fc region comprises aspartic acid (Asp) at position 239 according to EU numbering. In some embodiments, the first Fc region and / or the second Fc region comprises glutamic acid (Glu) at position 332 according to EU numbering.

[0383] In some embodiments, the VHH1 is connected to the N-terminus of the VHH3 via a first linker peptide sequence. In some embodiments, the VHH2 is connected to the N-terminus of the VHH4 via a second linker peptide sequence.

[0384] In some embodiments, the first joint peptide sequence and / or the second joint peptide sequence comprise a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) repeats of GGGGS. In some embodiments, the first joint peptide sequence and / or the second joint peptide sequence comprise a sequence selected from GSGGSGGSGGSG (SEQ ID NO: 202) and GSGGSGGSGGSGGSG (SEQ ID NO: 203).

[0385] Bispecific-V3 structure

[0386] like Fig. 13C As shown, a PD-L1 / VEGF bispecific antibody having a bispecific-V3 structure can be prepared. Specifically, the PD-L1 / VEGF bispecific antibody comprises (a) a first polypeptide, which comprises, from N-terminus to C-terminus: VHH3, VHH1, a first hinge region, and a first Fc region; (b) a second polypeptide, which comprises, from N-terminus to C-terminus: VHH4, VHH2, a second hinge region, and a second Fc region.

[0387] In some embodiments, VHH1 and VHH2 specifically bind to PD-L1. In some embodiments, VHH3 and VHH4 specifically bind to VEGF. In some embodiments, the sequences of VHH1 and VHH2 are identical. In some embodiments, the sequences of VHH3 and VHH4 are identical.

[0388] In some embodiments, the first polypeptide comprises a sequence at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 87. In some embodiments, the second polypeptide comprises a sequence at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 87.

[0389] In some embodiments, the first Fc region and / or the second Fc region comprises aspartic acid (Asp) at position 239 according to EU numbering. In some embodiments, the first Fc region and / or the second Fc region comprises glutamic acid (Glu) at position 332 according to EU numbering.

[0390] In some embodiments, the VHH3 is connected to the N-terminus of the VHH1 via a first linker peptide sequence. In some embodiments, the VHH4 is connected to the N-terminus of the VHH2 via a second linker peptide sequence.

[0391] In some embodiments, the first joint peptide sequence and / or the second joint peptide sequence comprise a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) repeats of GGGGS. In some embodiments, the first joint peptide sequence and / or the second joint peptide sequence comprise a sequence selected from GSGGSGGSGGSG (SEQ ID NO: 202) and GSGGSGGSGGSGGSG (SEQ ID NO: 203).

[0392] Bispecific-V4 structure

[0393] like Fig.13D As shown, a PD-L1 / VEGF bispecific antibody having a bispecific-V4 structure can be prepared. Specifically, the PD-L1 / VEGF bispecific antibody comprises (a) a first polypeptide, which comprises, from N-terminus to C-terminus: VHH3, a first hinge region, a first Fc region, and VHH1; and (b) a second polypeptide, which comprises, from N-terminus to C-terminus: VHH4, a second hinge region, a second Fc region, and VHH2.

[0394] In some embodiments, VHH1 and VHH2 specifically bind to PD-L1. In some embodiments, VHH3 and VHH4 specifically bind to VEGF. In some embodiments, the sequences of VHH1 and VHH2 are identical. In some embodiments, the sequences of VHH3 and VHH4 are identical.

[0395] In some embodiments, the PD-L1 / VEGF bispecific antibody comprises a knob-in-hole mutation. In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the first polypeptide comprises a sequence at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 88. In some embodiments, the second polypeptide comprises a sequence at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 88.

[0396] In some embodiments, the first Fc region and / or the second Fc region comprises aspartic acid (Asp) at position 239 according to EU numbering. In some embodiments, the first Fc region and / or the second Fc region comprises glutamic acid (Glu) at position 332 according to EU numbering.

[0397] In some embodiments, the VHH1 is connected to the C-terminus of the first Fc region via a first linker peptide sequence. In some embodiments, the VHH2 is connected to the C-terminus of the second Fc region via a second linker peptide sequence.

[0398] In some embodiments, the first joint peptide sequence and / or the second joint peptide sequence comprise a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) repeats of GGGGS. In some embodiments, the first joint peptide sequence and / or the second joint peptide sequence comprise a sequence selected from GSGGSGGSGGSG (SEQ ID NO: 202) and GSGGSGGSGGSGGSG (SEQ ID NO: 203).

[0399] Antibody characteristics

[0400] The anti-PD-L1, anti-VEGF or anti-PD-L1 / VEGF antigen binding protein constructs (e.g., antibodies, bispecific antibodies, trispecific antibodies, multispecific antibodies or antibody fragments thereof) may include an antigen binding site derived from any anti-PD-L1 antibody, anti-VEGF antibody or any antigen binding fragment thereof as described herein.

[0401] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are VEGF antagonists. In some embodiments, the antibodies or antigen-binding fragments thereof are VEGF agonists.

[0402] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can bind to PD-L1 and / or VEGF, thereby blocking the interaction of these receptors and their corresponding ligands; reducing phosphorylation of downstream signaling pathways (e.g., ERK and / or Akt pathways); and / or directly killing cancer cells through ADCC and / or CDC.

[0403] In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to PD-L1 with an EC50 of less than or about 3 nM, less than or about 2.5 nM, less than or about 2.0 nM, less than or about 1.5 nM, less than or about 1.1 nM, less than or about 1 nM, less than or about 0.8 nM, less than or about 0.7 nM, less than or about 0.6 nM, less than or about 0.5 nM, less than or about 0.4 nM, less than or about 0.3 nM, less than or about 0.2 nM, less than or about 0.1 nM, less than or about 0.09 nM, less than or about 0.08 nM, less than or about 0.05 nM, or less than or about 0.04 nM. In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to PD-L1 with an EC50 of about 0.04 nM to 0.1 nM, about 0.05 nM to 0.2 nM, about 0.08 nM to 0.3 nM, about 0.09 nM to 0.4 nM, about 0.1 nM to 0.5 nM, about 0.2 nM to 0.6 nM, about 0.3 nM to 0.7 nM, about 0.4 nM to 0.8 nM, about 0.5 nM to 1 nM, about 0.6 nM to 1.1 nM, or about 0.7 nM to 1.5 nM.

[0404] In some embodiments, the antibody (or antigen-binding fragment thereof) is activated in less than 0.1 s -1 Less than 0.01s -1 , less than 0.001s -1 , less than 0.0001s -1 or less than 0.00001s -1 The dissociation rate (koff) of the antibody specifically binds to an antigen (e.g., PD-L1 or VEGF). In some embodiments, the dissociation rate (koff) is greater than 0.01 s -1 , greater than 0.001s -1 , greater than 0.0001s -1 , greater than 0.00001s -1 or greater than 0.000001s -1 In some embodiments, the kinetic association rate (kon) is greater than 1x10 2 / Ms, greater than 1x10 3 / Ms, greater than 1x 10 4 / Ms, greater than 1x 10 5 / Ms, greater than 2x 10 5 / Ms, greater than 3x 10 5 / Ms, greater than 4x 10 5 / Ms, greater than 5x 10 5 / Ms or greater than 6x 10 5 / Ms. In some embodiments, the kinetic association rate (kon) is less than 1 x 10 5 / Ms, less than 1x 10 6 / Ms or less than 1x 10 7 / Ms.

[0405] Affinity can be derived from the quotient of the kinetic rate constants (Kd = koff / kon). In some embodiments, Kd is less than 1 x 10 -4 M, less than 1x 10 -5 M, less than 1x 10 -6 M, less than 1x 10 -7 M, less than 1x 10 -8 M, less than 1x 10 -9 M or less than 1x 10 -10 In some embodiments, the Kd is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. In some embodiments, the Kd is greater than 1 x 10 -4 M, greater than 1x 10 -5 M, greater than 1x 10 -6 M, greater than 1x 10 -7 M, greater than 1x 10 -8 M, greater than 1x10 -9 M, greater than 1x 10 -10 M, greater than 1x 10 -11 M or greater than 1x 10 -12 M.

[0406] In some embodiments, the binding affinity for PD-L1 or VEGF is carefully adjusted, for example, the Kd can be between 100nM and 0.1nM, between 100nM and 1nM, between 100nM and 10nM, between 10nM and 0.1nM, between 10nM and 1nM, or between 1nM and 0.1nM.

[0407] Common techniques for measuring the affinity of an antibody for an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR).

[0408] In some embodiments, the stability of the antibodies or antigen-binding fragments thereof described herein can be analyzed by SEC-HPLC to assess the amount of aggregation and degradation. In some embodiments, as shown by SEC-HPLC, the proportion of high molecular weight (HMW) species is less than 20%, 15%, 10%, 7.5%, 5%, 2.5%, 1% or 0.5%. In some embodiments, as shown by SEC-HPLC, the proportion of monomers exceeds 60%, 65%, 70%, 75%, 80%, 85%, 90%, 97.5%, 95%, 97.5% or 98%. In some embodiments, as shown by SEC-HPLC, the proportion of low molecular weight (LMW) species is less than 20%, 15%, 10%, 7.5%, 5%, 2.5%, 1% or 0.5%. In some embodiments, as shown by SEC-HPLC, the proportion of high molecular weight (HMW) species exceeds 20%, 15%, 10%, 7.5%, 5%, 2.5%, 1% or 0.5%. In some embodiments, as shown by SEC-HPLC, the monomer accounts for less than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 97.5%, 95%, 97.5% or 98%. In some embodiments, as shown by SEC-HPLC, the low molecular weight (LMW) species accounts for more than 20%, 15%, 10%, 7.5%, 5%, 2.5%, 1% or 0.5%.

[0409] In some embodiments, the tumor growth inhibition rate percentage (TGI%) of the antibody is greater 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 tumor growth inhibition rate percentage of the antibody is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200%. TGI% can be determined, for example, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days or 30 days after the start of treatment, or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months after the start of treatment. As used herein, tumor growth inhibition rate percentage (TGI%) is calculated using the following formula:

[0410] TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100

[0411] Ti is the mean tumor volume in the treatment group at day i. T0 is the mean tumor volume in the treatment group at day zero. Vi is the mean tumor volume in the control group at day i. V0 is the mean tumor volume in the control group at day zero.

[0412] In some embodiments, the antibodies or antigen-binding fragments thereof described herein have an ADCC of at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 100%, at least or about 110%, at least or about 120%, at least or about 130%, at least or about 140%, at least or about 150%, at least or about 200% compared to the antibody-dependent cell-mediated cytotoxicity (ADCC) of a reference antibody (e.g., IMM2510). In some embodiments, the antibodies or antigen-binding fragments thereof described herein can increase ADCC by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, or 100-fold compared to the antibody-dependent cell-mediated cytotoxicity (ADCC) of a nonspecific antibody control or an isotype antibody control.

[0413] In some embodiments, the cell killing IC50 of the antibodies or antigen-binding fragments thereof described herein is less than or about 1000 ng / ml, less than or about 500 ng / ml, less than or about 400 ng / ml, less than or about 300 ng / ml, less than or about 250 ng / ml, less than or about 200 ng / ml, less than or about 100 ng / ml. In some embodiments, the cell killing IC50 value of the antibodies or antigen-binding fragments thereof described herein is less than or about 50%, less than or about 60%, less than or about 70%, less than or about 80%, less than or about 90%, less than or about 100%, less than or about 110%, less than or about 120%, less than or about 130%, less than or about 140%, less than or about 150%, less than or about 200% compared to the cell killing IC50 value of a reference antibody (e.g., IMM2510). In some embodiments, the cell killing IC50 value of the antibodies or antigen-binding fragments thereof described herein is less than or about 20%, less than or about 10%, less than or about 8%, less than or about 5%, less than or about 3%, less than or about 1% compared to the cell killing IC50 value of an isotype control antibody.

[0414] In some embodiments, the antibody or antigen binding fragment has a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated 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.

[0415] In some embodiments, the antibody or antigen-binding fragment can induce apoptosis.

[0416] In some embodiments, the antibody or antigen-binding fragment does not have a functional Fc region. For example, the antibody or antigen-binding fragment is a Fab, Fab', F(ab')2, and Fv fragment.

[0417] In some embodiments, antibody or antigen binding fragment is a humanized antibody.Humanization percentage means as compared with the human antibody sequence in the International Immunogenetics Information System (International Immunogenetics Information System, IMGT) database, the identity percentage of heavy chain or light chain variable region sequence.Top hit means that compared to other species, heavy chain or light chain variable region sequence is closer to a specific species.For example, the top hit to mankind means that compared to other species, the sequence is closer to mankind.The top hit to mankind and cynomolgus monkey (Macaca fascicularis) means that the sequence has the same identity percentage to human sequence and cynomolgus monkey sequence, and compared to the sequence of other species, these identity percentages are the highest.In some embodiments, the humanization percentage is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%. Detailed descriptions of how to determine the percentage of humanization and how to determine the top hits are known in the art and are described in, for example, Jones et al. "The INNs and outs of antibody nonproprietary names". Monoclonal Antibodies (Mabs.) Vol. 8. No. 1. Taylor & Francis, 2016, which is incorporated herein by reference in its entirety. A high percentage of humanization generally has various advantages, for example, being safer and more effective in humans, more likely to be tolerated by human subjects, and / or less likely to have side effects.

