Bispecific antibodies and uses thereof

Abstract

To provide bispecific antibodies or antigen-binding fragments for the development of various therapeutics, and to provide methods for producing bispecific antibodies or antigen-binding fragments.SOLUTION: The present disclosure relates to bispecific antibodies or antigen-binding fragments thereof, wherein the bispecific antibodies or antigen-binding fragments thereof specifically bind to two different antigens with different binding affinities.SELECTED DRAWING: Figure 28

Description

Technical Field

[0001] Priority Claim This application claims the benefit of U.S. Provisional Patent Application No. 62 / 539,970, filed Aug. 1, 2017, and U.S. Provisional Patent Application No. 62 / 654,112, filed Apr. 6, 2018. The entire contents of the foregoing are incorporated herein by reference. <F

[0002] Technical Field The present disclosure relates to bispecific antibodies or antigen-binding fragments thereof.

Background Art

[0003] Background A bispecific antibody is an artificial protein that can simultaneously bind to two different antigens or two different epitopes. This dual specificity opens up a wide range of applications, including redirecting T cells to tumor cells, simultaneously blocking two different signaling pathways, dual targeting of different disease mediators, and delivery of payloads to target sites. The approval of catumaxomab (anti-EpCAM and anti-CD3) and blinatumomab (anti-CD19 and anti-CD3) has been a major milestone in the development of bispecific antibodies.

[0004] Since bispecific antibodies have various applications, it is necessary to continue the development of various therapies based on bispecific antibodies.

Summary of the Invention

[0005] Summary The present disclosure relates to an asymmetric bispecific antibody or antigen-binding fragment thereof, in which the bispecific antibody or antigen-binding fragment specifically binds to two different antigens with different binding affinities.

[0006] In some embodiments, the present disclosure relates to a bispecific antibody or antigen-binding fragment comprising a first heavy chain variable region, a second heavy chain variable region, a first light chain variable region, and a second light chain variable region, wherein the first heavy chain variable region and the first light chain variable region bind to each other to form a first antigen-binding region that specifically binds to a first antigen with a binding affinity higher than 10 7 M -1 、10 8 M -1 、10 9 M -1 、10 10 M -1 、10 11 M -1 、or 10 12 M -1 and the second heavy chain variable region and the second light chain variable region bind to each other to form a second antigen-binding region that specifically binds to a second antigen with a binding affinity lower than 10 9 M -1 、10 8 M -1 、10 7 M -1 、10 6 M -1 、10 5 M -1 、or 10 4 M -1 .

[0007] In some embodiments, the second antigen-binding region specifically binds to the second antigen with a binding affinity higher than 10 7 M -1 、10 6 M -1 、10 5 M -1 、or 10 4 M -1 .

[0008] In some embodiments, the binding affinity of the first antigen-binding region when binding to the first antigen is at least 100-fold, 1000-fold, or 10000-fold higher than the binding affinity of the second antigen-binding region when binding to the second antigen.

[0009] In some embodiments, the first light chain variable region and the second light chain variable region are at least 90%, 95%, 99%, or 100% identical.

[0010] In some aspects, the disclosure relates to a bispecific antibody or antigen-binding fragment thereof comprising a first arm comprising a first heavy chain variable region and a first light chain variable region, and a second arm comprising a second heavy chain variable region and a second light chain variable region, wherein the first arm specifically binds to a first antigen with a binding affinity higher than 10 7 M -1 、10 8 M -1 、10 9 M -1 、10 10 M -1 、10 11 M -1 、10 12 M -1 、and the second arm specifically binds to a second antigen with a binding affinity lower than 10 9 M -1 、10 8 M -1 、10 7 M -1 、10 6 M -1 、10 5 M -1 、or 10 4 M -1 .

[0011] In some embodiments, the second arm specifically binds to the second antigen with a binding affinity higher than 10 7 M -1 、10 6 M -1 、10 5 M -1 、or 10 4 M -1 .

[0012] In some embodiments, the binding affinity of the first arm when binding to the first antigen is at least 100-fold, 1000-fold, or 10,000-fold higher than the binding affinity of the second arm when binding to the second antigen.

[0013] In some embodiments, the first light chain variable region and the second light chain variable region are at least 90%, 95%, 99%, or 100% identical.

[0014] In some aspects, the present disclosure relates to a bispecific antibody or antigen-binding fragment thereof comprising a first heavy chain comprising a first heavy chain variable region, a second heavy chain comprising a second heavy chain variable region, a first light chain comprising a first light chain variable region, and a second light chain comprising a second light chain variable region, wherein the first heavy chain variable region and the first light chain variable region bind to each other to form a first antigen-binding region that specifically binds to a first antigen with a binding affinity higher than 10 7 M -1 、10 8 M -1 、10 9 M -1 、10 10 M -1 、10 11 M -1 、10 12 M -1 M, 10 9 M -1 、10 8 M -1 、10 7 M -1 、10 6 M -1 、10 5 M -1 、or 10 4 M -1 M or lower, and the second heavy chain variable region and the second light chain variable region bind to each other to form a second antigen-binding region that specifically binds to a second antigen with a binding affinity lower than 10

[0015] In some embodiments, the second antigen-binding region specifically binds to the second antigen with a binding affinity higher than 10 7 M -1 、10 6 M -1 、10 5 M -1 、or 4 M -1 M or higher.

[0016] In some embodiments, the binding affinity of the first antigen-binding region when binding to the first antigen is at least 100-fold, 1000-fold, or 10,000-fold higher than the binding affinity of the second antigen-binding region when binding to the second antigen.

[0017] In some embodiments, the first light chain and the second light chain are at least 90%, 95%, 99%, or 100% identical.

[0018] In some embodiments, the first heavy chain and the second chain bind to each other by the "knobs into holes" method.

[0019] In some embodiments, the first antigen is a cancer-specific antigen and the second antigen is CD3.

[0020] In some embodiments, the first antigen is CD20 and the second antigen is CD3.

[0021] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:1, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:2, and the first and second light chain variable regions comprise a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:3.

[0022] In some embodiments, the first antigen is a cancer-specific antigen and the second antigen is a cancer-related antigen.

[0023] In some embodiments, the first antigen is PD-L1 and the second antigen is CD55.

[0024] In some embodiments, the first heavy-chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:4, the second heavy-chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:5, and the first and second light-chain variable regions comprise a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:6 or SEQ ID NO:7.

[0025] In some aspects, the present disclosure relates to a method of making a bispecific antibody or antigen-binding fragment, the method comprising: selecting a first antigen and a second antigen and identifying a first antibody or antigen-binding fragment that binds to the first antigen and a second antibody or antigen-binding fragment that binds to the second antigen, wherein the first antibody or antigen-binding fragment comprises a first heavy-chain variable region (VHa) and a first light-chain variable region (VLa), and the second antibody or antigen-binding fragment comprises a second heavy-chain variable region (VHb) and a second light-chain variable region (VLb); determining the amino acid sequences of VHa, VLa, VHb, and VLb; aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is higher than 80%; designing a common light-chain variable region (VLc) that maintains the affinity for the first antigen when binding to VHa; redesigning the VHa and VHb sequences to obtain VHa' and VHb' that increase the difference in biochemical or biophysical properties between a first protein comprising two polypeptides each comprising VHa' and two polypeptides each comprising VLc and a second protein comprising two polypeptides each comprising VHb' and two polypeptides each comprising VLc; and Producing a bispecific antibody or antigen-binding fragment having two light chain variable regions and two heavy chain variable regions, wherein each of the two light chain variable regions comprises VLc, and the two heavy chain variable regions each comprise VHa’ and VHb’, said step comprising.

[0026] In some embodiments, in step (d), the binding affinity of VLc-VHb for the second antigen may be reduced.

[0027] In some embodiments, the method further comprises creating a buffer system for purifying the bispecific antibody or antigen-binding fragment.

[0028] In some aspects, the present disclosure relates to a method of making a bispecific antibody or antigen-binding fragment, the method comprising selecting a first antigen and a second antigen and identifying a first antibody or antigen-binding fragment that binds to the first antigen and a second antibody or antigen-binding fragment that binds to the second antigen, wherein the first antibody or antigen-binding fragment comprises a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or antigen-binding fragment comprises a second heavy chain variable region (VHb) and a second light chain variable region (VLb), said step; determining the amino acid sequences of VHa, VLa, and VLb; aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%; replacing all light chain variable regions in a phage display antibody library with VLa and panning against the second antigen to obtain a third heavy chain variable region (VHc); Redesign the VHa and VHc sequences, thereby obtaining VHa' and VHc' that increase the difference in biochemical or biophysical properties between a first protein comprising two polypeptides each comprising VHa' and two polypeptides each comprising VLa, and a second protein comprising two polypeptides each comprising VHc' and two polypeptides each comprising VLa; and Producing a bispecific antibody or antigen-binding fragment having two light chain variable regions and two heavy chain variable regions, each of the two light chain variable regions comprising VLa, and the two heavy chain variable regions comprising VHa' and VHc', respectively, said step comprising.

[0029] In some embodiments, the method further comprises creating a buffer system for purifying the bispecific antibody or antigen-binding fragment.

[0030] In some aspects, the present disclosure relates to a method of making a bispecific antibody or antigen-binding fragment, the method comprising: Selecting a first antigen and a second antigen and identifying a first antibody or antigen-binding fragment that binds to the first antigen and a second antibody or antigen-binding fragment that binds to the second antigen, wherein the first antibody or antigen-binding fragment comprises a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or antigen-binding fragment comprises a second heavy chain variable region (VHb) and a second light chain variable region (VLb), said step; Determining the amino acid sequences of VHa, VLa, VHb, and VLb; Aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%; Replacing all light chain variable regions in a phage display antibody library with a plurality of light chain variable regions that are at least 80%, 85%, 90%, 95%, or 99% identical to VLa or VLb, said step; Panning against the second antigen; Selecting a common light chain variable region (VLc) and a third heavy chain variable region (VHc), wherein VHa-VLc binds to a first antigen with a desired affinity and VHc-VLc binds to a second antigen with a desired affinity; Redesigning the VHa and VHc sequences, thereby obtaining VHa' and VHc' that increase the difference in biochemical or biophysical properties between a first protein comprising two polypeptides each comprising VHa' and two polypeptides each comprising VLc, and a second protein comprising two polypeptides each comprising VHc' and two polypeptides each comprising VLc; and Producing a bispecific antibody or antigen-binding fragment having two light chain variable regions and two heavy chain variable regions, each of the two light chain variable regions comprising VLc and the two heavy chain variable regions each comprising VHa' and VHc' comprising.

[0031] In some embodiments, in step (d), the plurality of light chain variable regions are produced by error-prone PCR.

[0032] In some embodiments, the method further comprises creating a buffer system for purifying the bispecific antibody or antigen-binding fragment.

[0033] In one aspect, the present disclosure provides a method of making a bispecific antibody or antigen-binding fragment thereof. The method includes one or more of the following steps: (a) Selecting a first antigen and a second antigen and identifying a first antibody or antigen-binding fragment thereof that binds to the first antigen and a second antibody or antigen-binding fragment thereof that binds to the second antigen. In some embodiments, the first antibody or antigen-binding fragment thereof comprises a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or antigen-binding fragment thereof comprises a second heavy chain variable region (VHb) and a second light chain variable region (VLb); (b) determining the amino acid sequences of VHa, VLa, VHb, and VLb; (c) aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%; (d) replacing all light chain variable regions in a phage display antibody library with a plurality of light chain variable regions. In some embodiments, the light chain variable regions are at least 80%, 85%, 90%, 95%, or 99% identical to VLa or VLb; (e) panning against a second antigen; (f) selecting a common light chain variable region (VLc) and a third heavy chain variable region (VHc). In some embodiments, VHc-VLc binds to the second antigen with a desired affinity; (g) determining that the homology between VLa and VLc is higher than 80%; (h) designing a common light chain variable region (VLd). In some embodiments, VLd maintains the affinity for the first antigen when binding to VHa and has the desired affinity for the second antigen when binding to VHc; (i) optionally, redesigning the VHa and VHc sequences, thereby obtaining VHa' and VHc' that increase the difference in biochemical or biophysical properties between a first protein comprising two polypeptides each comprising VHa' and two polypeptides each comprising VLd and a second protein comprising two polypeptides each comprising VHc' and two polypeptides each comprising VLd; and (j) optionally, producing a bispecific antibody or an antigen-binding fragment thereof having two light chain variable regions and two heavy chain variable regions. In some embodiments, each of the two light variable regions comprises VLd and the two heavy chain variable regions each comprise VHa' and VHc'.

[0034] In one aspect, the present disclosure provides a method for producing a bispecific antibody or an antigen-binding fragment thereof. This method includes one or more of the following steps: (a) Select a first antigen and a second antigen, and identify a first antibody or an antigen-binding fragment thereof that binds to the first antigen and a second antibody or an antigen-binding fragment thereof that binds to the second antigen. In some embodiments, the first antibody or an antigen-binding fragment thereof comprises a first heavy-chain variable region (VHa) and a first light-chain variable region (VLa), and the second antibody or an antigen-binding fragment thereof comprises a second heavy-chain variable region (VHb) and a second light-chain variable region (VLb); (b) Determine the amino acid sequences of VHa, VLa, VHb, and VLb; (c) Align the amino acid sequences of VLa and VLb and determine that the sequence homology between VLa and VLb is higher than 80%; (d) Design a common light-chain variable region (VLc). In some embodiments, VLc maintains the affinity for the first antigen when binding to VHa; and (e) Optionally, produce a bispecific antibody or an antigen-binding fragment thereof having two light-chain variable regions and two heavy-chain variable regions. In some embodiments, each of the two light variable regions comprises VLc, and the two heavy-chain variable regions comprise VHa and VHb, respectively.

[0035] In one aspect, the present disclosure also provides a method of producing a bispecific antibody or an antigen-binding fragment thereof. This method includes one or more of the following steps: (a) Select a first antigen and a second antigen, and identify a first antibody or an antigen-binding fragment thereof that binds to the first antigen and a second antibody or an antigen-binding fragment thereof that binds to the second antigen. In some embodiments, the first antibody or an antigen-binding fragment thereof comprises a first heavy-chain variable region (VHa) and a first light-chain variable region (VLa), and the second antibody or an antigen-binding fragment thereof comprises a second heavy-chain variable region (VHb) and a second light-chain variable region (VLb); (b) Determine the amino acid sequences of VHa, VLa, and VLb; (c) Aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%; (d) Replacing all light chain variable regions in the phage display antibody library with VLa and panning against the second antigen to obtain a third heavy chain variable region (VHc); and (e) Optionally, producing a bispecific antibody or an antigen-binding fragment thereof having two light chain variable regions and two heavy chain variable regions. In some embodiments, each of the two light variable regions comprises VLa, and the two heavy chain variable regions each comprise VHa and VHc.