[0418] The disclosure also provides antibodies or antigen binding fragments thereof that cross-compete with any antibody or antigen binding fragment as described herein. Cross-competition assays are known in the art and are described, for example, in Moore et al., "Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein". Journal of virology 70.3 (1996): 1863-1872, which is incorporated herein by reference in its entirety. On the one hand, the disclosure also provides antibodies or antigen binding fragments thereof that bind to the same epitope or region as any antibody or antigen binding fragment as described herein. Epitope binning assays are known in the art and are described, for example, in Estep et al., "High throughput solution-based measurement of antibody-antigen affinity and epitope binning," Monoclonal Antibodies, Vol. 5. No. 2. Taylor & Francis, 2013, which is incorporated herein by reference in its entirety.

[0419] Antibodies and antigen-binding fragments

[0420] The present disclosure provides antibodies and antigen-binding fragments thereof comprising the complementarity determining regions (CDRs), VHHs, heavy chain variable regions, light chain variable regions, heavy chains or light chains described herein.

[0421] Typically, antibodies (also known as immunoglobulins) consist of two types of polypeptide chains: light chains and heavy chains. Non-limiting antibodies of the present disclosure may be complete four immunoglobulin chain antibodies comprising two heavy chains and two light chains. The heavy chain of the antibody may be of any isotype (including IgM, IgG, IgE, IgA or IgD) or subisotype (including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc.). The light chain may be a kappa light chain or a lambda light chain. The antibody may contain two identical copies of a light chain and / or two identical copies of a heavy chain. The heavy chains (each containing a variable domain (or variable region, VH) and multiple constant domains (or constant regions)) are bound to each other by disulfide bonds within their constant domains to form the "stem" of the antibody. The light chains (each containing a variable domain (or variable region, VL) and a constant domain (or constant region)) are each bound to a heavy chain by a disulfide bond. The variable region of each light chain is aligned 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).

[0422] These hypervariable regions, known as complementarity determining regions (CDRs), form loops that comprise the major antigen binding surface of the antibody. The four framework regions largely adopt a β-sheet conformation, and the CDRs form loops that connect and in some cases form part of the β-sheet structure. The CDRs in each chain are tightly bound by the framework regions and, together with the CDRs in the other chain, contribute to the formation of the antigen binding region.

[0423] Methods for identifying the CDR regions of antibodies by analyzing the amino acid sequence of the antibodies are well known, and many definitions of CDR are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the position of the structural loop regions. The IMGT numbering of V-DOMAIN (IG and TR) is derived from the IMGT unique numbering of V-REGION.These methods and definitions are described in, for example, 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 of Enzymology 133: 214-227. Enzymol. 203:121-53 (1991); Morea et al., Biophys Chem. 68(1-3):9-16 (October 1997); Morea et al., J Mol Biol. 275(2):269-94 (January 1998); Chothia et al., Nature 342(6252):877-83 (December 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); Kontermann, R. and Dübel, S. (eds.). (2010). Antibody Engineering: Vol. 2. Springer; Lefranc M.-P., "The IMGT unique numbering for Immunoglobulins, T cell receptors and Ig-like domains," The Immunologist, 7, 132-136 (1999); each of which is incorporated herein by reference in its entirety. In some embodiments, the CDRs are based on the Kabat definition.In some embodiments, the CDRs are based on the Chothia definition.In some embodiments, the CDRs are the longest CDR sequences as determined by the Kabat, Chothia, AbM, IMGT or contact definitions.

[0424] CDR is important for recognizing antigen epitopes. As used herein, an "epitope" is the smallest portion of a target molecule that can be specifically bound by the antigen binding domain of an antibody. The minimum size of an epitope may be about three, four, five, six or seven amino acids, but these amino acids are not necessarily located in a continuous linear sequence of the primary structure of the antigen, because the epitope may depend on the three-dimensional configuration of the antigen based on the secondary and tertiary structures of the antigen.

[0425] In some embodiments, the antibody is a complete immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, distinguished by their constant regions, particularly their hinge and upper CH2 domains. The sequences and differences of IgG subclasses are 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 the references is incorporated herein by reference in its entirety.

[0426] Antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, rats, camelids). Antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies, and chimeric antibodies including immunoglobulin binding domains fused to another polypeptide. The term "antigen binding domain" or "antigen binding fragment" is a part of an antibody that retains the specific binding activity of a complete antibody, i.e., any antibody portion that can specifically bind to an epitope on a target molecule of a complete antibody. It includes, for example, variants of Fab, Fab', F(ab')2, VHH, and these fragments. Therefore, in some embodiments, an antibody or its antigen binding fragment can be, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, double antibodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed by antibody fragments, and any polypeptide comprising a binding domain that is an antibody binding domain or a binding domain homologous to an antibody binding domain. Non-limiting examples of antigen binding domains include, e.g., heavy and / or light chain CDRs of an intact antibody, heavy and / or light chain variable regions of an intact antibody, the full length heavy or light chain of an intact antibody, or a single CDR from a heavy or light chain of an intact antibody.

[0427] In some embodiments, scFV has two heavy chain variable domains and two light chain variable domains. In some embodiments, scFV has two antigen binding regions (antigen binding regions: A and B), and the two antigen binding regions can bind to corresponding target antigens with different affinities.

[0428] In some embodiments, the antigen binding fragment may form a part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single-chain variable fragment (scFv) or VHH as described herein with a CD3ζ transmembrane domain and an internal domain. In some embodiments, the chimeric antigen receptor also includes an intracellular signaling domain from a variety of costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor includes multiple signaling domains (e.g., CD3z-CD28-41BB or CD3z-CD28-OX40) to increase effectiveness. Therefore, on the one hand, the present disclosure also provides cells (e.g., T cells) expressing chimeric antigen receptors as described herein.

[0429] In some embodiments, the antibody or its antigen-binding fragment can bind to two different antigens or two different epitopes. In some embodiments, the antibody or its antigen-binding fragment can bind to three different antigens or three different epitopes.

[0430] Fv fragment is an antibody fragment containing complete antigen recognition and binding site. This region is composed of a dimer of a heavy chain variable domain and a light chain variable domain that are tightly associated, and the tight association can be covalent in nature, such as in scFv. In this configuration, the three CDRs of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. In short, six CDRs or their subsets jointly confer antigen binding specificity for antibody. However, even a single variable domain (or only half Fv comprising three CDRs specific to an antigen) can also have the ability to recognize and bind antigens, but usually the affinity is lower than the entire binding site.

[0431] Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form a desired structure for antigen binding.

[0432] In some embodiments, the scFv described herein comprises, from N-terminus to C-terminus: VH; a polypeptide linker; and VL. In some embodiments, the scFv described herein comprises, from N-terminus to C-terminus: VL; a polypeptide linker; and VH.

[0433] The Fab fragment contains the variable and constant domains of the light chain and the variable domain and the first constant domain (CH1) of the heavy chain. The F(ab')2 antibody fragment comprises a pair of Fab fragments, which are usually covalently linked by hinge cysteines between them near their carboxyl termini. Other chemical couplings of antibody fragments are also known in the art.

[0434] The antibodies and antibody fragments of the present disclosure can be modified in the Fc region to provide the desired effector function or serum half-life. In some embodiments, the Fc region in any of the antibodies or antigen-binding fragments described herein comprises aspartic acid (Asp) at position 239 according to EU numbering. In some embodiments, the Fc region in any of the antibodies or antigen-binding fragments described herein comprises glutamic acid (Glu) at position 332 according to EU numbering. In some embodiments, the Fc region described herein is any one of the Fc regions described herein, comprising aspartic acid (Asp) at position 239 according to EU numbering and / or glutamic acid (Glu) at position 332 according to EU numbering. In some embodiments, Asp239 and / or Glu332 described herein can increase the effector function (e.g., ADCC or CDC) of an antibody or its antigen-binding fragment by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to the effector function of a wild-type antibody or its antigen-binding fragment. Detailed information can be found in, e.g., Lazar, GA et al., "Engineered antibody Fc variants with enhanced effector function," Proceedings of the National Academy of Sciences 103.11 (2006): 4005-4010, which is incorporated herein by reference in its entirety.

[0435] In some embodiments, the Fc region in any of the antibodies or antigen-binding fragments described herein comprises a wild-type human IgG1 CH2 domain. In some embodiments, the Fc region in any of the antibodies or antigen-binding fragments described herein comprises a mutated human IgG1 CH2 domain.

[0436] Any of the antibodies or antigen-binding fragments described herein can be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or solution). Non-limiting examples of stabilizing molecules include: polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin such as human serum albumin). The conjugation of stabilizing molecules can increase the half-life of the antibody or antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in the human body) or extend its biological activity.

[0437] In some embodiments, the antibodies or antigen-binding fragments described herein (e.g., bispecific antibodies) can be conjugated to a therapeutic agent. Antibody drug conjugates comprising antibodies or antigen-binding fragments thereof can be covalently or non-covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids (e.g., DM-1 and DM-4), dione, mitoxantrone, mithramycin, actinomycin D, D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin and cyclophosphamide and the like).

[0438] In some embodiments, the multispecific antibodies or antigen-binding fragments thereof described herein (e.g., PD-L1 / VEGF bispecific antibodies) bind to an antigen (e.g., PD-L1) with about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, or about 200% of the binding ability of a heavy chain antibody (e.g., an anti-PD-L1 heavy chain antibody) comprising the same VHH of the multispecific antibody.

[0439] In some embodiments, the multispecific antibodies or antigen-binding fragments thereof described herein (e.g., PD-L1 / VEGF bispecific antibodies) bind to an antigen (e.g., VEGF) with about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, or about 200% of the binding ability of an antibody or antigen-binding fragment comprising the same VHH targeting VEGF of the multispecific antibody (e.g., anti-VEGF heavy chain antibody).

[0440] In some embodiments, the bispecific antibodies or antigen-binding fragments thereof described herein (e.g., PD-L1 / VEGF bispecific antibodies) mediate CDC or ADC by at least or about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 30-fold, 40-fold, or 50-fold compared to complement dependent cytotoxicity (CDC) or ADC mediated by an isotype control antibody.

[0441] Recombinant vector

[0442] The present disclosure also provides a recombinant vector (e.g., an expression vector) comprising an isolated polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein), a host cell into which the recombinant vector is introduced (i.e., such that the host cell contains the polynucleotide and / or a vector comprising the polynucleotide), and the production of a recombinant antibody polypeptide or fragment thereof by recombinant technology.

[0443] As used herein, "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. "Expression vector" is capable of delivering one or more polynucleotides of interest and expressing them as encoded polypeptides in a host cell into which the expression vector has been introduced. Therefore, by being operably connected to regulatory elements such as promoters, enhancers and / or poly-A tails in the vector or in the host cell genome at or near or on the flanks of the integration site of the polynucleotide of interest so that the polynucleotide of interest will be translated in the host cell introduced with the expression vector, the polynucleotide of interest is positioned in the vector for expression.

[0444] The vector can be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., with a recombinant virus). Therefore, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0445] In some embodiments, polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein) are introduced using a viral expression system (e.g., vaccinia or other poxviruses, retroviruses, or adenoviruses), which may involve the use of non-pathogenic (defective) replication-competent viruses, or replication-defective viruses may be used. In the latter case, viral propagation will generally occur only in complementing viral packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. 86:317-321; Flexner et al., 1989, Ann. NY Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89 / 01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91 / 02805; Berkner, Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science, 252:431-434; 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 of ordinary skill in the art. The DNA can also be "naked" as described in, for example, Ulmer et al., 1993, Science, 259: 1745-1749 and Cohen, 1993, Science, 259: 1691-1692. The uptake of naked DNA can be increased by coating the DNA onto biodegradable beads, which are efficiently transported into cells.

[0446] For expression, a DNA insert comprising a polynucleotide encoding an antibody or a polynucleotide encoding a polypeptide disclosed herein can be operably linked to an appropriate promoter (e.g., a heterologous promoter), such as the bacteriophage λ PL promoter, E. coli lac, trp and tac promoters, SV40 early and late promoters, and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan. The expression construct may further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct may include translation initiated at the beginning and a stop codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0447] As indicated, the expression vector may 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 culture in Escherichia coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells, such as Escherichia coli, Streptomyces and Salmonella typhimurium cells; fungal cells such as yeast cells; insect cells such as fruit flies S2 and noctuid Sf9 cells; animal cells such as CHO, COS, Bowes melanoma and HK293 cells; and plant cells. Suitable culture media and conditions for host cells described herein are known in the art.

[0448] 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 apparent to the skilled artisan.

[0449] 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 LTRs such as the promoter of Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0450] In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For review, 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).

[0451] The introduction of constructs into host cells may be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0452] Transcription of the DNA encoding the antibodies of the present disclosure by higher eukaryotic organisms can be increased by inserting an enhancer sequence into the vector. An enhancer is a cis-acting element of DNA, typically about 10 to 300 bp, that acts to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer, located on the rear side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the rear side of the replication origin, and adenovirus enhancers.

[0453] In order to secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, an appropriate secretion signal may be incorporated into the expressed polypeptide. The signal may be endogenous to the polypeptide, or the signal may be a heterologous signal.

[0454] Polypeptides (e.g., antibodies) can be expressed in modified forms such as fusion proteins (e.g., GST-fusions) or with histidine tags, and can include not only secretion signals, but also additional heterologous functional regions. For example, additional amino acids, particularly charged amino acid regions, can be added to the N-terminus of the polypeptide to improve stability and persistence in host cells during purification or during subsequent handling and storage. Similarly, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to the final preparation of the polypeptide. Adding peptide moieties to polypeptides to cause secretion or excretion, improve stability and facilitate purification, etc. is a well-known and conventional technique in the art.

[0455] 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%, 10 ... The present invention also provides the present invention relates to 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 of the amino acid sequences described herein.

[0456] The present disclosure also provides nucleic acids 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 as described herein. Sequences and 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% homologous to any of the amino acid sequences described herein.

[0457] In some embodiments, the disclosure relates to a nucleotide sequence encoding any peptide described herein, or any amino acid sequence encoded by any nucleotide sequence 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.

[0458] 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 of the sequences described herein.

[0459] 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 of the sequences described herein.

[0460] Methods for preparing antibodies

[0461] Isolated fragments of human proteins (e.g., PD-L1, VEGF, or cancer antigens) can be used as immunogens to produce antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be produced in animals by multiple injections (e.g., subcutaneous injections or intraperitoneal injections) of antigenic peptides or proteins. In some embodiments, the antigenic peptides or proteins are injected with at least one adjuvant. In some embodiments, the antigenic peptides or proteins can be conjugated with an agent that is immunogenic in the species to be immunized. The antigenic peptides or proteins can be injected more than once (e.g., twice, three times, or four times) into the animal.

[0462] The full-length polypeptide or protein can be used as an immunogen, or alternatively, an antigenic peptide fragment thereof can be used as an immunogen. The antigenic peptide of a protein comprises 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 the epitope of the protein, such that antibodies generated against the peptide form specific immune complexes with the protein.

[0463] Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic preparations may contain, for example, recombinantly expressed polypeptides or chemically synthesized polypeptides. The preparations may further include an adjuvant, such as Freund's complete or incomplete adjuvant or a similar immunostimulant.

[0464] As described above, polyclonal antibodies can be prepared by immunizing a suitable subject with a polypeptide or its antigenic peptide (e.g., a portion of a protein) as an immunogen. Antibody titers in immunized subjects can be monitored over time by standard techniques, such as an enzyme-linked immunosorbent assay (ELISA) using an immobilized polypeptide or peptide. If necessary, antibody molecules can be isolated from mammals (e.g., from blood) and further purified to obtain an IgG fraction by well-known techniques (e.g., protein A of protein G chromatography). At an appropriate time after immunization, for example, when specific antibody titers are 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 Kohle et al. (Nature 256:495-497, 1975), human B cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), or tri-hybridoma technique. Techniques for producing hybridomas are well known (see generally, Current Protocols in Immunology, 1994, Coligan et al. (eds.), John Wiley & Sons, New York, NY). Hybridoma cells producing monoclonal antibodies are detected by screening hybridoma culture supernatants for antibodies that bind to the polypeptide or epitope of interest, for example, using a standard ELISA assay.

[0465] VHH can also be obtained from a purebred or designed synthetic llama VHH library. PBMC can be obtained from llama, and RNA can be isolated to produce cDNA by reverse transcription. Then, the VHH gene can be amplified by PCR, and the gene can be cloned into a phage display vector to construct a natural VHH library. Synthetic (e.g., humanized) VHH libraries can be prepared by incorporating the reorganized VHHCDR1, 2, and 3 produced by overlapping PCR into modified human VH scaffolds to produce enhanced diversity and maintain low immunogenicity. The VHH library can then be panned for antigens to obtain VHH with desired binding capacity.

[0466] Variants of antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into DNA encoding human antibodies, humanized antibodies or chimeric antibodies or their antigen-binding fragments described herein or by peptide synthesis. Such variants include, for example, deletions, insertions or substitutions of residues in the amino acid sequence of the antigen-binding site or antigen-binding domain of the antibody. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein. Any combination of deletions, insertions and / or combinations can be performed to obtain antibodies or their antigen-binding fragments with increased binding capacity to the target. Amino acid changes introduced into antibodies or antigen-binding fragments can also change antibodies or antigen-binding fragments or introduce new post-translational modifications into antibodies or antigen-binding fragments, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation sites (e.g., changing the amino acid sequence so that different sugars are connected by enzymes present in the cell) or introducing new glycosylation sites.

[0467] The antibodies disclosed herein can be derived from any animal species, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates (e.g., monkeys and apes), cows, pigs, horses, sheep, camelids (e.g., camelids, alpacas 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.

[0468] Phage display (panning) can be used to optimize the antibody sequence with desired binding capacity. In this technology, the gene encoding single-chain Fv (comprising VH or VL) or VHH can be inserted into the phage coating protein gene, so that the phage "displays" scFv or VHH in its outside while containing the gene of protein in its inside, thereby causing the connection between genotype and phenotype. These display phages can then be screened for the target antigen to detect the interaction between the displayed antigen binding site and the target antigen. Therefore, it is possible to screen and amplify large protein libraries in a process called in vitro selection and to obtain antibody sequences with desired binding capacity.

[0469] Human antibodies and humanized antibodies include antibodies having variable regions and constant regions derived from human germline immunoglobulin sequences (or having an amino acid sequence identical to an amino acid sequence derived from human germline immunoglobulin sequences). Human antibodies may include, for example, amino acid residues in the CDRs that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random mutagenesis or site-specific mutagenesis or by in vivo somatic mutation).

[0470] Humanized antibodies generally have a human framework (FR) transplanted with non-human CDRs. Therefore, humanized antibodies have one or more amino acid sequences introduced thereto from a non-human source. These non-human amino acid residues are generally referred to as "import" residues, which are generally taken from the "import" variable domains. Humanization can be performed essentially by, for example, replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. These methods are described in, for example, Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988); Each document in the document is incorporated herein by reference in its entirety. Therefore, a "humanized" antibody is a chimeric antibody in which substantially less than a complete human V domain has been replaced by a corresponding sequence from a non-human species.

[0471] More importantly, antibody is humanized, retains high specificity and affinity to antigen and other favorable biological properties.In order to achieve this goal, humanized antibody can be prepared by using the three-dimensional model analysis of parental sequence and humanized sequence and the process of various conceptual humanized products.Three-dimensional immunoglobulin model is usually available, and is familiar to those skilled in the art.It is available to illustrate and display the computer program of the possible three-dimensional conformation structure of selected candidate immunoglobulin sequence.The inspection of these displays allows the possible effect of residue in the function of candidate immunoglobulin sequence to be analyzed, i.e. the residue that affects the ability of candidate immunoglobulin to combine its antigen is analyzed.In this way, FR residues can be selected and combined from acceptor sequence and input sequence, thereby realize desired antibody characteristics, such as the affinity to target antigen is increased.

[0472] Identity or homology with respect to the original sequence is typically the percentage of amino acid residues present in the candidate sequence that are identical to the sequence present in the human antibody, humanized antibody or chimeric antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity and not taking into account conservative substitutions as part of the sequence identity.

[0473] In some embodiments, antibodies or their antigen-binding fragments can be covalently modified. These covalent modifications can be performed by chemical synthesis or enzymatic synthesis or by enzymatic cleavage or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting the targeted amino acid residues of the antibody or fragment with an organic derivatizing agent that can react with selected side chains or N-terminal residues or C-terminal residues.

[0474] In some embodiments, antibody variants having a carbohydrate structure lacking fucose connected to the Fc region (directly or indirectly) are adopted. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%. For example, the amount of fucose is determined by calculating the average amount of the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) attached to Asn 297 by calculating the fucose at Asn297 in the sugar chain as measured by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues; or position 314 in Kabat numbering); however, due to minor sequence changes in antibodies, Asn297 can also be located at about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function.In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody may be further engineered to replace the asparagine at position 297 with alanine (N297A).

[0475] In certain embodiments, in order to promote production efficiency by avoiding Fab arm exchange, the Fc region of the antibody is further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P). A detailed description of the S228 mutation is described below, such as 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 as a whole.

[0476] In certain embodiments, the methods described herein are designed to prepare bispecific antibodies. Bispecific antibodies can be prepared by engineering the interface between 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 an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (such as tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (such as alanine or threonine), a compensation "cavity" of the same or similar size as one or more large side chains is produced on the interface of the second antibody molecule. This provides a mechanism to increase the output of heterodimers rather than other unwanted end products such as homodimers. For example, this method is described in WO 96 / 27011, which is incorporated by reference in its entirety.

[0477] In some embodiments, one or more amino acid residues in the CH3 portion of IgG are substituted. In some embodiments, one heavy chain has one or more of the following substitutions Y349C and T366W. Another heavy chain may have one or more of the following substitutions E356C, T366S, L368A and Y407V. In addition, substitutions (-ppcpScp-->-ppcpPcp-) may also be introduced at the hinge region of two substituted IgGs. In some embodiments, one heavy chain has a T366Y (knob) substitution and another heavy chain has a Y407T (hole) substitution (EU numbering).

[0478] One aspect of the present application provides a heteromultimer (e.g., heterodimer) protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first heavy chain constant domain 3 (CH3) domain, and the second polypeptide comprises a second CH3 domain, wherein the first CH3 domain comprises a substitution with a bulky hydrophobic amino acid at amino acid position 354 relative to a wild-type CH3 domain, and / or the second CH3 domain comprises a substitution with a negatively charged amino acid at amino acid position 347 relative to a wild-type CH3 domain, and wherein the amino acid residue numbering is based on EU numbering. In some embodiments, the bulky hydrophobic amino acid at amino acid position 354 forms a hydrophobic interaction with an amino acid residue in the second CH3 domain. In some embodiments, the second CH3 domain comprises a bulky hydrophobic residue at amino acid position 349 (e.g., Y349). In some embodiments, the negatively charged amino acid at amino acid position 347 forms an ionic bond with an amino acid residue in the first CH3 domain. In some embodiments, the first CH3 domain comprises a positively charged residue at amino acid position 360 (e.g., K360). In some embodiments, the first CH3 domain and the second CH3 domain are human CH3 domains. In some embodiments, the first CH3 domain comprises a substitution selected from the group consisting of: S354Y, S354F and S354W. In some embodiments, the first CH3 domain comprises S354Y. In some embodiments, the second CH3 domain does not comprise a compensatory substitution (e.g., a substitution at Y349) for replacing S354 in the first CH3 domain. In some embodiments, the second CH3 domain comprises a substitution selected from the group consisting of Q347E and Q347D. In some embodiments, the second CH3 domain comprises Q347E. In some embodiments of any one of the heteromultimeric proteins described above, the first CH3 domain and the second CH3 domain further comprise a knob-hole (KIH) residue. In some embodiments, the knob-hole residues are T366Y and Y407T. In some embodiments, the first CH3 domain comprises T366Y and S354Y, and the second CH3 domain comprises Y407T and Q347E. In some embodiments, the first CH3 domain comprises Y407T and S354Y, and the second CH3 domain comprises T366Y and Q347E. Details can be found in, e.g., PCT / US2020 / 025469, which is incorporated herein by reference.

[0479] Treatment

[0480] The methods described herein include methods for treating a disorder associated with cancer. Generally, the methods include administering a therapeutically effective amount of an engineered bispecific antibody, an antigen-binding fragment thereof, as described herein, to a subject in need or determined to be in need of such treatment.

[0481] As used in this context, "treating" means improving at least one symptom of a condition associated with cancer. Typically, cancer leads to death; therefore, treatment can increase life expectancy (e.g., by at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or by at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years). Administration of a therapeutically effective amount of an agent described herein (e.g., a bispecific antibody) for treating a condition associated with cancer will result in a reduction in the number of cancer cells and / or relief of symptoms.

[0482] As used herein, the term "cancer" refers to cells with autonomous growth capacity, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of the histopathological type or invasive stage. As used herein, the term "tumor" refers to cancer cells such as a large number of cancer cells. Cancers that can be treated or diagnosed using the methods described herein include malignant tumors of various organ systems, such as malignant tumors affecting the lungs, breasts, thyroids, lymphs, gastrointestinal tracts and urogenital tracts, and adenocarcinomas including malignant tumors, such as most colon cancers, renal cell carcinomas, prostate cancers and / or testicular tumors, non-small cell lung cancers, small intestinal cancers and esophageal cancers. In some embodiments, the agents described herein are designed to treat or diagnose cancer in a subject. The term "cancer" is recognized in the art and refers to malignant tumors of epithelial or endocrine tissues, including respiratory cancer, gastrointestinal cancer, urogenital cancer, testicular cancer, breast cancer, prostate cancer, endocrine cancer, and melanoma. In some embodiments, the cancer is renal cancer or melanoma. Exemplary cancers include cancers that form in the cervix, lung, prostate, breast, head and neck, colon, and ovarian tissue. The term also includes carcinosarcoma, for example, including malignancies composed of cancerous tissue and sarcoma tissue. "Adenocarcinoma" refers to a cancer that originates from glandular tissue or in which tumor cells form a recognizable glandular structure. The term "sarcoma" is recognized in the art and refers to a mesenchymal-derived malignancy.