[0036] In one aspect, the present disclosure further provides a method of making a bispecific antibody or an antigen-binding fragment thereof. The method includes one or more of the following steps: (a) Selecting a first antigen and a second antigen and identifying a first antibody or an antigen-binding fragment thereof that binds to the first antigen and a second antibody or an antigen-binding fragment thereof that binds to the second antigen. In some embodiments, the first antibody or an antigen-binding fragment thereof comprises a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or an antigen-binding fragment thereof comprises a second heavy chain variable region (VHb) and a second light chain variable region (VLb); (b) Determining the amino acid sequences of VHa, VLa, VHb, and VLb; (c) Aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%; (d) Replacing all light chain variable regions in the phage display antibody library with a plurality of light chain variable regions. In some embodiments, the light chain variable region is at least 80%, 85%, 90%, 95%, or 99% identical to VLa or VLb; (e) Panning against the first and / or second antigen; (f) Selecting a common light chain variable region (VLc) and a third heavy chain variable region (VHc). In some embodiments, VHa-VLc binds to a first antigen with a desired affinity, and VHc-VLc binds to a second antigen with a desired affinity; and, (g) Optionally, producing a bispecific antibody or an antigen-binding fragment thereof having two light chain variable regions and two heavy chain variable regions. In some embodiments, each of the two light variable regions comprises VLc, and the two heavy chain variable regions each comprise VHa and VHc.

[0037] In another aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to CD3, which is a heavy chain variable region (VH) comprising complementarity determining regions (CDR) 1, 2, 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and, is a light chain variable region (VL) comprising CDR1, 2, 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence wherein the selected VH CDR1, 2, 3 amino acid sequences and the selected VL CDR, 1, 2, 3 amino acid sequences are one of the following: The selected VH CDR1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22-24, respectively, and the selected VL CDR1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 28-30, respectively.

[0038] In some embodiments, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NO: 22, 23, and 24, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NO: 28, 29, and 30, respectively.

[0039] In some embodiments, the antibody or antigen-binding fragment specifically binds to human CD3.

[0040] In some embodiments, the antibody or antigen-binding fragment is a bispecific antibody.

[0041] In another aspect, the present disclosure also provides an antibody or antigen-binding fragment thereof that binds to PD-L1, which is a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and is a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence and wherein the selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR 1, 2, and 3 amino acid sequences are one of the following: (1) The amino acid sequences of the selected VH CDR1, 2, and 3 are set forth in SEQ ID NOs: 41-43, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are set forth in SEQ ID NOs: 53-55, respectively; (2) The amino acid sequences of the selected VH CDR1, 2, and 3 are set forth in SEQ ID NOs: 41-43, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are set forth in SEQ ID NOs: 59-61, respectively.

[0042] In some embodiments, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 41-43, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 59-61, respectively.

[0043] In some embodiments, the antibody or antigen-binding fragment specifically binds to human CD3. In some embodiments, the antibody or antigen-binding fragment is a bispecific antibody.

[0044] In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to CD55, which is a heavy chain variable region (VH) comprising complementarity-determining regions (CDR) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR3 amino acid sequence; and A light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence, said light chain variable region (VL) comprising, wherein the amino acid sequences of the selected VH CDR1, 2, 3 and the amino acid sequences of the selected VL CDR1, 2, 3 are one of the following: (1) The amino acid sequences of the selected VH CDR1, 2, 3 are respectively set forth in SEQ ID NO: 47-49, and the amino acid sequences of the selected VL CDR1, 2, 3 are respectively set forth in SEQ ID NO: 53-55; (2) The amino acid sequences of the selected VH CDR1, 2, 3 are respectively set forth in SEQ ID NO: 47-49, and the amino acid sequences of the selected VL CDR1, 2, 3 are respectively set forth in SEQ ID NO: 59-61.

[0045] In some embodiments, VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 47-49 respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 59-61 respectively.

[0046] In some embodiments, the antibody or antigen-binding fragment specifically binds to human CD3.

[0047] In some embodiments, the antibody or antigen-binding fragment is a bispecific antibody.

[0048] In one aspect, the present disclosure provides a nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising VL comprising CDR1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 53 to 55. In some embodiments, VL binds to PD-L1 when paired with VH comprising the amino acid sequence set forth in SEQ ID NO: 4 and / or binds to CD55 when paired with VH comprising the amino acid sequence set forth in SEQ ID NO: 5.

[0049] In one aspect, the present disclosure provides a nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising VL comprising CDR1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 59 to 61. In some embodiments, VL binds to PD-L1 when paired with VH comprising the amino acid sequence set forth in SEQ ID NO: 4 and / or binds to CD55 when paired with VH comprising the amino acid sequence set forth in SEQ ID NO: 5.

[0050] In some embodiments, the nucleic acid encodes a bispecific antibody. In some embodiments, the nucleic acid is cDNA.

[0051] In one aspect, the present disclosure provides a vector comprising one or more of the nucleic acids described herein.

[0052] In one aspect, the present disclosure provides a cell comprising the vector described herein. In some embodiments, the cell is a CHO cell.

[0053] In one aspect, the present disclosure provides a cell comprising one or more of the nucleic acids described herein.

[0054] In one aspect, the present disclosure provides a bispecific antibody or an antigen-binding fragment thereof that binds to CD20 and CD3, comprising a first polypeptide comprising a first heavy-chain variable region (VH) having an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, a second polypeptide comprising a second heavy-chain variable region (VH) having an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2, a third polypeptide comprising a first light-chain variable region (VL) having an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3, and a fourth polypeptide comprising a second light-chain variable region (VL) having an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3.

[0055] In some embodiments, the first heavy-chain variable region (VH) comprises SEQ ID NO:1, the second heavy-chain variable region (VH) comprises SEQ ID NO:2, the first light-chain variable region (VL) comprises SEQ ID NO:3, and the second light-chain variable region (VL) comprises SEQ ID NO:3.

[0056] In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:34, 35, or 36, the second polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:37, 38, or 39, the third polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:40, and the fourth polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:40.

[0057] In some embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:35, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:38.

[0058] In one aspect, the present disclosure provides a bispecific antibody or antigen-binding fragment thereof that binds to PD-L1 and CD55, comprising a first polypeptide comprising a first heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4, a second polypeptide comprising a second heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5, a third polypeptide comprising a first light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6 or 7, and a fourth polypeptide comprising a second light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6 or 7.

[0059] In some embodiments, the first heavy chain variable region (VH) comprises SEQ ID NO:4, the second heavy chain variable region (VH) comprises SEQ ID NO:5, the first light chain variable region (VL) comprises SEQ ID NO:7, and the second light chain variable region (VL) comprises SEQ ID NO:7.

[0060] In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:65, the second polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:66, the third polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:67 or 68, and the fourth polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:67 or 68.

[0061] In some embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:65, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:66, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO:68, and the fourth polypeptide comprises the amino acid sequence shown in SEQ ID NO:68.

[0062] In one aspect, the present disclosure provides an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof described herein covalently linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cytostatic agent.

[0063] In one aspect, the present disclosure provides a method of treating a subject having cancer. The method comprises administering to the subject a therapeutically effective amount of a composition comprising an antibody or an antigen-binding fragment thereof described herein, or an antibody-drug conjugate described herein. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is melanoma, pancreatic cancer, or a hematological malignancy. In some embodiments, the cancer is non-Hodgkin lymphoma, lymphoma, or chronic lymphocytic leukemia.

[0064] In one aspect, the present disclosure provides a method for reducing the rate of tumor growth. The method includes contacting tumor cells of a subject with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof described herein or an antibody-drug conjugate described herein.

[0065] In one aspect, the present disclosure provides a method for killing tumor cells. The method includes contacting tumor cells of a subject with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof described herein or an antibody-drug conjugate described herein.

[0066] In one aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier.

[0067] In one aspect, the present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate described herein and a pharmaceutically acceptable carrier.