[0483] In one aspect, the present disclosure also provides methods for treating cancer in a subject, methods for reducing the rate at which a subject's tumor volume increases over time, methods for reducing the risk of metastasis, or methods for reducing the risk of additional metastasis in a subject. In some embodiments, treatment can stop, slow, delay, or inhibit the progression of cancer. In some embodiments, treatment can result in a decrease in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0484] In one aspect, the disclosure features a method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof, or antibody drug conjugate disclosed herein to a subject in need thereof, e.g., a subject having, or identified or diagnosed as having, a cancer, such as breast cancer (e.g., triple-negative breast cancer), benign tumor cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or a hematological malignancy.

[0485] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification, and describe the animal, human or non-human being provided for treatment according to the method of the present invention. Veterinary and non-veterinary applications are contemplated in the present invention. Human patients can be adults or adolescents (e.g., people under the age of 18). In addition to people, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs and primates. Including, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, porcine animals (e.g., pigs, miniature pigs), horses, dogs, cats, cattle and other livestock animals, farm animals and zoo animals.

[0486] In some embodiments, the cancer is a cancer that expresses PD-L1. In some embodiments, the cancer is a cancer that expresses VEGF. In some embodiments, the cancer is a cancer that expresses both PD-L1 and VEGF.

[0487] In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer or metastatic hormone-refractory prostate cancer. In some embodiments, the subject suffers from a solid tumor. In some embodiments, the cancer is head and neck squamous cell carcinoma (SCCHN), renal cell carcinoma (RCC), triple negative breast cancer (TNBC) or colorectal cancer, cervical cancer, endometrial cancer or hepatocellular carcinoma. In some embodiments, the subject suffers from Hodgkin's lymphoma. In some embodiments, the subject suffers from triple negative breast cancer (TNBC), gastric cancer, urothelial carcinoma, Merkel cell carcinoma or head and neck squamous cell carcinoma. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, hematological malignancies, particularly non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia or advanced solid tumors.

[0488] In some embodiments, the cancer is colon cancer, rectal cancer, lung cancer, breast cancer, kidney cancer, hepatocellular carcinoma, renal cancer, endometrial cancer, pancreatic cancer, head and neck cancer, or an advanced solid tumor.

[0489] In some embodiments, the cancer cells described herein are cell lines. In some embodiments, the cancer cells have elevated levels of VEGF or PD-L1, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% higher than non-cancerous cells.

[0490] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for developing cancer.Cancer patients can be identified by various methods known in the art.

[0491] As used herein, "effective amount" means an amount or dosage sufficient to achieve a beneficial or desired result, including stopping, slowing, delaying or inhibiting the progression of a disease (e.g., cancer). The effective amount will vary depending on, for example, the age and weight of the subject to whom the antibody, antigen-binding fragment, antibody drug conjugate, polynucleotide encoding the antibody, vector comprising the polynucleotide, and / or a composition thereof is to be administered, the severity of the symptoms, and the route of administration, and thus administration can be determined on an individual basis.

[0492] The effective amount can be administered in one or more administrations. For example, the effective amount of an antibody, an Fab or an antibody drug conjugate is enough to improve, stop, stabilize, reverse, inhibit, slow down and / or delay the progress of a patient's disease (e.g., autoimmune disease or cancer), or is enough to improve, stop, stabilize, reverse, slow down and / or delay the amount of cell (e.g., any cancer cell or cell line (e.g., cancer cell line) in vitro propagation of a biopsy cell, a cancer cell described herein). As known in the art, the effective amount of an antibody, an Fab or an antibody drug conjugate can vary, particularly depending on the patient's medical history and other factors, such as the type (and / or dosage) of the antibody used.

[0493] The effective amount and regimen of administering the antibodies, polynucleotides encoding the antibodies, antibody drug conjugates and / or compositions disclosed herein can be determined empirically, and making such determinations is within the skill of the art. It will be appreciated by those skilled in the art that the dosage that must be administered will vary, for example, depending on the mammal to which the antibodies, polynucleotides encoding the antibodies, antibody drug conjugates and / or compositions disclosed herein will be administered, the route of administration, the specific type of antibodies, polynucleotides encoding the antibodies, antigen-binding fragments, antibody drug conjugates and / or compositions disclosed herein used, and other drugs administered to the mammal. Guidance for selecting appropriate dosages of antibodies or antigen-binding fragments can be found in the literature on the therapeutic use of antibodies and antigen-binding fragments, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, (1985), Chapter 22 and pages 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pages 365-389.

[0494] The typical daily dosage of an effective amount of an antibody is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dosage may 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 dosage may 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 dosage is about 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.

[0495] In any of the methods described herein, the at least one antibody, antigen binding fragment thereof, 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 may be administered to the 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 with the same composition (e.g., a liquid composition). In some embodiments, at least one antibody, antigen binding fragment, antibody drug conjugate, and at least one additional therapeutic agent are administered with the same composition (e.g., a liquid composition containing at least one antibody or antigen binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, the at least one additional therapeutic agent is administered in a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained-release oral formulation.

[0496] In some embodiments, one or more additional therapeutic agents may be administered to a 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, 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, or pharmaceutical compositions described herein) are administered to a subject such that the biological activity periods of the one or more additional therapeutic agents and at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject overlap.

[0497] 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) may 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 may determine the length of the treatment period using any of the methods described herein for diagnosing or tracking the effectiveness of treatment (e.g., observing at least one symptom of cancer). As described herein, a skilled medical professional may also change (e.g., increase or decrease) the identity and amount of the antibody or antigen-binding antibody fragment, antibody drug conjugate (and / or one or more additional therapeutic agents) administered to a subject, and may also adjust (e.g., increase or decrease) the dose or frequency of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to a subject based on an assessment of the effectiveness of treatment (e.g., using any of the methods described herein and known in the art).

[0498] In certain embodiments, one or more additional therapeutic agents may be administered to a subject. Additional therapeutic agents may include one or more inhibitors selected from the group consisting of: B-Raf inhibitors, VEGF inhibitors, PD-1 inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, dual PI3K / mTOR inhibitors, Bruton's tyrosine kinase (Bruton's tyrosine kinase, BTK) inhibitors and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In certain embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-1) (IDO1) (epacadostat).

[0499] In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of a HER3 inhibitor, a LSD1 inhibitor, a MDM2 inhibitor, a BCL2 inhibitor, a CHK1 inhibitor, an inhibitor of the activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.

[0500] In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of trabectedin, nab-paclitaxel, Trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, fluoropyrimidine, sutent, temsirolimus, sutent ... Limulus, sorafenib, Votrient, pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalidomide, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0501] In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, a tumor necrosis factor (TNF) α, 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.

[0502] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

[0503] In some embodiments, the additional therapeutic agent is an anti-VEGFR antibody, an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody.

[0504] Pharmaceutical compositions and routes of administration

[0505] Also provided herein is a pharmaceutical composition containing at least one (e.g., one, two, three, or four) of the antibodies, antigen binding fragments, or antibody drug conjugates described herein. Two or more (e.g., two, three, or four) of any of the antibodies, antigen binding fragments, or antibody drug conjugates described herein may be present in the pharmaceutical composition in any combination. The pharmaceutical composition may be formulated in any manner known in the art.

[0506] The pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous or intraperitoneal). The composition may include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvent, an antibacterial or antifungal agent such as benzyl alcohol or methyl paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate or phosphate; and an isotonic agent such as a sugar (e.g., dextrose), a polyol (e.g., mannitol or sorbitol) or a salt (e.g., sodium chloride) or any combination thereof. Liposomal suspensions may also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Pat. No. 4,522,811). The preparation of the composition may be formulated and packaged in an ampoule, a disposable syringe or a multidose vial. Where necessary (e.g., in injectable formulations), appropriate fluidity can be maintained, for example, by using a coating (such as lecithin) or a surfactant. Absorption of the antibody or its antigen-binding fragment can be extended by including agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled 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.).

[0507] Compositions containing one or more of any of the 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 dosage unit form (i.e., physically discrete units containing a predetermined amount of active compound to facilitate administration and uniformity of dosage).

[0508] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell culture or experimental animals (e.g., monkeys). For example, the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents that exhibit high therapeutic indexes are preferred. In the case where the agent exhibits undesirable side effects, care should be taken to minimize potential damage (i.e., reduce undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0509] The data obtained from cell culture assays and animal studies can be used to formulate the appropriate dose of any given agent for a subject (e.g., mankind). The therapeutically effective amount of one or more (e.g., one, two, three or four) antibodies or their antigen-binding fragments (e.g., any antibody or antibody fragment described herein) will be in a subject (e.g., identified as a human subject with cancer) or identified as a subject at risk of suffering from a disease (e.g., previously having suffered from cancer but now cured) to treat the subject's disease (e.g., killing cancer cells), reduce the severity of one or more symptoms of the disease of the subject (e.g., mankind), frequency and / or duration. The effectiveness and dosage of any antibody or antigen-binding fragment in the antibodies or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods known in the art and by observing one or more symptoms of the disease of the subject (e.g., mankind). Certain factors may affect the dosage and time course required for the effective treatment of a subject (e.g., the severity of the disease or illness, previous treatment, the overall health status of the subject and / or the age and the presence of other diseases).

[0510] Exemplary dosages include any of the antibodies or antigen-binding fragments or antibody drug conjugates described herein in milligram or microgram amounts 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 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 1 μg / kg to about 50 μg / kg). Although these dosages cover a wide range, it will be appreciated by those of ordinary skill in the art that the efficacy of therapeutic agents (including antibodies and antigen-binding fragments thereof) varies, and effective amounts can be determined by methods known in the art. Typically, a relatively low dose is first administered, and the attending health care professional or veterinary professional (in the case of therapeutic applications) or researcher (when still working in the research and development phase) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it should be understood that the specific dosage level for any particular subject will depend on a variety of factors, including the activity of the specific compound employed, the age, weight, general health, sex and diet of the subject, time of administration, route of administration, rate of excretion and half-life of the antibody or antibody fragment in vivo.

[0511] The pharmaceutical composition can be included in a container, pack, or dispenser together with instructions for administration. The present disclosure also provides methods for making antibodies or antigen-binding fragments thereof or antibody drug conjugates for various uses as described herein.

[0512] Examples

[0513] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0514] Example 1: Immunization, generation of anti-human PD-L1 VHH antibodies and hit identification

[0515] Antigens of recombinant proteins of the extracellular domain (ECD) of human, cynomolgus monkey and mouse PD-L1 were purchased from Sinobiological USA. Immunization with PD-L1 antigen was performed using 2-year-old virgin female alpacas. Serum antibody titers were measured by ELISA assay. After 4 rounds of immunization, high titers (1:100,000) of anti-h-PD-L1 were observed. 80 ml of whole blood was collected from the alpacas, and PBMCs were isolated. RNA was isolated from PBMCs.

[0516] The VHH (variable domain of the heavy chain of the heavy chain antibody) gene was amplified by PCR using 10 pairs of specially designed primers covering all alpaca germlines. The PCR product was purified by DNA agarose gel and gel extraction kit, constructed into the phagemid vector pADL-23c and transformed into TG1 electrocompetent cells by electroporation. The transformed TG1 cells were cultured in 2YT medium, and phages showing the target VHH were produced by adding helper phages and culturing overnight. The phages in the culture supernatant were harvested by precipitation with 4% PGE / 0.5M NaCl and high-speed centrifugation. Binders to human PD-L1 (h-PD-L1) or mouse PD-L1 (m-PD-L1) antigens were selected using streptavidin-coupled Dynabeads coated with biotinylated h-PD-L1ECD protein or m-PD-L1 ECD protein. After 2-3 rounds of panning, the binders of h-PD-L1 or m-PD-L1 were eluted and used to infect SS320 cells. Colonies of SS320 cells were picked and cultured in 2YT medium, and 0.5mM IPTG was added for secretion of VHH antibodies. Plates coated with h-PD-L1 ECD antigen were determined by ELISA, followed by flow cytometry whole-cell binding assays using 293T cells stably expressing h-PD-L1 to screen supernatants with VHH antibodies. Whole-cell binders were picked for sequencing. 54 clones with different sequences were obtained, many of which had only minor sequence differences in CDR or framework. 24 clones with more unique sequences were identified. CDR and framework sequences were obtained using IMGT (International Immunogenetics Information System) by analyzing the sequences.

[0517] Example 2: Characterization of functional PD-L1 VHH antibodies

[0518] 24 unique clones were used to prepare bivalent antibodies with human IgG1 Fc region (VHH-Fc) by adding the hinge region and domain of human IgG1 constant heavy chain 2 (CH2) and constant heavy chain 3 (CH3). The constructed bivalent VHH-Fc antibodies were transiently expressed in CHO cells, and the proteins in the supernatant were harvested and purified by protein A resin.

[0519] Binding of bivalent PD-L1 VHH-Fc clones to human PD-L1 in stably transfected cells was determined by flow cytometry whole cell binding (WCB) assay. Briefly, antibodies (at different concentrations) were incubated with cells stably expressing h-PD-L1 (0.2x10 6 / ml) were incubated in 96-well plates at 100ul / well in FACS buffer (PBS with 1.0% FBS) for 30 minutes. After washing, Alexaflour488-conjugated anti-human IgG Fc secondary antibody (Alexa 488AffiniPure goat anti-human IgG, Fcγ fragment specific, Jackson labs, 1:750 dilution) and incubated for 30 minutes. After washing, the median fluorescence intensity was measured by gating the live cell population using CytoFlex (Beckman Coulter) and using the FITC channel. The EC50 of binding was calculated using GraphPad Prism7.0. Figure 1A Representative results are shown in . An overview of the functional binding agents (fused to human IgG1 Fc) is shown in Table 1 below.

[0520] The function of PD-L1 VHH-Fc clones was determined by PD-L1 blocking assay. Briefly, CHO cells stably expressing h-PD-L1 were cultured overnight at 20k / well in a 96-well half-area cell culture plate. The culture medium was removed, and serial dilutions of PD-L1 antibodies were added at 20ul / well. Jurkat cells stably transfected with human PD1 and NFAT reporter genes were added at a density of 2 million / ml at 20ul / well. After incubation for 5 hours, 40ul of Bright-Glo luciferase assay buffer with substrate (Promega) was added, and luciferase activity was measured by chemiluminescent activity using plate reader EnSight (PerkinElmer). The efficacy of antibody blocking PD-L1 activity was calculated using nonlinear regression by GraphPad Prims7.0. Figure 1BRepresentative results are shown in . A summary of the potency (EC50 values ​​in nM) of the functional clones is shown in the table below. R1-R3 (clone names) represent panning rounds 1 to 3.

[0521] Table 1: Summary of EC50s in whole cell binding assay and PD-L1 blocking assay.

[0522]

[0523] Fig.28 and 30 And the sequences of functional binders are shown in the table below.

[0524] Table 2: Sequences of CDRs and frameworks of functional binders

[0525]

[0526]

[0527] Example 3: Characterization and selection of lead mouse PD-L1 binders

[0528] The affinity of antibodies (all antibodies fused to human IgG1 Fc) to human PD-L1 was determined by BLI (biolayer interferometry) kinetic assay using a Gator instrument and recombinant protein of human PD-L1-ECD. High affinity binders are shown in the table below. 5F8 has the highest affinity, as determined by the equilibrium dissociation constant (KD). Cross-binding of PD-L1 clones to mouse PD-L1 was determined by ELISA assay using mouse PD-L1-ECD antigen-coated plates, followed by whole-cell binding to 293T cells stably transfected with mouse PD-L1. Figure 2 Positive mouse PD-L1 binders are shown in .

[0529] Table 3: Affinity of the top PD-L1 clones in the kinetic binding assay

[0530]

[0531]

[0532] Clones 5F8 and 3H1 were selected for humanization based on their (1) binding affinity to human PD-L1 and (2) EC50 for whole-cell binding to mouse PD-L1. Figure 2). Humanization was performed by analyzing germline sequences using Igblast. The closest germline sequences were used to alter the frameworks of 5F8 and 3H1. For clone 5F8, eight humanized variants were made, and for clone 3H1, seven humanized variants were made, all fused to human IgG1 Fc. Variants 7 and 8 of 5F8 (5F8-hv7 and 8) and variant 7 of 3H1 (3H1-hv7) retained h-PD-L1 blocking function ( Figure 3A ), the blocking function had a similar potency to the reference antibody Envafolimab (KN035), a single-domain PD-L1 antibody. To select potent mouse PD-L1 binders from these humanized variants, whole-cell binding assays were performed using 293T mouse PD-L1 stably transfected cells. Figure 3B Binding activity is shown in . Among the humanized variants, 3H1-hv7 showed a binding ability similar to that of atezolizumab to mouse PD-L1. All other variants showed much weaker binding ability (data not shown). In 293T cells overexpressing mouse PD-L1, the effective human PD-L1 blockers 5F8-hv7 and hv8 showed much lower binding ability to mouse PD-L1 ( Figure 3B ). Hepa1-6 cells were used to further examine the binding of 3H1-hv7 and 5F8-hv8 to endogenously expressed mouse PD-L1 in mouse cancer cells. Figure 3C As shown, surprisingly, clone 3H1-hv7 showed much weaker binding ability to endogenously expressed mouse PD-L1, and clone 5F8-hv8 showed no binding ability. Fig.28 and 30 The sequences of the humanized variants and parental clones 5F8 and 3H1 are shown in the table below.

[0533] Table 4: Sequences of R3-5F8 humanized variants

[0534]

[0535]

[0536] Table 5: Sequences of R3-3H1 humanized variants

[0537]

[0538] To increase the binding of 3H1-hv7 to mouse PD-L1, affinity maturation was performed. Primers were designed to generate single mutations of amino acids in each CDR region. The library was prepared using assembly PCR and cloned into the phagemid vector pADL-23c. Helper phage was used to produce phage, and streptavidin-coupled Dynabeads coated with biotinylated mouse PD-L1-ECD were used for phage panning. After two rounds of panning, elution of the panning product was used to infect SS320 cells. Colonies were picked and cultured in 2YT medium with 0.5mM IPTG. The binding activity of VHH antibodies in the supernatant was determined by whole-cell binding assay using 293T cells stably transfected with mouse PD-L1. Binders with high binding capacity were selected for sequencing. Cloned genes with unique sequences were synthesized and cloned into pcDNA3.4 expression vectors fused to human IgG1 Fc. The binding of VHH-Fc clones to mouse PD-L1 was tested in whole cell binding assays using 293T cells or Hepa1-6 cells (mouse cell lines with endogenous PD-L1 expression) that stably express mouse PD-L1 (Table 6). The efficacy of VHH-Fc clones in blocking mouse PD-L1 activity was also tested in a mouse PD-L1 blocking reporter gene assay. Binding agents with high binding capacity and efficacy were selected. The EC50 in whole cell binding and the efficacy in blocking m-PD-L1 are shown in the table below.

[0539] Table 6: Whole cell binding capacity and potency of selected 3H1-hv7 affinity matured clones

[0540]

[0541] Fig.28 and 30 and the sequences are listed in the table below. The top clone 3B9 was selected as a surrogate molecule to construct a bispecific antibody for in vivo studies based on binding capacity and potency.

[0542] Table 7: Sequences of selected 3H1-hv7 affinity matured clones

[0543]

[0544]

[0545] Affinity maturation of 5F8-hv8 clones was also performed and clones with increased potency in blocking human PD-L1 are shown in the table below (all clones were fused to human IgG1 Fc).

[0546] Table 8: Whole cell binding capacity and potency of selected 5F8-hv8 affinity matured clones

[0547]

[0548] Fig.28 and 30 And the sequence is shown in the table below.

[0549] Table 9: Sequences of selected 5F8-hv8 affinity matured clones

[0550]

[0551]

[0552] Example 4: Characterization and selection of lead human PD-L1 functional binders, and hotspot correction and humanization of leading clones.

[0553] The ability of the lead human PD-L1 binder to bind to human PD-L1 expressed on human tumor cells was determined using T24 cells, a human bladder cancer cell line that expresses endogenous human PD-L1 at moderate levels. Surprisingly, compared to clones 5C12, 1F4, 4D2, and 4E2 (a group of clones with the same CDR3 ( Figure 4 As shown in the table below), the high affinity clone 5F8 had much lower binding capacity to endogenously expressed human PD-L1 in T24 cells in the kinetic assay. Therefore, further studies on this group were focused.

[0554] Table 10: EC50 of the top h-PD-L1 VHH-Fc clones in WCB with h-PD-L1 endogenously expressed in T24 cells

[0555] PD-L1 lead clone EC50 in WCB of T24, nM 5F8 0.503 5C12 0.078 1F4 0.059 4D2 0.041 4E2 0.050 KN35-hv 0.054 Avelumab 0.336 Atezolizumab 0.091

[0556] The CDR3 sequences of these 4 PD-L1 binders contain hot spots (NGS) that induce N-glycosylation and change the monomer ratio of the protein, as shown in SEC-HPLC analysis (Figure 5). To correct the hot spots in CDR3, three different designs were performed. N was changed to T, S or Q. Fig.28 and 30 And the sequence is shown in the table below.

[0557] Table 11: CDR3 of hotspot corrected 5C12 variants (other CDRs and FRs of variants 46-48 remain unchanged)

[0558] Clone Name CDR3 SEQ ID 5C12 Parent NALVWNGSSYNN 9 5C12-NQ NALVWQGSSYNN 46 5C12-NT NALVWTGSSYNN 47 5C12-NS NALVWSGSSYNN 48

[0559] The new variants with hotspot correction were analyzed by SEC-HPLC and examined in whole cell binding assays and PD-L1 blocking reporter gene assays. Changing N to T, S or Q corrected N-glycosylation, and SEC-HPLC demonstrated a much higher monomer ratio (Figure 5). The whole cell binding ability and potency in the blocking reporter gene assay were retained compared to its parental clone, as shown in the figure ( Fig. 6A and 6B ).

[0560] The 5C12 clone was humanized using Igblast by analyzing germline sequences. One of the closest human germlines of 5C12 is IGHV-3-11.01. In addition to most of the amino acid sequences in framework 2 (specifically the three marks of the alpaca / lamb sequence of QREF at 44, 45, and 47), the framework of the alpaca sequence of 5C12 was changed to the framework of IGHV-3-11.01, which was retained due to the influence of these amino acids in terms of stability and affinity. 9 different humanized variants were prepared with slightly different designs in the humanization of frameworks 2 and 3. Humanized VHH was fused to human IgG1 Fc, and protein was produced by transient transfection into ExpiCHO cells and purified by protein A resin. Whole cell binding assays were performed using cells stably expressing h-PD-L1. Fig. 7A Representative variants with high binding capacity to h-PD-L1 are shown in Figure 1. hv1, hv2, and hv7 showed similar binding capacity to the parental clone 5C12 (EC50 at 0.22 nM). Hotspot-corrected humanized variants were also prepared, and whole-cell binding to CHO cells stably expressing h-PD-L1 and human tumor cells NCI-H441 endogenously expressing h-PD-L1 was determined ( Figure 7B and 7C ). PD-L1 blockade function was determined by reporter gene assay ( Fig.7D ). The results showed that the 5C12-hv9-NT variant had the highest binding capacity and efficacy. Fig.28 and 30 And the sequences of 5C12 humanized variants and CDR3 hotspot corrected variants are shown in the table below.

[0561] Table 12: Sequences of 5C12 CDR3 hotspot corrected variants and humanized variants

[0562]

[0563] 1F4 was humanized using human germline IGHV-3-11.01 or IGHV-3-23.04 by the same strategy as used to clone 5C12. Twelve humanized variants were prepared (all humanized variants were fused to human IgG1 Fc) and their CDR3 hot spots (N to T) were corrected. Its binding ability to h-PD-L1 was examined by whole cell binding assay. Fig. 8A The results for clones with high binding capacity are shown in and their efficacy in blocking PD-L1 was tested in a PD-L1 blocking reporter gene assay, and Figure 8B The results are shown in . Fig.28 and 30 The sequences of the humanized 1F4 clones are shown in the table below. 1F4-hv7 and hv9 retained high binding capacity and potency compared to the parental clone 1F4.

[0564] Table 13: Sequences of 1F4 CDR3 hotspot correction variants and humanized variants

[0565]

[0566]

[0567] Humanization and CDR3 hotspot correction of clones 4D2 and 4E2 were performed following the same strategy used for 5C12. Fig.9A and 9B The whole cell binding of humanized variants (all variants fused to human IgG1 Fc) to CHO cells stably expressing h-PD-L1 and human tumor cells NCI-H441 endogenously expressing h-PD-L1 is shown in Figure 4. The efficacy of blocking human PD-L1 was determined, and Fig.10 The results are shown in Figure 9. The humanized variants with CDR3 hotspot correction can retain the binding activity to human PD-L1 and the efficacy of blocking human PD-L1, wherein high binding ability and efficacy were observed in 4D2-hv1-NT, 4E2-hv1-NT and 4E2-hv2-NT (Figures 9-10 and the table below).

[0568] Table 14: WCB of PD-L1 and efficacy of blocking PD-L1 of 4D2 and 4E2 humanized variants

[0569]

[0570] Fig.28 and 30 And the sequences of the parental alpaca clone and the humanized variants are shown in the table below.