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials for use in the present invention are described herein, other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0069] [Invention 1001] A first heavy chain variable region, A second heavy chain variable region, A first light chain variable region, and A second light chain variable region A bispecific antibody or an antigen-binding fragment thereof, comprising The first heavy chain variable region and the first light chain variable region bind to each other to form a first antigen-binding region that specifically binds to a first antigen with a binding affinity higher than 10 7 M -1 、10 8 M -1 、10 9 M -1 、10 10 M -1 、10 11 M -1 、 or 10 12 M -1 and the second heavy chain variable region and the second light chain variable region bind to each other to form a second antigen-binding region that specifically binds to a second antigen with a binding affinity lower than 10 M 9 、10 -1 、10 8 M -1 、10 7 M -1 、10 6 M -1 、10 5 M -1 、 or 10 4 M -1 of the bispecific antibody or an antigen-binding fragment thereof. The bispecific antibody or an antigen-binding fragment thereof according to the present invention 1002. [Invention 1002 of the present invention] The second antigen-binding region specifically binds to a second antigen with a binding affinity higher than 10 7 M -1 、10 6 M -1 、10 5 M -1 、 or 10 4 M -1 of the bispecific antibody or an antigen-binding fragment thereof according to the present invention 1001. [Invention 1003 of the present invention] The binding affinity of the first antigen-binding region when binding to a first antigen is at least 100-fold, 1000-fold, or 10000-fold higher than the binding affinity of the second antigen-binding region when binding to a second antigen, of the bispecific antibody or an antigen-binding fragment thereof according to the present invention 1001. [Invention 1004 of the present invention] The bispecific antibody or antigen-binding fragment thereof of the present invention 1001, wherein the first light chain variable region and the second light chain variable region are at least 90%, 95%, 99%, or 100% identical. [The present invention 1005] A first arm comprising a first heavy chain variable region and a first light chain variable region; and A second arm comprising a second heavy chain variable region and a second light chain variable region A bispecific antibody or antigen-binding fragment thereof, comprising: wherein the first arm specifically binds to a first antigen with a binding affinity higher than 10 7 M -1 、10 8 M -1 、10 9 M -1 、10 10 M -1 、10 11 M -1 、10 12 M -1 and the second arm specifically binds to a second antigen with a binding affinity lower than 10 9 M -1 、10 8 M -1 、10 7 M -1 、10 6 M -1 、10 5 M -1 、or 10 4 M -1 The bispecific antibody or antigen-binding fragment thereof. The bispecific antibody or antigen-binding fragment thereof. [The present invention 1006] wherein the second arm specifically binds to a second antigen with a binding affinity higher than 10 7 M -1 、10 6 M -1 、10 5 M -1 、or 10 4 M -1 The bispecific antibody or antigen-binding fragment thereof of the present invention 1005. [The present invention 1007] The bispecific antibody or antigen-binding fragment thereof of the present invention 1005, wherein the binding affinity of the first arm when binding to the first antigen is at least 100-fold, 1000-fold, or 10000-fold higher than the binding affinity of the second arm when binding to the second antigen. [The present invention 1008] The bispecific antibody or antigen-binding fragment thereof of the present invention 1005, wherein the first light chain variable region and the second light chain variable region are at least 90%, 95%, 99%, or 100% identical. [The present invention 1009] A first heavy chain comprising a first heavy chain variable region, A second heavy chain comprising a second heavy chain variable region, A first light chain comprising a first light chain variable region, and A second light chain comprising a second light chain variable region A bispecific antibody or antigen-binding fragment thereof, comprising: The first heavy chain variable region and the first light chain variable region bind to each other to form a first antigen-binding region that specifically binds to the first antigen with a binding affinity higher than 10 7 M -1 10 8 M -1 10 9 M -1 10 10 M -1 10 11 M -1 10 12 M -1 and a second antigen-binding region that specifically binds to the second antigen with a binding affinity lower than 10 The second heavy chain variable region and the second light chain variable region bind to each other to form a second antigen-binding region that specifically binds to the second antigen with a binding affinity lower than 10 9 M -1 10 8 M -1 10 7 M -1 10 6 M -1 10 5 M -1 or 10 4 M -1 The bispecific antibody or antigen-binding fragment thereof. [The present invention 1010] ​ The second antigen-binding region specifically binds to the second antigen with a binding affinity higher than 10 7 M -1 、10 6 M -1 、10 5 M -1 、 or 10 4 M -1 The bispecific antibody of the present invention 1009 or an antigen-binding fragment thereof that specifically binds to the second antigen with a binding affinity higher than 10 [The present invention 1011] The binding affinity of the first antigen-binding region when binding to the first antigen is at least 100-fold, 1000-fold, or 100,000-fold higher than the binding affinity of the second antigen-binding region when binding to the second antigen. The bispecific antibody of the present invention 1009 or an antigen-binding fragment thereof. [The present invention 1012] The first light chain and the second light chain are at least 90%, 95%, 99%, or 100% identical. The bispecific antibody of the present invention 1009 or an antigen-binding fragment thereof. [The present invention 1013] The first heavy chain and the second heavy chain are bound to each other by the knobs into holes method. The bispecific antibody of the present invention 1009 or an antigen-binding fragment thereof. [The present invention 1014] The first antigen is a cancer-specific antigen, and the second antigen is CD3. The bispecific antibody of any one of the present inventions 1001 to 1013 or an antigen-binding fragment thereof. [The present invention 1015] The first antigen is CD20, and the second antigen is CD3. The bispecific antibody of any one of the present inventions 1001 to 1013 or an antigen-binding fragment thereof. [The present invention 1016] A bispecific antibody or antigen-binding fragment thereof of the present invention 1015, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:1, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:2, and the first and second light chain variable regions comprise a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:3. [The present invention 1017] A bispecific antibody or antigen-binding fragment thereof according to any one of the present inventions 1001 to 1013, wherein the first antigen is a cancer-specific antigen and the second antigen is a cancer-related antigen. [The present invention 1018] A bispecific antibody or antigen-binding fragment thereof according to any one of the present inventions 1001 to 1013, wherein the first antigen is PD-L1 and the second antigen is CD55. [The present invention 1019] A bispecific antibody or antigen-binding fragment thereof of the present invention 1018, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:4, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:5, and the first and second light chain variable regions comprise a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:6 or SEQ ID NO:7. [The present invention 1020] A method for producing a bispecific antibody or antigen-binding fragment thereof, the method comprising: (a) selecting a first antigen and a second antigen and identifying a first antibody or antigen-binding fragment thereof that binds to the first antigen and a second antibody or antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or antigen-binding fragment thereof comprises a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or antigen-binding fragment thereof comprises a second heavy chain variable region (VHb) and a second light chain variable region (VLb); (b) determining the amino acid sequences of VHa, VLa, VHb, and VLb; (c) aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is higher than 80%; (d) designing a common light chain variable region (VLc), wherein the VLc maintains the affinity for a first antigen when binding to VHa; (e) redesigning the sequences of VHa and VHb, thereby obtaining VHa’ and VHb’ that increase the difference in biochemical or biophysical properties between a first protein comprising two polypeptides each containing VHa’ and two polypeptides each containing VLc, and a second protein comprising two polypeptides each containing VHb’ and two polypeptides each containing VLc; and (f) producing a bispecific antibody or an antigen-binding fragment thereof having two light chain variable regions and two heavy chain variable regions, wherein each of the two light chain variable regions contains VLc and the two heavy chain variable regions contain VHa’ and VHb’, respectively. [Invention 1021] The method of Invention 1020, wherein in step (d), the binding affinity of VLc-VHb for a second antigen may decrease. [Invention 1022] The method of Invention 1020, further comprising: (g) creating a buffer system for purifying the bispecific antibody or an antigen-binding fragment thereof. [Invention 1023] A method for producing a bispecific antibody or an antigen-binding fragment thereof, the method comprising: (a) Selecting a first antigen and a second antigen, and identifying a first antibody or an antigen-binding fragment thereof that binds to the first antigen and a second antibody or an antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or the antigen-binding fragment thereof comprises a first heavy-chain variable region (VHa) and a first light-chain variable region (VLa), and the second antibody or the antigen-binding fragment thereof comprises a second heavy-chain variable region (VHb) and a second light-chain variable region (VLb); (b) Determining the amino acid sequences of VHa, VLa, and VLb; (c) Aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%; (d) Replacing all light-chain variable regions in a phage display antibody library with VLa and panning against the second antigen to obtain a third heavy-chain variable region (VHc); (e) Redesigning the sequences of VHa and VHc, thereby obtaining VHa’ and VHc’ that increase the difference in biochemical or biophysical properties between a first protein comprising two polypeptides each comprising VHa’ and two polypeptides each comprising VLa, and a second protein comprising two polypeptides each comprising VHc’ and two polypeptides each comprising VLa; and (f) Producing a bispecific antibody or an antigen-binding fragment thereof having two light-chain variable regions and two heavy-chain variable regions, wherein each of the two light-chain variable regions comprises VLa and the two heavy-chain variable regions each comprise VHa’ and VHc’. [Invention 1024] The method of Invention 1023, further comprising: (g) Creating a buffer system for purifying the bispecific antibody or an antigen-binding fragment thereof. [Invention 1025] A method for producing a bispecific antibody or an antigen-binding fragment thereof, the method comprising: (a) Selecting a first antigen and a second antigen, and identifying a first antibody or an antigen-binding fragment thereof that binds to the first antigen and a second antibody or an antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or the antigen-binding fragment thereof comprises a first heavy-chain variable region (VHa) and a first light-chain variable region (VLa), and the second antibody or the antigen-binding fragment thereof comprises a second heavy-chain variable region (VHb) and a second light-chain variable region (VLb); (b) Determining the amino acid sequences of VHa, VLa, VHb, and VLb; (c) Aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%; (d) Replacing all light-chain variable regions in a phage display antibody library with a plurality of light-chain variable regions that are at least 80%, 85%, 90%, 95%, or 99% identical to VLa or VLb; (e) Panning against the second antigen; (f) Selecting a common light-chain variable region (VLc) and a third heavy-chain variable region (VHc) such that VHa-VLc binds to the first antigen with a desired affinity and VHc-VLc binds to the second antigen with a desired affinity; (g) Redesigning the sequences of VHa and VHc to obtain VHa' and VHc' that increase the difference in biochemical or biophysical properties between a first protein comprising two polypeptides each comprising VHa' and two polypeptides each comprising VLc and a second protein comprising two polypeptides each comprising VHc' and two polypeptides each comprising VLc; and (h) Producing a bispecific antibody or an antigen-binding fragment thereof having two light-chain variable regions and two heavy-chain variable regions, wherein each of the two light-chain variable regions comprises VLc and the two heavy-chain variable regions each comprise VHa' and VHc'. [Invention 1026] In step (d), a method of the present invention 1025 in which a plurality of light chain variable regions are produced by error-prone PCR. [The present invention 1027] The method of the present invention 1025 further comprising the following: (i) The step of creating a buffer system for purifying the bispecific antibody or antigen-binding fragment thereof. [The present invention 1028] A method for producing a bispecific antibody or antigen-binding fragment thereof, the method comprising the following: (a) Selecting a first antigen and a second antigen, and identifying a first antibody or antigen-binding fragment thereof that binds to the first antigen and a second antibody or antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or antigen-binding fragment thereof comprises a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or antigen-binding fragment thereof comprises a second heavy chain variable region (VHb) and a second light chain variable region (VLb); (b) Determining the amino acid sequences of VHa, VLa, VHb, and VLb; (c) Aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%; (d) Replacing all light chain variable regions in a phage display antibody library with a plurality of light chain variable regions that are at least 80%, 85%, 90%, 95%, or 99% identical to VLa or VLb; (e) Panning against the second antigen; (f) Selecting a common light chain variable region (VLc) and a third heavy chain variable region (VHc) such that VHc-VLc binds to the second antigen with a desired affinity; (g) Determining that the homology between VLa and VLc is higher than 80%; (h) Designing a common light chain variable region (VLd) such that VLd maintains the affinity for the first antigen when binding to VHa and has the desired affinity for the second antigen when binding to VHc; (i) Optionally, redesign the sequences of VHa and VHc, thereby obtaining VHa’ and VHc’ that increase the difference in biochemical or biophysical properties between a first protein comprising two polypeptides each comprising VHa’ and two polypeptides each comprising VLd, and a second protein comprising two polypeptides each comprising VHc’ and two polypeptides each comprising VLd; and (j) Optionally, producing a bispecific antibody or an antigen-binding fragment thereof having two light chain variable regions and two heavy chain variable regions, wherein each of the two light chain variable regions comprises VLd and the two heavy chain variable regions each comprise VHa’ and VHc’, said step. [Inventive Concept 1029] A method for producing a bispecific antibody or an antigen-binding fragment thereof, the method comprising: (a) Selecting a first antigen and a second antigen and identifying a first antibody or an antigen-binding fragment thereof that binds to the first antigen and a second antibody or an antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or an antigen-binding fragment thereof comprises a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or an antigen-binding fragment thereof comprises a second heavy chain variable region (VHb) and a second light chain variable region (VLb), said step; (b) Determining the amino acid sequences of VHa, VLa, VHb, and VLb; (c) Aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is higher than 80%; (d) Designing a common light chain variable region (VLc), said step wherein VLc maintains the affinity for the first antigen when binding to VHa; and (e) Optionally, producing a bispecific antibody or an antigen-binding fragment thereof having two light chain variable regions and two heavy chain variable regions, wherein each of the two light chain variable regions comprises VLc and the two heavy chain variable regions each comprise VHa and VHb, said step. [Inventive Concept 1030] A method for producing a bispecific antibody or an antigen-binding fragment thereof, the method comprising: (a) selecting a first antigen and a second antigen, and identifying a first antibody or an antigen-binding fragment thereof that binds to the first antigen and a second antibody or an antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or an antigen-binding fragment thereof comprises a first heavy-chain variable region (VHa) and a first light-chain variable region (VLa), and the second antibody or an antigen-binding fragment thereof comprises a second heavy-chain variable region (VHb) and a second light-chain variable region (VLb); (b) determining the amino acid sequences of VHa, VLa, and VLb; (c) aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%; (d) replacing all light-chain variable regions in a phage display antibody library with VLa and panning against the second antigen to obtain a third heavy-chain variable region (VHc); and (e) optionally, producing a bispecific antibody or an antigen-binding fragment thereof having two light-chain variable regions and two heavy-chain variable regions, each of the two light-chain variable regions comprising VLa, and the two heavy-chain variable regions comprising VHa and VHc, respectively. [Inventive Concept 1031] A method for producing a bispecific antibody or an antigen-binding fragment thereof, the method comprising: (a) selecting a first antigen and a second antigen, and identifying a first antibody or an antigen-binding fragment thereof that binds to the first antigen and a second antibody or an antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or an antigen-binding fragment thereof comprises a first heavy-chain variable region (VHa) and a first light-chain variable region (VLa), and the second antibody or an antigen-binding fragment thereof comprises a second heavy-chain variable region (VHb) and a second light-chain variable region (VLb); (b) determining the amino acid sequences of VHa, VLa, VHb, and / or VLb; (c) aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%; (d) replacing all light chain variable regions in a phage display antibody library with a plurality of light chain variable regions, wherein the light chain variable region is at least 80%, 85%, 90%, 95%, or 99% identical to VLa or VLb; (e) panning against the first and / or second antigen; (f) selecting a common light chain variable region (VLc) and a third heavy chain variable region (VHc), wherein VHa-VLc binds to the first antigen with a desired affinity and VHc-VLc binds to the second antigen with a desired affinity; and (g) optionally, preparing a bispecific antibody or an antigen-binding fragment thereof having two light chain variable regions and two heavy chain variable regions, wherein each of the two light chain variable regions comprises VLc and the two heavy chain variable regions comprise VHa and VHc, respectively. [Invention 1032] A heavy chain variable region (VH) comprising complementarity-determining regions (CDR) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and A light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence An antibody or an antigen-binding fragment thereof that binds to CD3, comprising The amino acid sequences of the selected VH CDR1, 2, and 3 are set forth in SEQ ID NOs: 22-24, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are set forth in SEQ ID NOs: 28-30, respectively, The antibody or antigen-binding fragment thereof. [Inventive Item 1033] The antibody or antigen-binding fragment thereof according to Inventive Item 1032, wherein VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively. [Inventive Item 1034] The antibody or antigen-binding fragment thereof according to any one of Inventive Items 1032-1033, which specifically binds to human CD3. [Inventive Item 1035] The antibody or antigen-binding fragment thereof according to any one of Inventive Items 1032-1034, which is a bispecific antibody. [Inventive Item 1036] A heavy chain variable region (VH) comprising complementarity-determining regions (CDR) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR2 amino acid sequence, and the VH CDR3 comprises an amino acid sequence that is at least 80% identical to the selected VH CDR3 amino acid sequence; and A light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR3 amino acid sequence An antibody or antigen-binding fragment thereof that binds to PD-L1, comprising The amino acid sequences of the selected VH CDR1, 2, and 3 and the amino acid sequences of the selected VL CDR1, 2, and 3 are (1) The amino acid sequences of the selected VH CDR1, 2, 3 are respectively described in SEQ ID NO: 41 - 43, and the amino acid sequences of the selected VL CDR1, 2, 3 are respectively described in SEQ ID NO: 53 - 55; (2) The amino acid sequences of the selected VH CDR1, 2, 3 are respectively described in SEQ ID NO: 41 - 43, and the amino acid sequences of the selected VL CDR1, 2, 3 are respectively described in SEQ ID NO: 59 - 61 and are one of the following The antibody or its antigen - binding fragment. [Invention 1037] The antibody or its antigen - binding fragment of Invention 1036, wherein VH contains CDR1, 2, 3 having amino acid sequences respectively described in SEQ ID NO: 41 - 43, and VL contains CDR1, 2, 3 having amino acid sequences respectively described in SEQ ID NO: 59 - 61. [Invention 1038] The antibody or its antigen - binding fragment of any one of Inventions 1036 - 1037, which specifically binds to human CD3. [Invention 1039] The antibody or its antigen - binding fragment of any one of Inventions 1036 - 1038, which is a bispecific antibody. [Invention 1040] A heavy - chain variable region (VH) comprising complementarity - determining regions (CDR) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR3 amino acid sequence; and A light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence, said light chain variable region (VL) An antibody or antigen-binding fragment thereof that binds to CD55 and comprises the amino acid sequences of the selected VH CDR1, 2, and 3 and the amino acid sequences of the selected VL CDR1, 2, and 3 are (1) The amino acid sequences of the selected VH CDR1, 2, 3 are respectively set forth in SEQ ID NO: 47-49, and the amino acid sequences of the selected VL CDR1, 2, 3 are respectively set forth in SEQ ID NO: 53-55; (2) The amino acid sequences of the selected VH CDR1, 2, 3 are respectively set forth in SEQ ID NO: 47-49, and the amino acid sequences of the selected VL CDR1, 2, 3 are respectively set forth in SEQ ID NO: 59-61 and is one of said antibody or antigen-binding fragment thereof. [Invention 1041] An antibody or antigen-binding fragment thereof of Invention 1040, wherein VH comprises CDR1, 2, 3 having amino acid sequences respectively set forth in SEQ ID NO: 47-49, and VL comprises CDR1, 2, 3 having amino acid sequences respectively set forth in SEQ ID NO: 59-61. [Invention 1042] An antibody or antigen-binding fragment thereof according to any one of Inventions 1040-1041 that specifically binds to human CD3. [Invention 1043] An antibody or antigen-binding fragment thereof according to any one of Inventions 1040-1042 that is a bispecific antibody. [Invention 1044] An immunoglobulin light chain or a fragment thereof comprising VLs containing CDR1, 2, and 3, each containing the amino acid sequences set forth in SEQ ID NOs: 53 to 55 A nucleic acid comprising a polynucleotide encoding a polypeptide comprising when the VL is paired with a VH containing the amino acid sequence set forth in SEQ ID NO: 4, binds to PD-L1 and / or when paired with a VH containing the amino acid sequence set forth in SEQ ID NO: 5, binds to CD55 said nucleic acid. [Invention 1045] An immunoglobulin light chain or a fragment thereof comprising VLs containing CDR1, 2, and 3, each containing the amino acid sequences set forth in SEQ ID NOs: 59 to 61 A nucleic acid comprising a polynucleotide encoding a polypeptide comprising when the VL is paired with a VH containing the amino acid sequence set forth in SEQ ID NO: 4, binds to PD-L1 and / or when paired with a VH containing the amino acid sequence set forth in SEQ ID NO: 5, binds to CD55 said nucleic acid. [Invention 1046] A nucleic acid according to any one of Inventions 1044 to 1045, encoding a bispecific antibody. [Invention 1047] A nucleic acid according to any one of Inventions 1044 to 1045, which is cDNA. [Invention 1048] A vector comprising one or more of the nucleic acids according to any one of Inventions 1044 to 1047. [Invention 1049] A cell comprising the vector of Invention 1048. [Invention 1050] The cell of Invention 1049, which is a CHO cell. [Invention 1051] A cell comprising one or more of the nucleic acids according to any one of Inventions 1044 to 1047. [Invention 1052] A first polypeptide comprising a first heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1; A second polypeptide comprising a second heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2; A third polypeptide comprising a first light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3; A fourth polypeptide comprising a second light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3 A bispecific antibody or antigen-binding fragment thereof that binds to CD20 and CD3. [Inventive Item 1053] Wherein the first heavy chain variable region (VH) comprises SEQ ID NO:1; Wherein the second heavy chain variable region (VH) comprises SEQ ID NO:2; Wherein the first light chain variable region (VL) comprises SEQ ID NO:3; and Wherein the second light chain variable region (VL) comprises SEQ ID NO:3, The bispecific antibody or antigen-binding fragment thereof of Inventive Item 1052. [Inventive Item 1054] Wherein the first polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:34, 35, or 36; Wherein the second polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:37, 38, or 39; Wherein the third polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:40; and The fourth polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:40, The bispecific antibody of the present invention 1052 or an antigen-binding fragment thereof. [The present invention 1055] The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:35; and The second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:38, The bispecific antibody of the present invention 1052 or an antigen-binding fragment thereof. [The present invention 1056] A first polypeptide comprising a first heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4; A second polypeptide comprising a second heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5; A third polypeptide comprising a first light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6 or 7; A fourth polypeptide comprising a second light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6 or 7 comprising a bispecific antibody that binds to PD-L1 and CD55 or an antigen-binding fragment thereof. [The present invention 1057] The first heavy chain variable region (VH) comprises SEQ ID NO:4; The second heavy chain variable region (VH) comprises SEQ ID NO:5; The first light chain variable region (VL) comprises SEQ ID NO:7; and The second light chain variable region (VL) comprises SEQ ID NO:7, The bispecific antibody of the present invention 1056 or an antigen-binding fragment thereof. [The present invention 1058] The first polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:65; The second polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:66; The third polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:67 or 68; and The fourth polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:67 or 68, The bispecific antibody of the present invention 1056 or an antigen-binding fragment thereof. [The present invention 1059] The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:65; The second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:66; The third polypeptide comprises the amino acid sequence set forth in SEQ ID NO:68; and The fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO:68, The bispecific antibody of the present invention 1056 or an antigen-binding fragment thereof. [The present invention 1060] An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof of any one of the present inventions 1001 to 1019, 1032 to 1043, and 1052 to 1059 covalently bound to a therapeutic agent. [The present invention 1061] The antibody-drug conjugate of the present invention 1060, wherein the therapeutic agent is a cytotoxic agent or a cytostatic agent. [The present invention 1062] A method for treating a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of a composition comprising any antibody or antigen-binding fragment thereof of the present invention 1001-1019, 1032-1043, and 1052-1059, or an antibody-drug conjugate of the present invention 1060 or 1061. [The present invention 1063] The method of the present invention 1062, wherein the subject has a solid tumor. [The present invention 1064] The method of the present invention 1062, wherein the cancer is melanoma, pancreatic cancer, or hematological malignancy. [The present invention 1065] The method of the present invention 1062, wherein the cancer is non-Hodgkin lymphoma, lymphoma, or chronic lymphocytic leukemia. [The present invention 1066] A method for reducing the rate of tumor growth, comprising contacting tumor cells of a subject with an effective amount of a composition comprising any antibody or antigen-binding fragment thereof of the present invention 1001-1019, 1032-1043, and 1052-1059, or an antibody-drug conjugate of the present invention 1060 or 1061. [The present invention 1067] A method for killing tumor cells, comprising contacting tumor cells of a subject with an effective amount of a composition comprising any antibody or antigen-binding fragment thereof of the present invention 1001-1019, 1032-1043, and 1052-1059, or an antibody-drug conjugate of the present invention 1060 or 1061. [The present invention 1068] A pharmaceutical composition comprising any antibody or antigen-binding fragment thereof of the present invention 1001-1019, 1032-1043, and 1052-1059 and a pharmaceutically acceptable carrier. [The present invention 1069] A pharmaceutical composition comprising an antibody-drug conjugate of the present invention 1060 or 1061 and a pharmaceutically acceptable carrier. Other features and advantages of the present invention will become apparent from the following detailed description, drawings, and claims. [[ID=3H]]BRIEF DESCRIPTION OF THE DRAWINGS