[0571] Table 15: Sequences of 4D2 and 4E2 humanized variants

[0572]

[0573]

[0574] BLI was used to determine the kinetic binding affinity of the selected lead humanized clones (all clones were fused to human IgG1 Fc). All lead clones showed good association curves with human PD-L1. The affinity of human PD-L1 calculated by Koff and Kon is shown in the table below.

[0575] Table 16: Binding affinity of lead PD-L1 clones to h-PD-L1

[0576] load koff(1 / second) kon(1 / millisecond) KD(M) 4D2-hv1-NT 1.74E-03 3.84E+05 4.54E-09 4E2-hv1-NT 2.25E-03 4.85E+05 4.64E-09 4E2-hv2-NT 2.48E-03 5.23E+05 4.75E-09 1F4-hv7-NT 3.41E-03 5.33E+05 6.39E-09 5C12-hv7-NT 4.37E-03 5.39E+05 8.12E-09

[0577] The following table shows the affinity of cynomolgus monkey PD-L1 (cyno-PD-L1) calculated by Koff and Kon.

[0578] Table 17: Binding affinity of lead PD-L1 clones to cyno-PD-L1

[0579] load koff(1 / second) kon(1 / millisecond) KD(M) 4D2-hv1-NT 2.06E-03 5.47E+05 3.76E-09 4E2-hv1-NT 2.10E-03 6.18E+05 3.40E-09 4E2-hv2-NT 2.10E-03 6.47E+05 3.24E-09 1F4-hv7-NT 2.34E-03 6.05E+05 3.87E-09 5C12-hv7-NT 2.57E-03 5.92E+05 4.34E-09

[0580] Example 5: Construction of synthetic VHH antibody library

[0581] Camel / alpaca VHH antibody is a heavy chain only antibody that retains binding (and function) to antigens similar to conventional antibodies containing both heavy and light chains. The unique feature of VHH is that it has a longer CDR3 compared to the CDR3 of the heavy chain of conventional antibodies. Recently, it has become more common to use "in silico" design to prepare synthetic VHH libraries. Based on the published information, a VHH synthetic library of its own VEGF binders was designed. A fixed length was used for CDR1 and 2, each containing 8 amino acids. For CDR3, 13 different lengths (10 to 22 amino acids) were designed and different ratios were used. The genes of the designed VHH library were synthesized and cloned into pADL-23c phagemid. The phagemid VHH library was used to transform TG1 cells. 1.5ug phagemid DNA produced> 2.5x 10 9 TG1 colonies. Twelve transformations were performed and the combined TG1 VHH library was approximately 3 x 10 10 The phage library was prepared by culturing 2 L of TG1 cells and 20 times of helper phage and purified according to standard protocols. The final titer of the phage library (measured at OD260) was 2.37 x 10 13 / mL.

[0582] Example 6: Panning of anti-VEGFA VHH antibodies

[0583] The antigen of recombinant human / cynomolgus monkey VEGF165 (isotype of h-VEGFA) was purchased from Sino-Bio Inc., USA and biotinylated with NHS-ester biotinylation reagent (EZ-Link Sulfo-NHS-SS-biotin, ThermoFisher). Dynabeads coupled with streptavidin coated with biotinylated VEGF165 were used to select the binding agent of human / cynomolgus monkey VEGF165 antigen. After 3 rounds of selection, the binding agent of VEGF165 was eluted and infected with SS320 cells. The colony of SS320 cells was picked and cultured in 2YT medium, and IPTG was added for secretion of VHH antibodies. The supernatant with VHH antibodies was screened by ELISA assay using a high binding plate coated with VEGF165. 9 plates (about 864 samples) were screened, and 176 ELISA positive binding agents were obtained. The inhibition of the reporter gene activity mediated by VHH supernatant to VEGF165 was measured, and the cross-binding with mouse VEGF164 (isotype of mouse VEGFA) was determined. Both human VEGFA blockers and mouse VEGFA blockers (72 clones in total) were selected for sequencing, and 42 sequence-unique clones were obtained. The cross-binding of sequence-unique clones with the subtypes of VEGF B, C, D and PIGF was checked, and cross-binding agents were eliminated. 19 unique clones were selected to prepare bivalent IgG1 or IgG4Fc clones.

[0584] Example 7: Affinity and potency of bivalent anti-VEGF VHH-Fc clones

[0585] Anti-VEGFA VHH clones are fused to human IgG4 Fc (hinge+CH2+CH3), cloned into pcDNA3.4 vectors, and transfected into CHO cells for protein production. VHH-Fc protein is purified by protein A resin. Luciferase reporter gene assay is used to determine the blocking function of VHH-Fc clones to human VEGFA, wherein stable cell lines overexpress VEGFA receptor KDR and NFAT-luciferase reporter genes. Fig.11 The results of the first four clones blocking human VEGFA are shown in Figure 2. Clones 7H3 and 1C8 have similar potency to the reference antibody Ramucirumab. The results of these four clones blocking mouse VEGFA ( Fig.12 ) showed that 7H3 was more potent than ramucirumab. BLI kinetic assay was also used to determine the binding affinity of the top 4 VHH-Fc clones to human VEGFA (see table below). 7H3 showed the highest affinity to human VEGFA.

[0586] Table 18: Binding affinity to human VEGFA

[0587] clone koff(1 / second) kon(1 / millisecond) KD(M) 7H3 2.71E-04 1.30E+05 2.08E-09 1C8 1.77E-04 6.80E+04 2.61E-09 4B11 6.52E-04 2.31E+05 2.82E-09 1G1 2.62E-03 1.82E+05 1.44E-08

[0588] Fig.29 and 30 The sequences of these four clones are shown in the table below.

[0589] Table 19: Sequences of the top 4 anti-VEGF clones

[0590]

[0591] Example 8: Construction of bispecific anti-PD-L1 x anti-VEGF antibodies and the efficacy of bispecific antibodies in blocking PD-L1 and VEGFA

[0592] The lead humanized anti-human PD-L1 clone was used to prepare bispecific antibodies with the lead anti-VEGF clone 7H3. First, 4 different formats of BsAb were prepared using the 4E2-hv2-NT clone for the anti-PD-L1 arm and 7H3 for the anti-VEGFA arm. IgG1 hinge and CH2+CH3 were used for all these constructions. Figures 13A-13D A diagram is shown in , and the sequences are shown in the following four tables.

[0593] Table 20: Sequence of 4E2_Fc_7H3 (SEQ ID NO: 85)

[0594]

[0595] Table 21: Sequence of 4E2+7H3_Fc (SEQ ID NO: 86)

[0596]

[0597] Table 22: Sequence of 7H3+4E2_Fc (SEQ ID NO: 87)

[0598]

[0599] Table 23: Sequence of 7H3_Fc_4E2 (SEQ ID NO: 88)

[0600]

[0601] The anti-PD-L1 arm was tested for its ability to block human PD-L1. Fig.14A The results are shown in the following table. Among these different formats of BsAb, the 4E2_Fc_7H3 format showed the highest efficacy in blocking h-PD-L1.

[0602] The efficacy of its anti-VEGFA arm was also tested, and the results were similar to those of the anti-PD-L1 arm. The 4E2_Fc_7H3 format showed the highest efficacy in blocking h-VEGFA, as Fig. 14B and as shown in the following table.

[0603] Table 24: Efficacy of different BsAb formats in blocking h-PD-L1 and h-VEGFA and their SEC-HPLC curves

[0604]

[0605] No = No activity. ND = Not determined

[0606] These BsAbs were tested for simultaneous binding to both h-PD-L1 and h-VEGFA antigens using a dual binding assay (Figure 15). All BsAbs were able to bind to both h-PD-L1 and h-VEGFA. In contrast, the parental monospecific antibodies (4E2-hv2-NT_Fc or 7H3_Fc) only bound to h-PD-L1 or h-VEGFA. The reference anti-PD-L1 antibody Avelumab only bound to h-PD-L1.

[0607] The anti-PD-L1_Fc_anti-VEGF format can retain the efficacy of two arms against PD-L1 or VEGF and has the highest monomer ratio in SEC (Figure 14 and Table 24). Therefore, this format was used to construct all lead PD-L1 clones of BsAb (Figure 16). The sequences are shown in the following four tables.

[0608] Table 25: Sequence of 5C12-hv7-NT_Fc_7H3 (SEQ ID NO: 89)

[0609]

[0610] Table 26: Sequence of 1F4-hv7-NT_Fc_7H3 (SEQ ID NO: 90)

[0611]

[0612] Table 27: Sequence of 4D2-hv1-NT_Fc_7H3 (SEQ ID NO: 91)

[0613]

[0614] Table 28: Sequence of 4E2-hv1-NT_Fc_7H3 (SEQ ID NO: 92)

[0615]

[0616] Table 29: Sequence of 4E2-hv2-NT_Fc_7H3 (SEQ ID NO: 93)

[0617]

[0618] The binding abilities of these BsAbs and their parental monospecific anti-PD-L1 bivalent antibodies (all antibodies fused to human IgG1 Fc) to human PD-L1 expressed in T24 tumor cells were determined by whole-cell binding assays, and Fig.17A The results are shown in Figure 2. The potency of these BsAbs and their parental monospecific anti-PD-L1 bivalent antibodies in blocking h-PD-L1 was also determined, and Fig. 17B The results are shown in . The binding capacity of the BsAb and its potency in blocking h-PD-L1 were retained to the greatest extent compared to its parental monospecific bivalent format. Fig. 17C The efficacy of blocking h-VEGFA is shown in the following table.

[0619] Table 30: Whole cell binding capacity to h-PD-L1 and efficacy in blocking h-PD-L1 and h-VEGFA

[0620]

[0621] No = Inactive

[0622] Example 9: Construction of alternative anti-PD-L1 x anti-VEGF BsAbs and efficacy of BsAbs in blocking mouse PD-L1 and mouse VEGFA

[0623] Because the lead anti-PD-L1 clone does not bind / block mouse PD-L1, clone 3B9 was selected as an alternative PD-L1 blocker due to its potency in blocking mouse PD-L1, as shown in Example 3. First, a new bivalent 7H3-Fc clone was prepared by converting 7H3-IgG4-Fc to 7H3-IgG1-Fc, and then a tandem format BsAb 3B9+7H3_Fc and an N-terminal or C-terminal format BsAb3B9_Fc_7H3 (Figure 18) were constructed. The sequences are shown in the following two tables.

[0624] Table 31: Sequence of 3B9+7H3_Fc (SEQ ID NO: 94)

[0625]

[0626] Table 32: Sequence of 3B9_Fc_7H3 (SEQ ID NO: 95)

[0627]

[0628] To measure the blocking function of BsAb in VEGFA-mediated NFAT luciferase reporter activity in mice, and Fig.19A The results are shown in . The tandem BsAb retains the blocking function against VEGFA with potency similar to that of the bivalent parental clone 7H3 or the reference antibody ramucirumab. Interestingly, when the anti-VEGFA 7H3 was fused to the C-terminus of the BsAb, 3B9_Fc_7H3 showed significantly higher potency in blocking m-VEGFA compared to the bivalent 7H3-Fc parental clone, with potency increased by more than 7-fold. The potency of the BsAb (both formats) in blocking mouse PD-L1 was also examined. Fig.19B As shown, two BsAbs blocked mouse PD-L1 activity, of which 3B9_Fc_7H3 was slightly more effective than 3B9+7H3_Fc, and showed similar efficacy to the parental clone bivalent 3B9. Based on the function of the two arms of anti-PD-L1 and anti-VEGFA, 3B9_Fc_7H3 was selected as an alternative BsAb candidate for in vivo studies. The stability of 3B9_Fc_7H3 in mouse serum was tested by incubating 3B9_Fc_7H3 with 66.6% mouse serum at 37°C for different time periods. Check the function of blocking mouse PD-L1 and mouse VEGFA. Fig. 20A and 20B 3B9_Fc_7H3 was shown to be stable under mouse serum treatment at 37°C for up to 6 days.

[0629] Example 10: In vivo efficacy study of surrogate anti-PD-L1 x anti-VEGF BsAb and monospecific anti-PD-L1 and anti-VEGF antibodies in the MC38 syngeneic mouse model

[0630] The efficacy of BsAb3B9_Fc_7H3 was studied using a C57BL / 6N mouse model with MC38 murine colon cancer to compare the efficacy of monospecific antibodies 3B9_Fc (anti-PD-L1) or 7H3_Fc (anti-VEGF). MC38 tumor cells were implanted one week before treatment. When the tumor reached approximately 46 mm 3 Treatment was started at 6 mg / kg or equivalent molar amount, and the drug was intraperitoneally administered three times a week for 3 weeks, as shown in Figure 21, for a total of 8 doses. Compared with the negative control (inactive anti-PD-L1 variant 5F8-hv2), both 3B9_Fc and 7H3_Fc significantly reduced tumor growth, with tumor growth inhibition rates (TGI) of 62.7% and 60.9% (19 days after treatment start, respectively). Fig.21Aand the following table). Compared with the monospecific antibodies, BsAb 3B9_Fc_7H3 had better efficacy, with TGI reaching 76.9% on day 19 after treatment. Statistical analysis using two-way ANOVA Tukey's multiple comparisons test showed that tumor inhibition was significant for all three treatment groups compared to the negative control, P < 0.0001. Tumor inhibition of 3B9_Fc_7H3 was also significant compared to the TGI of 3B9_Fc or 7H3_Fc, P = 0.0259 or 0.0089, respectively. Fig. 22 Individual tumor volumes for each treatment group are shown in . No significant differences in body weight were observed among mice from the different study groups, indicating that the drug was well tolerated ( Fig. 21B ).