[0070]

Figure 1

Figure 2A

Figure 2B

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21A

Figure 21B

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26A

Figure 26B

Figure 27A

Figure 27B

Figure 28

Figure 29

Modes for Carrying Out the Invention

[0071] Detailed Description A bispecific antibody or antigen-binding fragment thereof is an artificial protein that can simultaneously bind to two different antigens. In some embodiments, a bispecific antibody or antigen-binding fragment thereof can have two arms (arms A and B). Each arm contains one heavy chain variable region and one light chain variable region.

[0072] Bispecific antibodies or antigen-binding fragments thereof can be IgG-like or non-IgG-like. An IgG-like bispecific antibody can have two Fab arms and one Fc region, with the two Fab arms binding to different antigens. A non-IgG-like bispecific antibody or antigen-binding fragment can be, for example, a chemically linked Fab (e.g., the two Fab regions are chemically linked) or a single-chain variable fragment (scFV). For example, an scFV can have two heavy chain variable regions and two light chain variable regions.

[0073] In an unbalanced bispecific antibody or antigen-binding fragment thereof, the two arms (arms: A and B) or two antigen-binding regions (antigen-binding regions: A and B) can bind to their respective target antigens with different affinities. The binding affinity can be expressed by the association constant (Ka) as follows: Ka = [antibody-antigen] / [antibody] [antigen]

[0074] High affinity antibodies are usually 10 7 M -1 The Ka for one arm or one antigen-binding region is 10 5 M -1 , 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M-1 or 10 12 M -1 can be higher than. In some embodiments, Ka is 10 5 M -1 10 6 M -1 10 7 M -1 10 8 M -1 10 9 M -1 10 10 M -1 10 11 M -1 or 10 12 M -1 can be lower than.

[0075] The binding affinity (A) of the first arm or the first antigen - binding region can be higher than the binding affinity (B) of the second arm or the second antigen - binding region. Bispecific antibodies with unbalanced affinities can have various advantages. For example, bispecific antibodies with unbalanced affinities can be used to target a cancer - specific antigen on cancer cells and CD3 on T cells. In this case, the high affinity for the cancer - specific antigen can lead to better capture of cancer cells by T cells, and the low affinity for CD3 can avoid the induction of T - cell signals by CD3 (Figure 28). T cells are activated and can kill target cancer cells only when the bispecific antibody is presented to T cells by target cancer cells in a more multivalent manner. Furthermore, bispecific antibodies with unbalanced affinities can also be used to target a cancer - specific antigen and a cancer - associated antigen (Figure 29). In this case, the bispecific antibody binds weakly to non - cancer cells that express low levels of the cancer - associated antigen, but binds strongly to cancer cells that express both the cancer - specific antigen and high levels of the cancer - associated antigen.

[0076] For a bispecific antibody with unbalanced affinity, the Ka (A) of the first arm or the first antigen - binding region is 10 7 M -1 10 8 M -1 109 M -1 、 10 10 M -1 、 10 11 M -1 or 10 12 M -1 may be higher. In some embodiments, the Ka(A) of the first arm or the first antigen-binding region may be 10-fold, 100-fold, 1000-fold, 10,000-fold, or 100,000-fold higher than the Ka(B) of the second arm or the second antigen-binding region. Thus, in some embodiments, the Ka(B) of the second arm or the second antigen-binding region is 10 5 M -1 、 10 6 M -1 、 10 7 M -1 、 10 8 M -1 or 10 9 M -1 may be lower. In some embodiments, the Ka(B) of the second arm or the second antigen-binding region still binds specifically to the target antigen with a reasonable affinity (e.g., higher than 10 4 M -1 、 10 5 M -1 or 10 6 M -1 ).

[0077] The binding affinity can also be represented by the dissociation constant (Kd). Kd = [antibody] [antigen] / [antibody-antigen]

[0078] The Kd can be 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M may be lower. In some embodiments, the Kd is 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10-9 M, 10 -10 M, 10 -11 M or 10 -12 It can be higher than M.

[0079] In some embodiments, the binding affinity (A) of the first arm or the first antigen - binding region can be higher than the binding affinity (B) of the second arm or the second antigen - binding region. For example, the Kd (B) of the second arm or the second antigen - binding region can be 10 - fold, 100 - fold, 1000 - fold, 10000 - fold or 100000 - fold higher (and thus lower in affinity) than the Kd (A) of the first arm or the first antigen - binding region. Thus, in some embodiments, the Kd (A) of the first arm or the first antigen - binding region is 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 It can be lower than M. And the Kd (B) of the second arm or the second antigen - binding region is 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M or 10 -9 It can be higher than M.

[0080] In some embodiments, the bispecific antibody or its antigen - binding fragment comprises two light chains and two heavy chains. Each of the two light chains has one light - chain variable region (VL) and one light - chain constant region (CL). Each of the two heavy chains has one heavy - chain variable region (VH) and three heavy - chain constant regions (CH1, CH2, CH3). In some embodiments, the two light chains of arm A and arm B are the same. Thus, the CDRs within the VL of the two light chains can be the same. In some embodiments, the two heavy chains within the bispecific antibody or its antigen - binding fragment are different. Thus, the CDRs within the VH of the two heavy chains are different.

[0081] When producing bispecific antibodies, various methods can be utilized to ensure that the same heavy chains do not bind to each other. For example, the "knobs-into-holes" method introduces mutations of amino acids with large side chains into one heavy chain and mutations of amino acids with small side chains into the other heavy chain. Therefore, the same heavy chains are less likely to bind to each other, and two different heavy chains are more likely to bind to each other. The "knobs-into-holes" method is described, for example, in Ridgway, John BB, Leonard G. Presta, Paul Carter, ""‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization." Protein Engineering, Design and Selection" 9.7 (1996), which is hereby incorporated by reference in its entirety.

[0082] Unbalanced bispecific antibodies that bind to T cell-specific antigens and cancer antigens Bispecific antibodies (BsAb or BsMab) with binding arms for T cell-specific antigens (e.g., CD3, CD4, or CD8) that can mobilize and activate T cells have been widely studied for cancer treatment. However, many effector functions of such bispecific antibodies have been excluded for safety concerns. Since effector functions of antibodies such as ADCC and CDC have been shown to play important roles in the killing of cancer cells, maintaining the "safety" of the effector functions of antibodies improves the cancer-killing function of the antibodies and expands the mechanism of action of therapeutic antibodies. To maintain the "safety" of the effector functions and expand the application of such bispecific antibodies, a platform for unbalanced bispecific antibody technology has been developed based on computer-aided antibody design.

[0083] In this design, the first antigen-binding region targets a cancer-specific antigen, and the second antigen-binding region targets a T cell-specific antigen (e.g., CD3, CD4, or CD8) to form T cells for attacking cancer with the cancer-specific antigen (Figure 28).

[0084] As used herein, the term "cancer-specific antigen" refers to an antigen that is specifically expressed on the surface of cancer cells. These antigens can be used to identify tumor cells. Normal cells rarely express cancer-specific antigens. Some typical cancer-specific antigens include, for example, CD20, PSA, PSCA, PD-L1, Her2, Her3, Her1, β-catenin, CD19, CEACAM3, EGFR, c-Met, EPCAM, PSMA, CD40, MUC1, and IGF1R, etc. PSA is mainly expressed in prostate cancer cells, and Her2 is mainly expressed in breast cancer cells.

[0085] Bispecific antibodies that bind to CD20 and CD3 are described herein. This bispecific antibody can be applied to target a plurality of CD20-positive cancers such as CD20-positive non-Hodgkin lymphoma (NHL), and thus can be used for the treatment of non-Hodgkin lymphoma in a subject. The bispecific antibody conforms to a different mechanism of action for treating cancer compared to the target CD20 alone of the therapeutic antibody, and thus can be applied as a complementary treatment for CD20-positive cancer, especially for CD20-positive cancer that does not respond well to current CD20 therapies (such as rituximab-resistant NHL). [[ID=IO]]

[0086] Antibodies with high affinity for CD3 can induce T cell signals and may cause unwanted immune responses. Therefore, to reduce the risk of CD3-induced T cell signaling while "safely" maintaining the effector function of the antibody, a low affinity for CD3 (e.g., Ka is 10 5 M -1 、10 6 M -1 or 10 7 M -1(which may be less than) is required. As used herein, the term "safely maintaining the effector function of an antibody" means that the antibody does not induce ADCC or CDC in normal cells (e.g., non-cancer cells). When multiple bispecific antibodies are presented on target cancer cells (e.g., within a cluster) and bridge the interaction between cancer cells and T cells, these bispecific antibodies can induce T cell signaling via CD3 in a multivalent form, and the activated T cells kill the target cancer cells.

[0087] Accordingly, the present specification discloses a bispecific antibody or an antigen-binding fragment thereof comprising two heavy chain variable regions and two light chain variable regions, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:1, and the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2. Also, the first and second light chain variable regions comprise a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3.

[0088] In some embodiments, the CDR sequences for binding to CD20 include the CDRs of the heavy chain variable domain, SEQ ID NOs: 16-18, and the CDRs of the light chain variable domain, SEQ ID NOs: 28-30 (defined by Kabat numbering). In Chothia numbering, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 19-21, and the CDRs of the light chain variable domain are shown in SEQ ID NOs: 31-33.

[0089] In some embodiments, the sequences of the CDRs for binding to CD3 include the CDRs of the heavy chain variable domain, SEQ ID NOs: 22-24, and the CDRs of the light chain variable domain, SEQ ID NOs: 28-30 (defined by Kabat numbering). In Chothia numbering, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 25-27, and the CDRs of the light chain variable domain are shown in SEQ ID NOs: 31-33.