[0631] Table 33: Summary of efficacy in the MC38 syngeneic model

[0632]

[0633] a Mean ± SEM

[0634] b Comparison with negative control by two-way ANOVA Tukey's multiple comparison test

[0635] c Comparison with 3B9_Fc by two-way ANOVA Tukey's multiple comparison test

[0636] d Comparison with 7H3_Fc by two-way ANOVA Tukey's multiple comparison test

[0637] Example 11: Comparative study of lead BsAb with other anti-PD-L1 / PD1 x anti-VEGF bifunctional drug candidates IMM2510 or AK112 in cell-based functional assays

[0638] Currently, there are two main bifunctional drug candidates in clinical trials: IMM2510 and AK112. IMM2510 binds to VEGF at the N-terminus of the heavy chain. Trap Traditional anti-PD-L1 antibody fused to VEGF Trap It is a fusion protein that combines the ligand-binding elements from the extracellular components of human VEGF receptor I (domain 2 of VEGFRI) and human VEGF receptor II (domain 3 of VEGFRII). TrapIt binds to its ligand VEGFA with high affinity, thereby blocking the function of VEGFA. Currently, IMM2510 is in Phase I clinical trials. AK112 is a BsAb constructed using Avastin (Roche's anti-VEGF antibody) and fused to the scFv (single-chain variable fragment) of the anti-PD1 antibody in the C-terminus of the heavy chain of Avastin. Currently, AK112 is in Phase III clinical trials.

[0639] Comparative studies by whole-cell binding assay were performed using CHO cells stably transfected with human PD-L1 (see Fig.23 and Table 34). Compared with IMM2510, the lead BsAbs of the present invention all showed much higher binding ability, and their EC50 was about 1 / 10 of the EC50 of IMM2510. Whole cell binding assays of AK112 and PD1 stably transfected Jurkat cells were also performed, and the EC50 data are shown in Table 34. In the human PD1 / PD-L1 blocking assay, the lead BsAbs of the present invention showed more effective function in blocking PD1 / PD-L1 compared with IMM2510 and AK112 (see Fig.24 The EC50 of BsAb was 2.04nM and 8.08nM, respectively. In the human VEGFA blocking assay, the potency of BsAb and IMM2510 was similar, but IMM2510 was slightly more effective. AK112 was weak in blocking VEGFA and gradually lost its inhibitory function even when the concentration increased to above 10nM ( Fig.25 ).

[0640] SEC-HPLC was also performed to compare IMM2510 and AK112 with the lead BsAb of the present invention, as shown in Table 34. In the SEC-HPLC assay, the BsAb had more than 98% monomers, whereas IMM2510 had only 92.9% monomers, with 6.8% degraded fragments showing as low molecular weight (LMW) species. AK112 was the molecule with the worst stability, having only 65.8% monomers and having 21.2% aggregates showing as high molecular weight (HMW) species and 12.9% degraded fragments showing as LMW species. All protein samples were purified using protein A resin by the same purification method.

[0641] In general, the dual-armed anti-human PD1 / PD-L1 or VEGFA of the lead anti-PD-L1 x anti-VEGF BsAb of the present invention is more effective than AK112 in cell-based functional assays. In addition, the lead BsAb of the present invention is more effective than IMM2510 in blocking human PD-L1 and is as effective as IMM2510 in blocking human VEGFA. In addition, compared to both IMM2510 and AK112, the lead BsAb of the present invention has a better SEC curve, is a more stable molecule, and has much better developability in terms of developing a drug for cancer patients.

[0642] Table 34: Comparison of Lead BsAbs with Reference BsAbs in Whole Cell Binding, Human PD1 / PD-L1 Blockade, Human VEGFA Blockade, and SEC-HPLC Assays

[0643]

[0644] No = no activity.

[0645] *Not as Fig.25 Good nonlinear regression fit shown.

[0646] Example 12: In vivo efficacy study of lead anti-PD-L1 x anti-VEGF BsAb using MC38-hPD-L1 murine colon cancer cell model in C57BL / 6N syngeneic mice

[0647] MC38-hPD-L1 (MC38 CD274 ) is a murine colon cancer cell line (MC38) engineered to express the human PD-L1 gene (CD274) by knocking out the mouse PD-L1 gene and knocking in the human PD-L1 (hPD-L1) transgene into the mouse PD-L1 locus. Using C57BL / 6N mice with the MC38-hPD-L1 murine colon cancer model, the efficacy of anti-PD-L1 x anti-VEGF BsAb 1F4-hv7-NT_Fc_7H3 was evaluated and compared with the efficacy of monospecific antibodies 1F4-hv7-NT_Fc (anti-PD-L1), 7H3_Fc (anti-VEGF) and reference BsAb IMM2510. MC38-hPD-L1 tumor cells (1x 10 6 The average tumor volume reached about 105 mm 3Treatment was started at 6 mg / kg or an equivalent molar amount three times a week. The drug was administered intraperitoneally for 3 weeks. Statistical analysis of tumor volume (by two-way ANOVA and Tukey's multiple comparison test) showed that all treatment groups significantly inhibited tumor growth compared with the negative control (inactive anti-PD-L1 variant 5F8-hv2) (Table 35). Compared with the negative control, both monospecific antibodies 1F4-hv7-NT_Fc and 7H3_Fc significantly reduced tumor growth, with tumor growth inhibition rates (TGI) of 24.25% and 30.33%, respectively, on day 28 after treatment began. Fig.26A and Table 35). Compared with the monospecific antibodies, BsAb 1F4-hv7-NT_Fc_7H3 showed stronger efficacy, with a TGI of 81.32% at day 28 after treatment. It was also observed that BsAb 1F4-hv7-NT_Fc_7H3 had a significant increase in tumor inhibition compared to the reference BsAb IMM2510, P<0.013 (TGI: 81.32% vs. 55.62%).

[0648] No significant changes in body weight were observed in mice from the different study groups, suggesting that the drug was well tolerated ( Fig.26B ). Fig. 27 Individual tumor volume data for each treatment group are shown in .

[0649] Table 35: Summary of efficacy of the M38-PD-L1 tumor model in C57BL / 6N syngeneic mice

[0650]

[0651] a Mean ± SEM

[0652] b Comparison with negative control

[0653] c Compare with 1F4-hv7-NT_Fc or 7H3_Fc.

[0654] d Compare with IMM2510.

[0655] Statistical analysis was performed by two-way ANOVA and Tukey's multiple comparison test.

[0656] Other embodiments

[0657] It should be understood that although the invention has been described in conjunction with specific embodiments of the invention, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

1. An antibody or an antigen-binding fragment thereof, which binds to programmed death-ligand 1 (PD-L1), wherein the antibody or the antigen-binding fragment thereof comprises: a heavy chain antibody variable domain (VHH), said VHH comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein said VHH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR1 amino acid sequence, said VHH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR2 amino acid sequence, and said VHH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR3 amino acid sequence; wherein the selected VHH CDR 1, 2 and 3 amino acid sequences are as described in one of the following: (1) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 96, 97 and 98, respectively; (2) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 99, 100 and 101, respectively; (3) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 102, 103 and 104, respectively; (4) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 105, 106 and 107, respectively; (5) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 108, 109 and 110, respectively; (6) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 111, 112 and 113, respectively; (7) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 114, 115 and 116, respectively; (8) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 117, 118 and 119, respectively; (9) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 120, 121, and 122, respectively; (10) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 123, 124 and 125, respectively; (11) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 126, 127, and 128, respectively; (12) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 129, 130 and 131, respectively; (13) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 132, 133, and 134, respectively; (14) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 135, 136 and 137, respectively; (15) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 138, 139 and 140, respectively; (16) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 141, 142 and 143, respectively; (17) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 144, 145 and 146, respectively; (18) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 147, 148 and 149, respectively; (19) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 150, 151 and 152, respectively; (20) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 153, 154 and 155, respectively; (21) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 156, 157 and 158, respectively; (22) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 159, 160 and 161, respectively; (23) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 162, 163 and 164, respectively; (24) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 165, 166 and 167, respectively; (25) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 168, 169 and 170, respectively; (26) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 171, 172 and 173, respectively; (27) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 174, 175 and 176, respectively; (28) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 177, 178 and 179, respectively; (29) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 180, 181 and 182, respectively; and (30) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 183, 184 and 185, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VHH comprises CDRs 1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 183, 184 and 185, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VHH comprises CDRs 1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 171, 172, and 173, respectively.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VHH comprises CDRs 1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 177, 178 and 179, respectively.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VHH comprises CDRs 1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 180, 181 and 182, respectively.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VHH comprises CDRs 1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 132, 133, and 134, respectively.

7. An antibody or antigen-binding fragment thereof, which binds to PD-L1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain antibody variable domain (VHH), wherein the VHH comprises an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is selected from the group consisting of SEQ ID NO: 1-80.

8. The antibody or antigen-binding fragment thereof according to claim 8, wherein the VHH comprises the sequence of SEQ ID NO:

79.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the VHH comprises the sequence of SEQ ID NO:

60.

10. The antibody or antigen-binding fragment thereof according to claim 8, wherein the VHH comprises the sequence of SEQ ID NO:

68.

11. The antibody or antigen-binding fragment thereof according to claim 8, wherein the VHH comprises the sequence of SEQ ID NO:

74.

12. The antibody or antigen-binding fragment thereof according to claim 8, wherein the VHH comprises the sequence of SEQ ID NO:

78.

13. The antibody or antigen-binding fragment thereof according to claim 8, wherein the VHH comprises the sequence of SEQ ID NO:

33.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the antibody or antigen-binding fragment specifically binds to PD-L1.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof. 16 . An antibody or an antigen-binding fragment thereof, comprising the VHH CDRs 1, 2, and 3 of the antibody or antigen-binding fragment thereof according to claim 1 .

17. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the antibody or antigen-binding fragment comprises human IgG Fc.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, wherein the antibody or antigen-binding fragment comprises two or more heavy chain antibody variable domains.

19. An antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18.

20. An antibody or an antigen-binding fragment thereof, which binds to VEGF (vascular endothelial growth factor), wherein the antibody or the antigen-binding fragment thereof comprises: a heavy chain antibody variable domain (VHH), said VHH comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein said VHH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR1 amino acid sequence, said VHH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR2 amino acid sequence, and said VHH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR3 amino acid sequence; wherein the selected VHH CDR 1, 2 and 3 amino acid sequences are as described in one of the following: (1) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 186, 187 and 188, respectively; (2) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 189, 190 and 191, respectively; (3) the selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 192, 193 and 194, respectively; and (4) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 195, 196 and 197, respectively.

21. The antibody or antigen-binding fragment thereof according to claim 20, wherein the VHH comprises CDRs 1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 186, 187 and 188, respectively.

22. The antibody or antigen-binding fragment thereof according to claim 20, wherein the VHH comprises CDRs 1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 189, 190 and 191, respectively.

23. The antibody or antigen-binding fragment thereof according to claim 20, wherein the VHH comprises CDRs 1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 192, 193 and 194, respectively.

24. The antibody or antigen-binding fragment thereof according to claim 20, wherein the VHH comprises CDRs 1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 195, 196 and 197, respectively.

25. An antibody or antigen-binding fragment thereof, which binds to VEGF, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain antibody variable domain (VHH), wherein the VHH comprises an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is selected from the group consisting of SEQ ID NOs: 81-84.

26. The antibody or antigen-binding fragment thereof of claim 25, wherein the VHH comprises the sequence of SEQ ID NO:

81.

27. The antibody or antigen-binding fragment thereof of claim 25, wherein the VHH comprises the sequence of SEQ ID NO:

82.

28. The antibody or antigen-binding fragment thereof of claim 25, wherein the VHH comprises the sequence of SEQ ID NO:

83.

29. The antibody or antigen-binding fragment thereof of claim 25, wherein the VHH comprises the sequence of SEQ ID NO:

84.

30. The antibody or antigen-binding fragment thereof according to any one of claims 20 to 29, wherein the antibody or antigen-binding fragment specifically binds to VEGF.

31. The antibody or antigen-binding fragment thereof according to any one of claims 20 to 30, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.

32. An antibody or antigen-binding fragment thereof, comprising the VHH CDRs 1, 2, 3 of the antibody or antigen-binding fragment thereof according to any one of claims 20 to 31.

33. The antibody or antigen-binding fragment thereof according to any one of claims 20 to 32, wherein the antibody or antigen-binding fragment comprises human IgG Fc.