[0090] In some embodiments, the bispecific antibody or its antigen-binding fragment comprises a first heavy chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 34, 35 or 36; a second heavy chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37, 38 or 39; a first light chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 40; and a second light chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 40. In some embodiments, the first light chain amino acid sequence and the second light chain amino acid sequence are identical.

[0091] An unbalanced bispecific antibody that binds to a cancer-specific antigen and a cancer-related antigen Also disclosed herein is an unbalanced bispecific antibody having a first antigen-binding region that targets a cancer-specific antigen and a second antigen-binding region that targets a cancer-related antigen.

[0092] As used herein, the term "cancer-associated antigen" refers to an antigen that is expressed at a relatively high level on cancer cells, but may also be expressed at a relatively low level on normal cells. Various adhesion molecules such as CD55, CD59, CD46, and N-cadherin, VE-cadherin, NCAM, Mel-CAM, ICAM, NrCAM, VCAM1, ALCAM, MCAM are cancer-associated antigens. While both cancer-specific antigens and cancer-associated antigens are expressed on the surface of cancer cells, the difference between cancer-specific antigens and cancer-associated antigens is that cancer-associated antigens are also expressed on normal cells, but at a relatively low level compared to the level on cancer cells. On the other hand, cancer-specific antigens are rarely expressed on normal cells, and even if they are expressed on normal cells, the amount is extremely low. Antibodies targeting cancer-specific antigens usually do not induce antibody-dependent cell cytotoxicity (ADCC) or complement-dependent cell cytotoxicity (CDC) on normal cells. In contrast, antibodies that target cancer-associated cells with high affinity may cause a cytotoxic effect on normal cells. Therefore, it is important that the bispecific antibody binds to the cancer-associated antigen with a relatively low affinity (Figure 29).

[0093] Bispecific antibodies that bind to PD-L1 and CD55 are described in the examples. This antibody can be used to treat subjects with PD-L1 and CD55-positive cancers through ADCC or CDC, as well as by blocking the PD-L1 / PD1 interaction to activate the T cell-dependent immune response and reducing the suppression of CD55 against CDC. Furthermore, since cancer cells may become resistant to PD-L1 antibodies, the binding between the second arm and CD55 on cancer cells can provide an additional therapeutic effect.

[0094] Accordingly, the present disclosure discloses a bispecific antibody or an antigen-binding fragment thereof that includes two heavy-chain variable regions and two light-chain variable regions, wherein the first heavy-chain variable region includes a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4, the second heavy-chain variable region includes a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5, and the first and second light-chain variable regions include a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:6 or 7.

[0095] In some embodiments, the CDR sequences for binding to PD-L1 include the CDRs of the heavy-chain variable domain (SEQ ID NOs: 41-43) and the CDRs of the light-chain variable domain (SEQ ID NOs: 53-55 or 59-61) (defined by Kabat numbering). In Chothia numbering, the CDR sequences of the heavy-chain variable domain are shown in SEQ ID NOs: 44-46, and the CDRs of the light-chain variable domain are shown in SEQ ID NOs: 56-58 or 62-64.

[0096] In some embodiments, the CDR sequences for binding to CD55 include the CDRs of the heavy-chain variable domain, SEQ ID NOs: 47-49, and the CDRs of the light-chain variable domain, SEQ ID NOs: 53-55 or 59-61 (defined by Kabat numbering). In Chothia numbering, the CDR sequences of the heavy-chain variable domain are shown in SEQ ID NOs: 50-52, and the CDRs of the light-chain variable domain are shown in SEQ ID NOs: 56-58 or 62-64.

[0097] In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a first heavy chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:65; a second heavy chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:66; a first light chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:67 or 68; and a second light chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:67 or 68. In some embodiments, the first light chain amino acid sequence and the second light chain amino acid sequence are identical.

[0098] Producing an unbalanced bispecific antibody or antigen, or antigen-binding fragment thereof The bispecific antibody or antigen, or antigen-binding fragment thereof, can be produced by the following method: (1) Select two target antigens, and determine the sequence of the heavy chain variable region (VHa) and the sequence of the light chain variable region (VLa) of an antibody (antibody A) that binds to the first antigen, and determine the sequence of the heavy chain variable region (VHb) and the sequence of the light chain variable region (VLb) of an antibody (antibody B) that binds to the second antigen. (2) Align VLa and VLb, and when the sequence homology exceeds 80%, use computer modeling tools (such as BioLuminate from Schrödinger in Cambridge, Massachusetts) to design a common VL. During the design process, strive to maintain the affinity of VLa, but it may be necessary to sacrifice the affinity of VLb to some extent. The common VL can be VLa or VLb itself, or it can be a new VLc whose sequence shares high homology with VLa and VLb. The three-dimensional structures of VLa and VLb can be determined from, for example, structure modeling or crystal structures. This process can start with the sequence of VLa. When based on the three-dimensional structure, amino acids within the light chain are identified as important for binding to the second antigen (e.g., when combined with VHb) and not involved in binding to the first antigen (e.g., when combined with VHa), and the amino acids within VLa can be changed to the corresponding amino acids within VLb. After repeating this process several times, a common VLc can be obtained. (3) When the homology between VLa and VLb is less than 80%, create a human ScFV or Fab phage library by replacing the VL of an existing human naive ScFV library with the VL of antibody A, then use error-prone PCR to induce nucleotide mutations of less than 20% within the VL, pan against the antigen of antibody B, and obtain a new antibody B' that has VLa or its homolog (homology exceeding 80%) as its VL. If the VL is not VLa but a VLa homolog (e.g., having homology exceeding 80%), repeat step (2) to design a common VL. (4) To increase the differences in the biochemical and biophysical properties (such as three-dimensional isoelectric point (PI), etc.) between A and B, use computer modeling tools to redesign the sequences of VHa and VHb respectively. During this process, the affinity of A cannot decrease, and the affinity of B can decrease to a certain extent. (5) Create a buffer system and purify the unbalanced bispecific antibody.

[0099] The isoelectric point (PI) of a peptide is the pH at which, on statistical average, a particular molecule has no net charge. The amino acids that make up a peptide can be of positive, negative, neutral, or polar nature, and when combined, they give the protein its overall charge. However, certain amino acids within the protein are buried within the protein and do not interact with the surrounding solution. The three-dimensional PI takes into account the three-dimensional structure of the protein and provides a better estimate of the pH value at which the correctly folded protein has no net charge on statistical average. (Since the inventors used a gradient pH buffer from published literature, the buffer is not an invention of the inventors. However, the purification process still needs to be optimized.)

[0100] In some embodiments, the bispecific antibody or antigen, or antigen-binding fragment thereof, can also be made by the following method: (a) selecting a first antigen and a second antigen, and identifying a first antibody or antigen-binding fragment thereof that binds to the first antigen and a second antibody or antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or antigen-binding fragment thereof comprises a first heavy-chain variable region (VHa) and a first light-chain variable region (VLa), and the second antibody or antigen-binding fragment thereof comprises a second heavy-chain variable region (VHb) and a second light-chain variable region (VLb); (b) determining the amino acid sequences of VHa, VLa, and VLb; (c) aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%; (d) replacing all light-chain variable regions in a phage display antibody library with VLa and panning against the second antigen to obtain a third heavy-chain variable region (VHc); (e) Redesigning the sequences of VHa and VHc, thereby obtaining VHa’ and VHc’ that increase the difference in biochemical or biophysical properties between a first protein comprising two polypeptides each comprising VHa’ and two polypeptides each comprising VLa, and a second protein comprising two polypeptides each comprising VHc’ and two polypeptides each comprising VLa; and (f) Producing a bispecific antibody or an antigen-binding fragment thereof having two light chain variable regions and two heavy chain variable regions, wherein each of the two light chain variable regions comprises VLa, and the two heavy chain variable regions each comprise VHa' and VHc', said step.

[0101] In some embodiments, the bispecific antibody or antigen, or an antigen-binding fragment thereof, can also be produced by the following method: (a) Selecting a first antigen and a second antigen and identifying a first antibody or an antigen-binding fragment thereof that binds to the first antigen and a second antibody or an antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or an antigen-binding fragment thereof comprises a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or an antigen-binding fragment thereof comprises a second heavy chain variable region (VHb) and a second light chain variable region (VLb), said step; (b) Determining the amino acid sequences of VHa, VLa, VHb, and VLb; (c) Aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%; (d) Replacing all light chain variable regions in a phage display antibody library with a plurality of light chain variable regions that are at least 80%, 85%, 90%, 95%, or 99% identical to VLa or VLb, said step; (e) Panning against the first and / or second antigen (e.g., the second antigen); (f) Selecting a common light chain variable region (VLc) and a third heavy chain variable region (VHc), wherein VHa-VLc binds to a first antigen with a desired affinity and VHc-VLc binds to a second antigen with a desired affinity; (g) Redesigning the sequences of VHa and VHc, thereby obtaining VHa’ and VHc’ that increase the difference in biochemical or biophysical properties between a first protein comprising two polypeptides each comprising VHa’ and two polypeptides each comprising VLc and a second protein comprising two polypeptides each comprising VHc’ and two polypeptides each comprising VLc; and (h) Producing a bispecific antibody or an antigen-binding fragment thereof having two light chain variable regions and two heavy chain variable regions, wherein each of the two light chain variable regions comprises VLc and the two heavy chain variable regions comprise VHa' and VHc', respectively.

[0102] In some embodiments, additional procedures can be performed if VHa-VLc cannot bind to the first antigen with the desired affinity. For example, if VLc is at least 80% identical to VLa, a new common light chain can be designed. In some embodiments, the process begins with VLa and based on the methods described herein (e.g., based on the three-dimensional structures of VLa and VLc), amino acids can be mutated to the amino acids within VLc.

[0103] In some embodiments, the design of the common light chain variable region involves aligning VLa and VLb and studying the different residues between VLa and VLb at the same Kabat positions. If the different residues on VLb do not contact CDRs, interface residues, canonical residues, or residues in the Vernier zone on the B Fv structure, the residues on VLb are mutated to the residues at the same Kabat positions on VLa. Otherwise, the residues on VLb are retained.

[0104] In some embodiments, the redesign of the heavy chain variable region involves calculating the 3D PIs of Fv A and Fv B using BioLuminate, and mutating residues in non-CDR, non-canonical, non-interface, and non-vernier zones to make Fvs with high 3D PIs higher and Fvs with low 3D PIs lower.

[0105] The use of BioLuminate is described, for example, in the BioLuminate user guide for reference, which is hereby incorporated by reference in its entirety.

[0106] Antibodies and antigen-binding fragments The present disclosure provides antibodies and antigen-binding fragments thereof that include complementarity-determining regions (CDRs), heavy chain variable regions, light chain variable regions, heavy chains, or light chains described herein. In some embodiments, the antibodies and antigen-binding fragments thereof are unbalanced bispecific antibodies and antigen-binding fragments thereof.

[0107] Generally, an antibody (also called an immunoglobulin) is composed of two classes of polypeptide chains, a light chain and a heavy chain. Non-limiting antibodies in the present disclosure can be intact four-immunoglobulin chain antibodies consisting of two heavy chains and two light chains. The heavy chain of this antibody can be an isotype including IgM, IgG, IgE, IgA, or IgD, or a subtype including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a κ light chain or a λ light chain. An antibody can be composed of two identical copies of the light chain and / or two identical copies of the heavy chain, each containing one variable domain (or variable region, VH), and a plurality of constant domains (or constant regions), which are bound to each other via disulfide bonds within their constant domains to form the "stem" of the antibody. Each light chain contains one variable domain (or variable region, VL) and one constant domain (or constant region), and each is bound to one heavy chain via a disulfide bond. The variable region of each of these light chains is aligned with the variable region of the heavy chain to which it binds. The variable regions of both these light chains and heavy chains contain three hypervariable regions flanked by more conserved framework regions (FR).

[0108] These hypervariable regions, known as these complementarity-determining regions (CDR), form loops that include the major antigen-binding surface of the antibody. These four framework regions accept mostly β-sheet conformations, and the CDR form loops that bind to this β-sheet structure, or in some cases loops that form a part of it. The CDR of each chain are held in close proximity by the framework regions and the CDR of the other chains and contribute to the formation of the antigen-binding region.

[0109] Methods for identifying the CDR regions of an antibody by analyzing its amino acid sequence are well known, and a set of CDR definitions are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the positions of structural loop regions. These methods and definitions are described, for example, in Martin, “Protein sequence and structure analysis of antibody variable domains,” Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan, et al. “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains,” Molecular immunology 45.14(2008):3832-3839; Wu, T.T. and Kabat, E.A.(1970) J. Exp. Med. 132:211-250;Martin et al.,MethodsEnzymol. 203:121-53(1991); Morea et al., Biophys Chem. 68(1-3):9-16(Oct. 1997); Morea et al., J Mol Biol. 275(2):269-94(Jan. 1998); Chothia et al., Nature 342(6252):877-83(Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64(2007); and the entire contents of each cited reference are hereby incorporated by reference into this application. In the present disclosure, unless otherwise indicated, Kabat numbering is used as the default.

[0110] CDRs are important for recognizing epitopes of antigens. As used herein, an "epitope" is the minimal portion of a target molecule that has the ability to be specifically bound by the antigen-binding domain of an antibody. The minimal size of an epitope is generally 3, 4, 5, 6, or 7 amino acids, but because epitopes can be based on the three-dimensional configuration based on the secondary and tertiary structures of the antigen, these amino acids in the primary structure of the antigen do not have to be in a continuous linear arrangement.

[0111] In some embodiments, these antibodies can be intact immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved and differ in their constant regions, particularly in their hinge and upper CH2 domains. The sequences and differences of the IgG subclasses are well known in the art and are described, for example, in Vidarsson, et al, “IgG subclasses and allotypes:from structure to effector functions.” Frontiers in immunology 5(2014); Irani, et al. “Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases.” Molecular immunology 67.2(2015):171-182; Shakib, Farouk, ed. The human IgG subclasses:molecular analysis of structure, function and regulation. Elsevier, 2016; and the entire contents of each cited reference are hereby incorporated by reference into this application.

[0112] The antibody may be an immunoglobulin molecule derived from any species (e.g., human, rodent, mouse, rat, camelids). Antibodies disclosed herein include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies, and chimeric antibodies in which the immunoglobulin binding domain is fused to another polypeptide. The term "antigen-binding domain" or "antigen-binding fragment" is a portion of an antibody that retains the specific binding activity of the intact antibody, i.e., any portion of an antibody that has the ability to specifically bind to an epitope on a target molecule of the intact antibody. They are, for example, Fab, Fab’, F(ab’)2, and variants of these fragments. Thus, in some embodiments, the antibody or its antigen-binding fragment may be, for example, scFv, aFv, aFd, adAb, bispecific antibody, bispecific scFv, diabody, linear antibody, single-chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide that is an antibody-binding domain or a binding domain homologous to an antibody-binding domain. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy and / or light chains of an intact antibody, or the CDRs from either the heavy and / or light chains of an intact antibody.

[0113] In some embodiments, the scFV has two heavy chain variable domains and two light chain variable domains. In some embodiments, this scFV has two antigen-binding regions (antigen-binding regions: A and B), and these two antigen-binding regions can bind to their respective target antigens with different affinities.

[0114] In some embodiments, these antigen-binding fragments can form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single-chain variable fragment (scFv) disclosed herein fused to the CD3-ζ (zeta) transmembrane and intracellular regions. In some embodiments, the chimeric antigen receptor also includes intracellular signaling domains from various co-stimulatory 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, in increasing order of strength. Thus, in one aspect, the disclosure further provides cells (e.g., T cells) that express the chimeric antigen receptors described herein.

[0115] In some embodiments, the antibody or antigen-binding fragment thereof can bind to two different antigens or two different epitopes.

[0116] In some embodiments, the antibody or antigen-binding fragment thereof can include one, two, or three heavy-chain variable region CDRs selected from Tables 1, 2, 11, and 12. In some embodiments, the antibody or antigen-binding fragment thereof can include one, two, or three light-chain variable region CDRs selected from Tables 3, 13, and 14.

[0117] In some embodiments, the antibody is A heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 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 the amino acid sequence of the selected VHCDR1, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VHCDR2, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VHCDR3, said heavy chain variable region (VH), and A light chain variable region (VL) comprising CDRs 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 the selected VLCDR1 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 the selected VLCDR2 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 the selected VLCDR3 amino acid sequence, said light chain variable region (VL) can have. The amino acid sequences of the selected VHCDR1, 2, 3 are shown in Tables 1, 2, 11 and 12, and the amino acid sequences of the selected VLCDR1, 2, 3 are shown in Tables 3, 13 and 14. [[ID=...]]

[0118] In some embodiments, the antibody or antigen-binding fragment described herein can comprise a heavy chain variable domain comprising one, two, or three CDRs selected from Tables 1, 2, 11 and 12, and can include 0, 1 or 2 amino acid insertions, deletions, or substitutions.

[0119] In some embodiments, the antibody or antigen-binding fragment described herein comprises a light chain variable domain comprising one, two, or three CDRs selected from Table 3, Table 13, and Table 14, and can include 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0120] The insertions, deletions, and substitutions can be within the CDR sequences or at one or both ends of the CDR sequences.

[0121] The Fc region of the antibodies and antibody fragments of the present disclosure can be modified to provide desired effector functions or plasma half-lives.

[0122] Antibody multimerization can be achieved via natural aggregation of the antibody or through chemical or recombinant ligation techniques. For example, a few percent of a purified antibody preparation (e.g., purified IgG1 molecules) spontaneously form protein aggregates that include antibody homodimers and other higher-order antibody multimers.

[0123] Any antibody or antigen-binding fragment described herein can be linked to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or its antigen-binding fragment 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). Linking to a stabilizing molecule increases the half-life or extends the biological activity 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 a human).

[0124] In some embodiments, the antibodies or antigen-binding fragments described herein (e.g., bispecific antibodies) can be conjugated to a therapeutic agent. An antibody-drug conjugate comprising such an antibody or antigen-binding fragment thereof can bind to a therapeutic agent covalently or non-covalently. In some embodiments, the therapeutic agent is a cytotoxic agent or a cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin, maytansinoids such as DM-1 and DM-4, dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and congeners).

[0125] Antibody Properties The antibodies or antigen-binding fragments thereof described herein (e.g., bispecific antibodies) can enhance an immune response. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can increase an immune response, the activity or number of T cells (e.g., CD3+ cells, CD8+ and / or CD4+ cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

[0126] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can decrease the activity or number of T cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

[0127] In some embodiments, the antibodies or antigen-binding fragments thereof described herein do not induce an immune response in normal cells (e.g., non-tumor cells) or in the absence of tumor cells.

[0128] In some embodiments, the antibody or its antigen-binding fragment (e.g., bispecific antibody) can bind to PD-L1 or PD-L2. Accordingly, the antibodies or antigen-binding fragments described herein can block the binding between PD-1 and PD-L1 and / or the binding between PD-1 and PD-L2. In some embodiments, by binding to PD-L1 or PD-L2, this antibody can inhibit the PD-1 signaling pathway and upregulate the immune response. Accordingly, in some embodiments, the antibodies or antigen-binding fragments described herein are PD-1 antagonists. In some embodiments, the antibody or its antigen-binding fragment is a PD-1 agonist.

[0129] In some embodiments, the antibody or its antigen-binding fragment (e.g., bispecific antibody) can bind to CD3. Accordingly, the antibodies or antigen-binding fragments described herein can recruit T cells to target cells.

[0130] In some embodiments, the antibody (or its antigen-binding fragment) has a dissociation rate (koff) of less than 0.1 s -1 less than, 0.01 s -1 less than, 0.001 s -1 less than, 0.0001 s -1 less than, or 0.00001 s -1 and specifically binds to an antigen (e.g., human protein, monkey protein, and / or mouse protein) that is less than. In some embodiments, the dissociation rate (koff) is greater than 0.01 s -1 greater than, 0.001 s -1 greater than, 0.0001 s -1 greater than, 0.00001 s -1 greater than, or 0.000001 s -1 greater than, or 0.000001 s. In some embodiments, the kinetic binding rate (kon) is greater than 1x10 2 / Ms greater than, 1x10 3 / Ms greater than, 1x10 4 / Ms greater than, 1x10 5 / Ms greater than, or 1x106 exceeds / Ms. In some embodiments, the kinetic association rate (kon) is 1x10 5 less than / Ms, less than 1x10 6 less than / Ms, or less than 1x10 7 less than / Ms.

[0131] Affinity can be estimated from the quotient of the kinetic rate constants (Kd = koff / kon). In some embodiments, Kd is less than 1x10 -4 M, less than 1x10 -5 M, less than 1x10 -6 M, less than 1x10 -7 M, less than 1x10 -8 M, less than 1x10 -9 M, or less than 1x10 -10 M. In some embodiments, 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, or 1 nM. In some embodiments, Kd exceeds 1x10 -4 M, exceeds 1x10 -5 M, exceeds 1x10 -6 M, exceeds 1x10 -7 M, exceeds 1x10 -8 M, exceeds 1x10 -9 M, exceeds 1x10 -10 M, exceeds 1x10 -11 M, or exceeds 1x10 -12 M. Further, Ka can be calculated from Kd by the formula Ka = 1 / Kd.

[0132] Common techniques for measuring the affinity of an antibody for an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR).

[0133] In some embodiments, the thermal stability is determined. The antibodies or antigen-binding fragments described herein can have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.

[0134] Since IgG can be described as a multi-domain protein, the melting curve can sometimes show two transitions, or three transitions, with a first denaturation temperature, Tm D1, a second denaturation temperature Tm D2, and optionally a third denaturation temperature Tm D3.

[0135] In some embodiments, the antibodies or antigen-binding fragments described herein have a Tm D1 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. In some embodiments, the antibodies or antigen-binding fragments described herein have a Tm D2 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. In some embodiments, the antibodies or antigen-binding fragments described herein have a Tm D3 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.

[0136] In some embodiments, Tm, Tm D1, Tm D2, Tm D3 are less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.

[0137] In some embodiments, the antibodies or antigen-binding fragments described herein do not initiate aggregate formation at temperatures less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. In some embodiments, Tagg266 or Tagg473 is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.

[0138] In some embodiments, the antibodies or antigen-binding fragments described herein have a pI greater than 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, or 9.9. In some embodiments, the antibodies or antigen-binding fragments described herein have a pI less than 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, or 9.9.

[0139] In some embodiments, the antibody has a tumor growth inhibition percentage (TGI%) of more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the antibody has a tumor growth inhibition percentage of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. This TGI% can be determined, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the start of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. The tumor growth inhibition percentage (TGI%) as used herein is calculated using the following formula: TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100

[0140] Ti is the average tumor volume of the treatment group on day i. T0 is the average tumor volume of the treatment group on day 0. Vi is the average tumor volume of the control group on day i. V0 is the average tumor volume of the control group on day 0.

[0141] In some embodiments, the antibody or antigen-binding fragment can increase complement-dependent cytotoxicity (CDC) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

[0142] In some embodiments, the antibody or antigen-binding fragment can increase antibody-dependent cell-mediated cytotoxicity (ADCC) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

[0143] In some embodiments, the antibody or antigen-binding fragment can increase the internalization rate by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

[0144] In some embodiments, the antibody or antigen-binding fragment can increase the phagocytosis rate by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

[0145] In some embodiments, the antibody or antigen-binding fragment can enhance T cell function, for example, by an increase in effector T cell proliferation and / or an increase in gamma interferon production by effector T cells (e.g., as compared to proliferation and / or cytokine production prior to treatment with the antibody or antigen-binding fragment).

[0146] In some embodiments, the antibody or antigen-binding fragment can enhance CD4+ effector T cell function, for example, by increasing CD4+ effector T cell proliferation and / or increasing gamma interferon production by CD4+ effector T cells (e.g., as compared to proliferation and / or cytokine production prior to treatment with the antibody or antigen-binding fragment). In some embodiments, the cytokine is gamma interferon. In some embodiments, the antibody or antigen-binding fragment increases the number of intratumoral (infiltrating) CD4+ effector T cells (e.g., the total number of CD4+ effector T cells, or for example, the percentage of CD4+ cells among CD45+ cells) as compared to the number of intratumoral (infiltrating) CD4+ T cells prior to treatment with the antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment increases the number of intratumoral (infiltrating) CD4+ effector T cells that express gamma interferon (e.g., the total number of gamma interferon-expressing CD4+ cells, or for example, the percentage of gamma interferon-expressing CD4+ cells among the total CD4+ cells) as compared to the number of intratumoral (infiltrating) CD4+ T cells that express gamma interferon prior to treatment, e.g., with the antibody.

[0147] In some embodiments, the antibody or antigen-binding fragment increases the number of intratumoral (infiltrating) CD8+ effector T cells (e.g., the total number of CD8+ effector T cells, or for example, the percentage of CD8+ cells among CD45+ cells) as compared to the number of intratumoral (infiltrating) CD8+ T cells prior to treatment with the antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment increases the number of intratumoral (infiltrating) CD8+ effector T cells that express gamma interferon (e.g., the percentage of gamma interferon-expressing CD8+ cells among the total CD8+ cells) as compared to the number of intratumoral (infiltrating) CD8+ T cells that express gamma interferon prior to treatment, e.g., with the antibody.

[0148] In some embodiments, the antibody or antigen-binding fragment enhances memory T cell function, for example, by increasing memory T cell proliferation and / or by increasing cytokine (e.g., interferon-γ) production by memory cells.

[0149] 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.

[0150] In some embodiments, the antibody or antigen-binding fragment can induce apoptosis.

[0151] 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.

[0152] In some embodiments, the antibody or antigen-binding fragment is a humanized antibody. The percentage of humanization refers to the percentage identity when comparing the heavy or light chain variable region sequences to human antibody sequences in the International Immunogenetics Information System (IMGT) database. In some embodiments, the percentage of humanization exceeds 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. Detailed descriptions regarding methods for determining the percentage of humanization are well-known in the art and are described, for example, in Jones, Tim D., et al. “The INNs and outs of antibody nonproprietary names.” MAbs. Vol. 8. No. 1. Taylor & Francis, 2016, which is hereby incorporated by reference in its entirety. A high percentage of humanization often has various advantages, such as being safer and more effective in humans, having a higher likelihood of being more durable in human subjects, and / or having a lower likelihood of side effects. In some embodiments, the antibody or antigen-binding fragment is a human antibody.

[0153] Recombinant vector 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 has been introduced (i.e., such that the host cell contains the polynucleotide and / or the vector containing the polynucleotide), and the production of a recombinant antibody polypeptide or a fragment thereof by recombinant techniques.

[0154] As used herein, a "vector" is any construct that, when introduced into a host cell, has the ability to deliver one or more polynucleotides of interest to the host cell. An "expression vector" has the ability to deliver one or more polynucleotides of interest and express them as the encoded polypeptide when introduced into a host cell. Thus, in an expression vector, the polynucleotide of interest is placed in the vector for expression by functionally linking the polynucleotide of interest to regulatory elements such as promoters, enhancers and / or polyA tails, either in the vector or in the host cell genome, in or near or flanking the insertion site of the polynucleotide of interest, such that the polynucleotide of interest is translated in the host cell into which the expression vector has been introduced.

[0155] Vectors can be introduced into host cells by methods well known in the art such as, for example, electrophoresis, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or introduction (e.g., by recombinant virus). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors complexed with cationic condensing agents.

[0156] In some embodiments, the polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein) are introduced using a viral expression system (e.g., vaccinia or other poxviruses, retroviruses, or adenoviruses), and these viruses can include the use of non-pathogenic (defective), replicable viruses, or non-replicable viruses. In the latter case, viral growth generally occurs only in complementing virus packaging cells. Suitable systems are described, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. N.Y. 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; International Publication No. 89 / 01973; U.S. Patent Application No. 4,777,127; British Patent Application No. 2,200,651; European Patent Application No. 0,345,242; International Publication No. 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 may be "naked" as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749 and Cohen, 1993, Science, 259:1691-1692.Uptake of naked DNA can be enhanced by coating the DNA with biodegradable beads that are efficiently transported into cells.

[0157] For expression, the DNA insert is operably linked to a suitable promoter (e.g., a heterologous promoter) such as the phage λPL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters, and the promoters of retroviral LTRs. Suitable promoters are well known to those skilled in the art. Expression of the construct further includes a transcription initiation and termination site, and in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript by the construct may include a translation start at the beginning and, at the end of the polypeptide to be translated, a properly placed stop codon (UAA, UGA, or UAG).