34. The antibody or antigen-binding fragment thereof according to any one of claims 20 to 33, wherein the antibody or antigen-binding fragment comprises two or more heavy chain antibody variable domains.

35. An antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof according to any one of claims 20 to 34.

36. A multispecific antibody or an antigen-binding fragment thereof, comprising a first VHH (VHH1) that specifically binds to VEGF and a second VHH (VHH2) that specifically binds to PD-L1.

37. The multispecific antibody or antigen-binding fragment thereof according to claim 36, further comprising a third VHH (VHH3) that specifically binds to VEGF and a fourth VHH (VHH4) that specifically binds to PD-L1.

38. A multispecific antibody or antigen-binding fragment thereof according to claim 36 or 37, wherein the VHH1 and / or the VHH3 comprises complementarity determining regions (CDR) 1, 2 and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected CDR3 amino acid sequence; wherein the selected CDR 1, 2 and 3 amino acid sequences are listed in Figure 28.

39. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 36 to 38, wherein the VHH1 and / or the VHH3 comprises an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is selected from the group consisting of SEQ ID NO: 1-80.

40. The multispecific antibody or antigen-binding fragment thereof of any one of claims 36 to 39, wherein the VHH2 and / or the VHH4 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected CDR3 amino acid sequence; wherein the selected CDR 1, 2, and 3 amino acid sequences are listed in Figure 29.

41. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 36 to 40, wherein the VHH3 and the VHH4 comprise an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is selected from the group consisting of SEQ ID NOs: 81-84.

42. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 36 to 41, wherein the multispecific antibody or antigen-binding fragment thereof comprises human IgG Fc.

43. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 36 to 42, wherein the VHH1 and the VHH3 are linked to the N-terminus or the C-terminus of the human IgG Fc.

44. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 36 to 43, wherein the VHH2 and the VHH4 are linked to the N-terminus or the C-terminus of the human IgG Fc.

45. A polypeptide complex comprising (a) a first polypeptide, which comprises, from N-terminus to C-terminus: a first heavy chain antibody variable domain (VHH1), a first hinge region, a first Fc region, and a second VHH (VHH2); and (b) a second polypeptide, which comprises, from N-terminus to C-terminus: a third VHH (VHH3), a second hinge region, a second Fc region and a fourth VHH (VHH4), wherein the VHH1 and the VHH3 specifically bind to PD-L1, and the VHH2 and the VHH4 specifically bind to VEGF.

46. ​​The polypeptide complex of claim 45, wherein the first polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:85, 89, 90, 91, 92, 93 or 95; and / or wherein the second polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:85, 89, 90, 91, 92, 93 or 95. The polypeptide complex according to claim 45 or 46, wherein the VHH2 is connected to the C-terminus of the first Fc region via a first linker peptide sequence.

48. The polypeptide complex according to any one of claims 45 to 47, wherein the VHH4 is linked to the C-terminus of the second Fc region via a second linker peptide sequence.

49. The polypeptide complex according to claim 47 or 48, wherein the first linker peptide sequence and / or the second linker peptide sequence is at least 80% identical to SEQ ID NO: 202 or 203.

50. A polypeptide complex comprising (a) a first polypeptide, which comprises, from N-terminus to C-terminus: VHH1, VHH2, a first hinge region, and a first Fc region; and (b) a second polypeptide, which comprises, from N-terminus to C-terminus: VHH3, VHH4, a second hinge region, and a second Fc region, wherein the VHH1 and the VHH3 specifically bind to PD-L1, and the VHH2 and the VHH4 specifically bind to VEGF. The polypeptide complex according to claim 50 , wherein the VHH1 is connected to the N-terminus of the VHH2 via a first linker peptide sequence.

52. The polypeptide complex according to claim 50 or 51, wherein the VHH3 is connected to the N-terminus of the VHH4 via a second linker peptide sequence.

53. The polypeptide complex of any one of claims 50 to 52, wherein the first polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 86 or 94; and / or wherein the second polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 86 or 94.

54. The polypeptide complex according to any one of claims 51 to 53, wherein the first linker peptide sequence and / or the second linker peptide sequence is at least 80% identical to SEQ ID NO: 202 or 203.

55. A polypeptide complex comprising (a) a first polypeptide, which comprises, from N-terminus to C-terminus: VHH2, VHH1, a first hinge region, and a first Fc region; and (b) a second polypeptide, which comprises, from N-terminus to C-terminus: VHH4, VHH3, a second hinge region, and a second Fc region, wherein the VHH1 and the VHH3 specifically bind to PD-L1, and the VHH2 and the VHH4 specifically bind to VEGF. The polypeptide complex according to claim 55 , wherein the VHH2 is connected to the N-terminus of the VHH1 via a first linker peptide sequence.

57. The polypeptide complex according to claim 55 or 56, wherein the VHH4 is connected to the N-terminus of the VHH3 via a second linker peptide sequence.

58. The polypeptide complex of any one of claims 55 to 57, wherein the first polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 87; and / or wherein the second polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:

87.

59. The polypeptide complex according to any one of claims 56 to 58, wherein the first linker peptide sequence and / or the second linker peptide sequence is at least 80% identical to SEQ ID NO: 202 or 203.

60. A polypeptide complex comprising (a) a first polypeptide, which comprises, from N-terminus to C-terminus: VHH2, a first hinge region, a first Fc region and VHH1; and (b) a second polypeptide, which comprises, from N-terminus to C-terminus: VHH4, a second hinge region, a second Fc region and VHH3, wherein the VHH1 and the VHH3 specifically bind to PD-L1, and the VHH2 and the VHH4 specifically bind to VEGF.

61. The polypeptide complex of claim 60, wherein the first polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:88; and / or wherein the second polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:

88. The polypeptide complex according to claim 60 or 61 , wherein the VHH1 is connected to the C-terminus of the first Fc region via a first linker peptide sequence.

63. The polypeptide complex according to any one of claims 60 to 62, wherein the VHH3 is linked to the C-terminus of the second Fc region via a second linker peptide sequence.

64. The polypeptide complex according to claim 62 or 63, wherein the first linker peptide sequence and / or the second linker peptide sequence is at least 80% identical to SEQ ID NO: 202 or 203.

65. A polypeptide complex according to any one of claims 45 to 64, wherein the VHH1 and / or the VHH3 comprises complementarity determining regions (CDR) 1, 2 and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected CDR3 amino acid sequence; wherein the selected CDR 1, 2 and 3 amino acid sequences are listed in Figure 28.

66. The polypeptide complex according to any one of claims 45 to 65, wherein the VHH1 and / or the VHH3 comprises an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is selected from the group consisting of SEQ ID NO: 1-80.

67. A polypeptide complex according to any one of claims 45 to 66, wherein the VHH2 and / or the VHH4 comprises complementarity determining regions (CDR) 1, 2 and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected CDR3 amino acid sequence; wherein the selected CDR 1, 2 and 3 amino acid sequences are listed in Figure 29.

68. The polypeptide complex according to any one of claims 45 to 67, wherein the VHH2 and / or the VHH4 comprises an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is selected from the group consisting of SEQ ID NOs: 81-84.

69. An antibody or antigen-binding fragment thereof, which binds to programmed death-ligand 1 (PD-L1), wherein the antibody or antigen-binding fragment thereof comprises: A heavy chain antibody variable domain (VHH), the VHH comprising complementarity determining regions (CDR) 1, 2 and 3, wherein the VHH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR1 amino acid sequence, the VHH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR2 amino acid sequence, and the VHH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR3 amino acid sequence, wherein the selected VHH CDR 1, 2 and 3 amino acid sequences are as described in one of the following: (1) The selected VHH CDR3 is NARX1TTIY (SEQ ID NO: 198), wherein X1 = K, T or R; the selected VHH CDR1 is selected from the group consisting of: SEQ ID NO: 96, 99, 102, 105, 108, 111, 126 and 129; and the selected VHH CDR2 is selected from the group consisting of: SEQ ID NO: 97, 100, 103, 106, 109, 112, 127 and 130; (2) The selected VHH CDR3 is NALVWX2GSSYNN (SEQ ID NO: 199), wherein X2=Q, T, S or N; the selected VHH CDR1 is selected from the group consisting of: SEQ ID NO: 114, 117, 120, 123, 168, 171, 174, 177, 180 and 183; and the selected VHH CDR2 is selected from the group consisting of: SEQ ID NO: 115, 118, 121, 124, 169, 172, 175, 178, 181 and 184; and (3) the selected VHH CDR3 is selected from the group consisting of SEQ ID NO: 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164 and 167; the selected VHH CDR1 is selected from the group consisting of SEQ ID NO: 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162 and 165; and the selected VHH CDR2 is selected from the group consisting of SEQ ID NO: 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163 and 166.

70. The antibody or antigen-binding fragment thereof of claim 69, wherein the selected VHH CDR 1, 2 and 3 amino acid sequences are as described in one of the following: (1) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 96, 97 and 98, respectively; (2) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 99, 100 and 101, respectively; (3) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 102, 103 and 104, respectively; (4) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 105, 106 and 107, respectively; (5) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 108, 109 and 110, respectively; (6) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 111, 112 and 113, respectively; (7) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 114, 115 and 116, respectively; (8) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 117, 118 and 119, respectively; (9) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 120, 121, and 122, respectively; (10) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 123, 124 and 125, respectively; (11) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 126, 127, and 128, respectively; (12) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 129, 130 and 131, respectively; (13) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 132, 133, and 134, respectively; (14) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 135, 136 and 137, respectively; (15) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 138, 139 and 140, respectively; (16) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 141, 142 and 143, respectively; (17) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 144, 145 and 146, respectively; (18) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 147, 148 and 149, respectively; (19) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 150, 151 and 152, respectively; (20) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 153, 154 and 155, respectively; (21) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 156, 157 and 158, respectively; (22) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 159, 160 and 161, respectively; (23) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 162, 163 and 164, respectively; (24) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 165, 166 and 167, respectively; (25) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 168, 169 and 170, respectively; (26) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 171, 172 and 173, respectively; (27) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 174, 175 and 176, respectively; (28) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 177, 178 and 179, respectively; (29) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 180, 181 and 182, respectively; and (30) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 183, 184 and 185, respectively.

71. A nucleic acid comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 35, 69 and 70, the multispecific antibody or antigen-binding fragment thereof according to any one of claims 36 to 44, or the polypeptide complex according to any one of claims 45 to 68.

72. The nucleic acid of claim 71, wherein the nucleic acid is DNA (eg, cDNA) or RNA (eg, mRNA).

73. A vector comprising one or more nucleic acids of the nucleic acid according to claim 71 or 72.

74. A cell comprising the vector of claim 73.

75. The cell of claim 74, wherein the cell is a CHO cell.

76. A cell comprising one or more nucleic acids of the nucleic acid of claim 71 or 72.

77. A method for producing an antibody or an antigen-binding fragment thereof, the method comprising (c) culturing the cell under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment according to any one of claims 74 to 76; and (d) collecting the antibody or the antigen-binding fragment produced by the cell.

78. An antibody drug conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 35, 69 and 70, the multispecific antibody or antigen-binding fragment thereof according to any one of claims 36 to 44, or the polypeptide complex according to any one of claims 45 to 68, wherein the antibody drug conjugate is covalently bound to a therapeutic agent.

79. The antibody drug conjugate of claim 78, wherein the therapeutic agent is a cytotoxic agent or a cytostatic agent.

80. A method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 35, 69 and 70, the multispecific antibody or antigen-binding fragment thereof according to any one of claims 36 to 44, or the polypeptide complex according to any one of claims 45 to 68, or the antibody drug conjugate according to claims 78 or 79.

81. The method of claim 80, wherein the subject has a cancer that expresses PD-L1.

82. The method of claim 80 or 81, wherein the subject has a cancer that expresses VEGF.

83. The method of any one of claims 80 to 82, wherein the cancer is colon cancer, rectal cancer, lung cancer, breast cancer, kidney cancer, hepatocellular carcinoma, renal cancer, endometrial cancer, pancreatic cancer, head and neck cancer, or an advanced solid tumor.

84. The method of claim 83, wherein the cancer is non-small cell lung cancer (NSCLC).

85. A method for reducing tumor growth rate, the method comprising The tumor cells are contacted with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 35, 69 and 70, the multispecific antibody or antigen-binding fragment thereof according to any one of claims 36 to 44, or the polypeptide complex according to any one of claims 45 to 68, or the antibody drug conjugate according to claim 78 or 79.

86. A method for killing tumor cells, the method comprising The tumor cells are contacted with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 35, 69 and 70, the multispecific antibody or antigen-binding fragment thereof according to any one of claims 36 to 44, or the polypeptide complex according to any one of claims 45 to 68, or the antibody drug conjugate according to claim 78 or 79.

87. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 35, 69 and 70, the multispecific antibody or antigen-binding fragment thereof according to any one of claims 36 to 44, the polypeptide complex according to any one of claims 45 to 68, or the antibody-drug conjugate according to claim 78 or 79, and a pharmaceutically acceptable carrier.

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