[0158] As shown, the expression vector may include at least one selectable marker. Such markers include dihydrofolate reductase for eukaryotic cell cultures, or neomycin resistance, and the tetracycline or ampicillin resistance genes in cultures in Escherichia coli (E. coli) and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces; Salmonella typhimurium cells; fungal cells such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK293 cells; and plant cells. Suitable culture media and conditions for the host cells described herein are well known in the art.

[0159] Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9 available from Qiagen; pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

[0160] Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, λPR and PL promoters and trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, promoters of retroviral LTRs such as Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0161] In yeast, Saccharomyces cerevisiae, a series of vectors containing constitutive or inducible promoters such as the α-factor, alcohol oxidase, and PGH can be used. For reviews, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., and Grant et al., Method Enzymol., 153:516-544 (1997).

[0162] Introduction of the construct into the host cell can be achieved by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in standard laboratory manuals such as Davis et al., Basic Methods In Molecular Biology (1986), the entirety of which is incorporated herein by reference.

[0163] Transcription of the DNA encoding the antibodies of the present disclosure by higher eukaryotes can be increased by insertion of enhancer sequences into the vector. Enhancers are cis-acting elements of DNA, usually about 10 - 300 bp, and act to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV4o enhancer located downstream of the origin of replication at base pairs 100 - 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer downstream of the origin of replication, and the adenovirus enhancer.

[0164] For secretion of 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. These signals may be endogenous to the polypeptide or they may be heterologous signals.

[0165] The polypeptide (e.g., antibody) can be expressed in a modified form, such as with a fusion protein (e.g., GST-fusion) or a histidine tag, and can include not only a secretion signal but also other heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell during purification or subsequent processing or storage. Further, a peptide moiety can be added to the polypeptide to facilitate purification. Such regions can be removed prior to the final purification of the polypeptide. The addition of peptide moieties to the polypeptide to improve stability and facilitate purification for secretion or excretion is, inter alia, a well-known and routine technique in the art.

[0166] The present disclosure provides nucleic acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any nucleotide sequence described herein, and also provides amino acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any amino acid sequence described herein.

[0167] The present disclosure also provides nucleic acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to any nucleotide sequence described herein, and amino acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to any amino acid sequence described herein.

[0168] In some embodiments, the present disclosure relates to a nucleotide sequence encoding any peptide described herein, or to 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.

[0169] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence that is any one of the sequences described herein.

[0170] In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence that is any one of the sequences described herein.

[0171] For the determination of the percent identity of two amino acid sequences, or two nucleic acid sequences, these sequences are aligned for optimal comparison purposes (e.g., for comparison purposes, gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences so that an optimal alignment and non-homologous sequences can be ignored). The length of the reference sequence aligned for comparison purposes is at least 80% of the length of that reference sequence, and in some embodiments at least 90%, 95%, or 100%. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. If the position of the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position of the second sequence, then the molecule is identical at that position (as used herein, "identity" of an amino acid or nucleic acid is equal to "homology" of the amino acid or nucleic acid). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of the gaps that need to be introduced for the optimal alignment of the two sequences. For the purposes of the present invention, the comparison of sequences and determination of percent identity between two sequences can be achieved using the Blossum62 scoring matrix, a 12 gap penalty, a 4 gap extend penalty, and a 5 frameshift gap penalty.

[0172] The percentage of sequence identity (e.g., amino acid sequence identity or nucleic acid identity) can also be determined. Methods for determining the percentage of sequence identity are well known in the art. In some embodiments, amino acid residues that are conserved with similar physicochemical properties (percent identity), e.g., leucine and isoleucine, can be used to calculate sequence similarity. Families of amino acid residues with similar physicochemical properties are defined in the art. These families include, for example, amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the percentage of homology is higher than the percentage of identity.

[0173] Method for producing an antibody Isolated fragments of human proteins (e.g., CD55, CD3, cancer specific antigen or cancer associated antigen) can be used as immunogens for generating antibodies using standard techniques for the preparation of polyclonal and monoclonal antibodies. Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal injection) of an antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected together with at least one adjuvant. In some embodiments, the antigenic peptide or protein is conjugated to an agent that is immunogenic in the species to be immunized. The animals can be injected with the antigenic peptide or protein more than once (e.g., 2, 3, or 4 times).

[0174] A full-length polypeptide or protein can be used, or alternatively, its antigenic peptide fragment can be used as an immunogen. The antigenic peptide of a protein contains at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of this protein and encompasses an epitope of the protein such that an antibody raised against the peptide can form a specific immune complex with the protein.

[0175] An immunogen is 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 can include, for example, recombinantly expressed or chemically synthesized polypeptides. The preparation can further include an adjuvant such as Freund's complete adjuvant or incomplete adjuvant, or a similar immunostimulant.

[0176] As described above, polyclonal antibodies can be prepared by immunizing a suitable subject with a polypeptide, or an antigenic peptide thereof (e.g., a portion of a protein), as an immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques such as enzyme-linked immunosorbent assay (ELISA) using the immobilized polypeptide or peptide. If desired, the antibody molecules are isolated from mammals (e.g., their blood) and further purified by well-known techniques such as protein A of protein G chromatography to obtain the IgG fraction. After immunization, after an appropriate time has elapsed, for example, when the antibody titer is at its highest, antibody-producing cells are obtained from the subject and monoclonal antibodies are prepared using standard techniques such as the hybridoma technique first described by Kohler et al. (Nature 256:495-497, 1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), or the trioma technique. Techniques for producing hybridomas are well known (see generally Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY). Hybridoma cells producing monoclonal antibodies can be detected by screening the supernatant of the hybridoma culture for antibodies that bind to the polypeptide or epitope of interest, for example, using an ELISA assay.

[0177] The mutations of the antibodies or antigen-binding fragments described herein can be introduced into DNA encoding a human, humanized, or chimeric antibody, or an antigen-binding fragment thereof described herein with appropriate nucleotide changes, or can be prepared by peptide synthesis. Such mutations include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence forming the antigen-binding site or antigen-binding domain of the antibody. In such a population of mutations, some antibodies or antigen-binding fragments have an increased affinity for the target protein. Any combination of deletions, insertions, and / or combinations can be made to reach an antibody or an antigen-binding fragment thereof having an increased binding affinity for the target. Amino acid changes introduced into an antibody or antigen-binding fragment can change the post-translational modification of the antibody or antigen-binding fragment, such as a change in the number of glycosylation sites (e.g., increase or decrease), a change in the glycosylation site (e.g., a change in the amino acid sequence such that a different sugar is bound by an enzyme present in the cell), or the introduction of a new glycosylation site, or can introduce a new post-translational modification.

[0178] The antibodies disclosed herein can be derived from animals of any species, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates such as monkeys and apes, cows, pigs, horses, sheep, camels (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits) that have been genetically engineered to produce human antibodies.

[0179] The phage display method (panning) can be used to optimize antibody sequences with desired binding affinities. In this technique, a gene encoding a single-chain Fv (including VH or VL) can be inserted into the phage coat protein gene, thereby presenting the scFv on the outside of the phage while the gene for said protein is contained inside the phage, resulting in a linkage between genotype and phenotype. These displayed phages can then be screened against the target antigen to detect the interaction between the displayed antigen-binding site and the target antigen. Thus, a large library of proteins can be screened and amplified in a process called in vitro selection to obtain antibody sequences with desired binding affinities.

[0180] Human and humanized antibodies have variable and constant regions that are derived from human germline immunoglobulin sequences (or have amino acid sequences identical to those derived therefrom). Human antibodies can contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., by in vitro, random, or site-directed mutagenesis, or by somatic mutations in vivo), for example, in the CDRs.

[0181] Humanized antibodies typically have a human framework (FR) onto which non-human CDRs are grafted. Thus, humanized antibodies have one or more amino acid sequences introduced into them from non-human sources. These non-human amino acid residues are often referred to as "imported" residues, and these are typically taken from the "imported" variable domain. Humanization can essentially be done, for example, by replacing the corresponding sequences of a human antibody with, for example, rodent CDRs or CDR sequences. These methods are described, for example, in 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 of which is incorporated herein by reference. Thus, "humanized" antibodies are chimeric antibodies in which significantly fewer than the complete human V domains are replaced by the corresponding sequences from non-human species. In practice, humanized antibodies are typically mouse antibodies in which some CDR residues, and some FR residues, are replaced by residues from the analogous sites of human antibodies.

[0182] It is even more important that the antibody be humanized while retaining a high degree of specificity and affinity for the antigen, and other desirable biological properties. To achieve this goal, humanized antibodies can be prepared by an analytical process of the parental and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and well known to those skilled in the art. Computer programs are available that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays enables analysis of the possible role of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of the residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues are selected from the recipient and combined with the imported sequences to achieve the desired antibody properties such as increased affinity for the antigen.

[0183] Identity or homology with respect to an original sequence is the percentage of amino acid residues present in a candidate sequence that are identical to those present in a sequence in a human, humanized, or chimeric antibody or fragment, with the sequences aligned to achieve the maximum percent sequence identity and gaps introduced if necessary, and no regard being given to any conventional substitutions as part of the sequence identity.

[0184] In some embodiments, covalent modifications can be made to an antibody or antigen-binding fragment thereof. These covalent modifications can be generated by chemical or enzymatic synthesis or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment introduced into the molecule can be induced by reacting the target amino acid residue of the antibody or fragment with an organic derivatizing agent capable of reacting with a selected side chain or N- or C-terminal residue.

[0185] In some embodiments, an antibody variant is provided having a carbohydrate structure lacking fucose that binds (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody can be 1% - 80%, 1% - 65%, 5% - 65% or 20% - 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297 compared to the total of all sugar structures (e.g., complex, hybrid and high mannose structures) bound to Asn297, measured by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. For example, Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering); Asn297 can be located approximately ±3 amino acids upstream or downstream of position 297 due to minor sequence variations in the antibody, i.e., between positions 294 and 300. Such fucosylation variants improve the ADCC function. In some embodiments, the Fc region of the antibody can be further engineered to substitute the asparagine at position 297 with alanine (N297A) to reduce glycan heterogeneity.

[0186] In some embodiments, to promote production efficiency by avoiding Fab-arm exchange, the Fc region of the antibody can be further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P). A detailed description of the S228 mutation can be found, for example, in Silva et al., “The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation.” Journal of Biological Chemistry 290.9 (2015): 5462 - 5469, which is incorporated herein by reference in its entirety.

[0187] In some embodiments, the methods described herein are designed to generate bispecific antibodies. Bispecific antibodies can be generated by engineering the interface between pairs of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface can include at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). A compensatory “hole” of the same or similar size for the larger side chain is created by replacing a large amino acid side chain on the interface of the second antibody molecule with a smaller one (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers compared to other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated herein by reference in its entirety.

[0188] In some embodiments, one or more amino acid residues of the CH3 portion of IgG are substituted. In some embodiments, one heavy chain has one or more of the following substitutions: Y349C and T366W. The other heavy chain has one or more of the following substitutions: E356C, T366S, L368A, and Y407V. Additionally, the substitution (-ppcpScp-->-ppcpPcp-) can also be introduced into both substituted IgG hinge regions. In some embodiments, one heavy chain has a T366Y (knob) substitution, and the other heavy chain has a Y407T (hole) substitution.

[0189] Furthermore, anion exchange chromatography can be used in the production of bispecific antibodies. Anion exchange chromatography is a process of separating substances using an ion exchange resin containing a positively charged group such as a diethylaminoethyl (DEAE) group based on their charge. In solution, this resin is covered by positively charged counterions (cations). The anion exchange resin binds to negatively charged molecules and replaces the counterions. Anion exchange chromatography can be used to purify proteins based on the isoelectric point (pI). The isoelectric point is defined as the pH at which a protein has no net charge overall. When pH > pI, the protein has an overall negative charge, and when pH < pI, the protein has an overall positive charge. Thus, in some embodiments, different amino acid substitutions can be introduced into the two heavy chains, and the pI for the homodimer containing two arm A's and the pI for the homodimer containing two arm B's are different. The pI for the bispecific antibody having arm A and arm B is somewhere between the two pI's of the homodimers. Thus, the two homodimers and the bispecific antibody are eluted under different pH conditions. The present disclosure shows that substitution of some amino acid residues can introduce pI adjustment into the heavy chain.

[0190] Accordingly, in some embodiments, the amino acid residue at position 83 of the Kabat numbering is lysine, arginine, or histidine. In some embodiments, the amino acid residue at one or more positions of positions 1, 6, 43, 81, and 105 (Kabat numbering) is aspartic acid or glutamic acid.

[0191] In some embodiments, one or more of the amino acid residues at positions 13 and 105 (Kabat numbering) is aspartic acid or glutamic acid. In some embodiments, one or more of the amino acid residues at positions 13 and 42 (Kabat numbering) is lysine, arginine, histidine, or glycine.

[0192] Bispecific antibodies can include, for example, cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be linked to avidin, and the other can bind to biotin. Heteroconjugate antibodies can also be made using any convenient cross-linking method. Suitable cross-linking agents and cross-linking techniques are well known in the art and are described in U.S. Patent No. 4,676,980, which is hereby incorporated by reference in its entirety.

[0193] Methods for generating bispecific antibodies from antibody fragments are also well known in the art. For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al. (Science 229:81, 1985) describe a procedure for proteolytically cleaving intact antibodies to generate F(ab’)2 fragments. These fragments are reduced in the presence of a dithiol complexing agent, sodium arsenite, to prevent stabilization of adjacent dithiols and formation of intermolecular disulfide bonds. The resulting Fab’ fragments are then converted to thionitrobenzoic acid (TNB) derivatives. One of the Fab’TNB derivatives is then reconverted to Fab’ thiol by reduction with mercaptoethylamine, and equimolar amounts of another Fab’TNB derivative are mixed to form a bispecific antibody.

[0194] Treatment method The methods described herein include methods for the treatment of diseases associated with cancer. Generally, the method comprises administering a therapeutically effective amount of an engineered bispecific antibody (e.g., an unbalanced bispecific antibody) or an antigen-binding fragment thereof described herein to a subject in need thereof, or a subject determined to be in need of such treatment.

[0195] As used in this context, "treat" means to ameliorate at least one symptom of a disease associated with cancer. Often, cancer results in death; thus, treatment results in an increased mean life span (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years). Administration of a therapeutically effective amount of an agent (e.g., an unbalanced bispecific antibody) described herein for the treatment of a cancer-related medical condition results in a reduced number of cancer cells and / or alleviated symptoms.

[0196] As used herein, the term "cancer" refers to an abnormal state or condition characterized by cells having the ability of autonomous growth, i.e., rapidly proliferating cell growth. This term means to include all types of cancerous growth or carcinogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, regardless of histopathological type or invasive stage. The term "tumor" as used herein refers to a mass of cancerous cells, e.g., cancerous cells. By using the methods described herein, cancers that can be treated or diagnosed include, simultaneously with malignant tumors of various organ systems affecting the lung, breast, thyroid, lymph, digestive, and urogenital tracts, most colon cancers, renal cell carcinomas, prostate cancers and / or testicular tumors, non-small cell lung cancers, cancers of the small intestine, and adenocarcinomas such as esophageal cancer. In some embodiments, the agents described herein are designed to treat or diagnose cancer in a subject. "Cancer" is approved in the art and refers to malignant tumors of epithelial or endocrine tissues, including respiratory system cancer, digestive system cancer, urogenital system cancer, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. In some embodiments, the cancer is renal cancer or melanoma. Exemplary cancers include those formed from tissues of the neck, lung, prostate, breast, head and neck, colon, and ovary. This term also includes carcinosarcomas, which include, for example, malignant tumors composed of cancerous and sarcomatous tissues. "Adenocarcinoma" refers to a cancer derived from glandular tissue or in which tumor cells form recognizable glandular structures therein. The term "sarcoma" is approved in the art and refers to a malignant tumor derived from mesenchyme.

[0197] In some embodiments, the cancer is rituximab (Rituxan®)-resistant cancer.

[0198] In one aspect, the present disclosure also provides a method for treating cancer in a subject, a method for reducing the rate of increase of a tumor over time in a subject, a method for reducing the risk of developing metastases, or a method for reducing the risk of developing additional metastases in a subject. In some embodiments, the treatment can arrest, slow down, delay, or inhibit the progression of cancer. In some embodiments, the treatment can result in a decrease in one or more of the number, severity, and / or duration of symptoms of cancer in the subject.

[0199] In one aspect, the present disclosure features administering to a subject in need thereof a therapeutically effective amount of an antibody or an antigen-binding fragment thereof, or an antibody-drug conjugate disclosed herein, for example, a subject having, identified, or diagnosed with cancer such as breast cancer (e.g., triple-negative breast cancer), carcinoid 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, gastrointestinal cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematological malignancies.

[0200] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and refer to an animal, human or non-human, to which treatment according to the methods of the invention is provided. Veterinary and non-veterinary applications are contemplated by the present invention. A human patient can be an adult human or a juvenile human (e.g., a human less than 18 years old). Patients include, in addition to humans, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates, among others. Included are, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, suids (e.g., pigs, mini-pigs), equids, canids, felids, bovines, and other livestock, and zoo animals.

[0201] In some embodiments, the cancer is unresectable or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-resistant prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colorectal cancer. In some embodiments, the subject has Hodgkin lymphoma. In some embodiments, the subject has triple-negative breast cancer (TNBC), gastrointestinal cancer, urothelial cancer, Merkel cell carcinoma, or head and neck cancer. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, hematologic malignancy, particularly non-Hodgkin lymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumor.

[0202] In some embodiments, the compositions and methods disclosed herein can be used for the treatment of patients at risk of cancer and cancer patients, which can be identified by various methods well known in the art.

[0203] As used herein, "effective amount" means an amount or dose sufficient to achieve a beneficial or desired result, including, for example, arrest, slowdown, delay, or inhibition of the progression of a disease, such as cancer. The effective amount depends, for example, on the age and weight of the subject to whom the antibody, antigen-binding fragment, antibody-drug conjugate, antibody-encoding polynucleotide, vector containing the polynucleotide, and / or their compositions are administered, the severity of the symptoms, and the route of administration, and thus the administration is determined based on individual criteria.

[0204] An effective amount can be administered in one or more administrations. By way of example, an effective amount of an antibody, antigen-binding fragment, or antibody-drug conjugate is an amount sufficient to effect remission, arrest, stabilization, reversal, inhibition, slowdown, and / or delay of the progression of a patient's autoimmune disease or cancer, or in vitro, to effect remission, arrest, stabilization, reversal, slowdown, and / or delay of the proliferation of cells (e.g., biopsied cells, any cancer cells described herein, or cell lines (e.g., cancer cell lines)). As will be appreciated in the art, the effective amount of an antibody, antigen-binding fragment, or antibody-drug conjugate can vary depending, inter alia, on the patient's medical history and other factors such as the type (and / or dosage) of antibody being used.

[0205] The effective amounts and schedules for administration of the antibodies, antibody-encoding polynucleotides, antibody-drug conjugates, and / or compositions disclosed herein can be determined empirically and are within the skill of the art to make such determinations. The skilled artisan will understand that the dosage to be administered will vary depending, for example, on the mammal receiving the antibodies, antibody-encoding polynucleotides, antibody-drug conjugates, and / or compositions disclosed herein, the route of administration, the particular type of antibodies, antibody-encoding polynucleotides, antigen-binding fragments, antibody-drug conjugates and / or compositions being used, and other agents being administered to the mammal. Guidance for selecting an appropriate dosage for an antibody or antigen-binding fragment 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, N.J., 1985, ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp. 365-389.

[0206] The daily dose of the effective amount of the antibody is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose is less than 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dose exceeds 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or 0.01 mg / kg. In some embodiments, the dose is about 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.

[0207] In any method described herein, at least one antibody, an antigen-binding fragment thereof, an antibody-drug conjugate, or a pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, antibody-drug conjugates, or pharmaceutical compositions described herein), and optionally, at least one additional therapeutic agent can be administered to 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 can be administered in 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 can be administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent can be in two different compositions (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, at least one additional therapeutic agent can be administered as a pill, tablet, or capsule. In some embodiments, at least one additional therapeutic agent is administered as a sustained-release oral formulation.

[0208] In some embodiments, the one or more additional therapeutic agents can be administered to the subject prior to or subsequent to the administration of at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, the one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) can be administered to the subject such that 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) have an overlapping biological activity period in the subject's body.

[0209] In some embodiments, at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) can be administered to the subject over an extended period of time (e.g., at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional can determine the treatment period by using any of the diagnostic methods described herein or by pursuing the effectiveness of the treatment (e.g., by observing at least one symptom of cancer). A skilled medical professional, as described herein, can also vary the uniqueness and number (e.g., increase or decrease) of the antibody or antigen-binding antibody fragment, antibody-drug conjugate (and / or one or more additional therapeutic agents) administered to the subject, and can adjust the dosage (e.g., increase or decrease) or frequency of administration of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using the methods described herein and methods well known in the art).

[0210] In some embodiments, one or more additional therapeutic agents can be administered to the subject. This additional therapeutic agent can include one or more inhibitors selected from the following: B-Raf inhibitor, EGFR inhibitor, MEK inhibitor, ERK inhibitor, K-Ras inhibitor, c-Met inhibitor, anaplastic lymphoma kinase (ALK) inhibitor, phosphatidylinositol-3-kinase (PI3K) inhibitor, Akt inhibitor, mTOR inhibitor, PI3K / mTOR dual inhibitor, Bruton's tyrosine kinase (BTK) inhibitor, and isocitrate dehydrogenase 1 (IDH1) inhibitor and / or isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase-1) (IDO1) inhibitor (e.g., epacadostat).

[0211] In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of a HER3 inhibitor, an LSD1 inhibitor, an 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.

[0212] In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of trabectedin, nanoparticle albumin-bound paclitaxel (nab-paclitaxel), trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, reolysin, alimta, zykadia, sutent, temsirolimus, axitinib, everolimus, sorafenib, votrient, pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, temazolomide, IL-2, IFNα, vinblastine, thalidomide, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomib, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

[0213] 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, 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 therapeutic target CX3CL1, a therapeutic target CXCL9, a therapeutic target CXCL10, a therapeutic target CCL5, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0214] In some embodiments, carboplatin, nanoparticle albumin-bound paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

[0215] In some embodiments, the additional therapeutic agent is 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.

[0216] Pharmaceutical compositions and routes of administration Further provided herein is a pharmaceutical composition comprising at least one (e.g., one, two, three, or four) of the antibodies, antigen-binding fragments, or antibody-drug conjugates described herein. Any two or more (e.g., two, three, or four) of the antibodies, antigen-binding fragments, or antibody-drug conjugates described herein can be present in any combination in the pharmaceutical composition. The pharmaceutical composition can be formulated by any method well known in the art.

[0217] The pharmaceutical composition is formulated to be compatible with the 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, glycerin, propylene glycol or other synthetic solvents, an antibacterial or antifungal agent such as benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, an antioxidant such as ascorbic acid or sodium bisulfite, a chelating agent such as ethylenediaminetetraacetic acid, a buffer such as acetate, citrate, phosphate, and an isotonic agent such as a sugar (e.g., dextrose), a polyalcohol (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride), or any combination thereof. Liposome suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811). The formulation of the composition can be encapsulated in ampoules, disposable syringes, or multi-dose vials. If necessary (e.g., as in injectable formulations), proper fluidity can be maintained by coatings such as lecithin or surfactants. The absorption of the antibody or its antigen-binding fragment can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems that may include biodegradable biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0218] A composition comprising one or more of any of the antibodies, antigen-binding fragments, antibody-drug conjugates described herein can be formulated in unit dosage forms (i.e., physically discrete units containing a predetermined amount of the active compound for ease of administration and uniformity of dosage) for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration.

[0219] The toxicity and therapeutic efficacy of the compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). The LD50 (the dose lethal to 50% of the population) and the ED50 (the therapeutically effective dose in 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents with a high therapeutic index are preferred. When an agent exhibits undesirable side effects, care must be taken to minimize the potential damage (i.e., reduce the undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0220] Data obtained from cell culture assays and animal studies are used to formulate the appropriate dosage of any given agent to be used in a subject (e.g., a human). A therapeutically effective amount of one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) is an amount that treats (e.g., kills cancer cells) a disease in a subject (e.g., a human subject identified as having cancer) or reduces the symptoms of one or more diseases in a subject (e.g., a human) in terms of the severity, frequency, and / or duration of the subject (e.g., a human) where the subject is identified as having a risk of developing the disease (e.g., a subject who previously had cancer but is now cured). The effectiveness or dosage of any of the antibodies or antigen-binding fragments described herein can be determined by a medical or veterinary professional using methods well known in the art and simultaneously by observing one or more symptoms of the disease in a subject (e.g., a human). Certain factors can affect the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or illness, previous treatments, the general health and / or age of the subject, and the presence of other diseases).

[0221] Exemplary dosages include the antibodies or antigen-binding fragments, or antibody-drug conjugates described herein in milligrams or micrograms per kilogram of the subject's body weight (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; or about 1 μg / kg to about 50 μg / kg). These dosages cover a wide range, but those skilled in the art will understand that the effective amount of therapeutic agents, including antibodies and their antigen-binding fragments, whose titers vary, can be determined by methods well known in the art. Typically, relatively low dosages are administered first, and the attending physician or veterinary specialist (in the case of therapeutic use) or researcher (if still working in the development stage) will subsequently increase the dosage gradually until an appropriate response is obtained. Moreover, it is understood that the specific dosage level for any particular subject depends on various factors, including the activity of the specific compound used, age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the excretion rate, and the half-life of the antibody or antibody fragment in vivo.

[0222] The pharmaceutical composition can be included in a container, pack, or dispenser together with instructions for administration. The present disclosure also provides methods for manufacturing antibodies or their antigen-binding fragments, or antibody-drug conjugates for various uses described herein.

Example

[0223] The present invention is further described in the following examples, which do not limit the scope of the present invention described in the claims.

[0224] Example Ⅰ: Methods and Materials The following assays are used in the examples.

[0225] Binding Assay (a) Dispense 5x10 5 cells in 50 μL of medium into each well of a 96-well plate. (b) Add 100 μL of the antibody into the wells at different dilutions. (c) Incubate the plate at room temperature (RT) for 60 minutes. (d) Pellet the cells by centrifugation and wash three times with phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA). (e) Resuspend the cell pellet in 100 μL of PBS with 0.1% BSA containing a 1:500 Cy3-conjugated goat anti-human IgG antibody. (f) Incubate in the dark at room temperature for 30 minutes. (g) Wash the cells three times and resuspend in fluorescence-activated cell sorting (FACS) buffer. (h) Analyze the cells using a flow cytometer.

[0226] Antibody-dependent cell-mediated cytotoxicity (ADCC) assay (a) Wash the target cells once with PBS before calcein AM labeling. (b) Use a 1:333 2.5 mM calcein AM stock solution for labeling the target cells. (c) Incubate the cells for 30 minutes at 37 °C in the dark. (d) Wash the cells three times with PBS. (e) 1x10 4 Dispense 50 μL of the calcein AM-labeled target cells into each well. (f) Add 100 μL of the diluted antibody to the wells. (g) Incubate the plate at room temperature for 60 minutes. (h) 5x10 4 Add PBMCs (which affect the cells) at a ratio of 5 (E / T ratio = 5) in 50 μL of medium in each well. (i) Incubate this plate at 37 °C for 4 hours. (j) Pellet the cells by centrifugation and transfer 180 μL of the supernatant to another 96-well plate with a translucent bottom and black walls. (k) Read the plate at wavelengths of 485 excitation and 520 emission.

[0227] Complement-dependent cytotoxicity (CDC) assay (a) Collect target cells and stain with calcein AM as an ADCC (assay with only calcein release). (b) Seed 50 μL of target cells (1x10 5 cells) into each well of a 96-well plate. (c) Add 100 μL of antibody at different concentrations into the wells. [[ID=IO]](d) Incubate the plate at room temperature for 15 minutes. (e) Add 50 μL of 10% complement-concentrated human serum to each well (final concentration 5%). (f) Incubate the plate at room temperature for 45 minutes. (g) Transfer 180 μL of the supernatant to another 96-well plate with a translucent bottom and black walls (for the calcein release assay only). (h) Wash the cells three times with PBS containing 0.1% BSA. (i) Stain the cells with 2 μL of 7-aminoactinomycin D (7AAD) per well for 15 minutes at room temperature in the dark. (j) Wash the cells three times and analyze the cells by flow cytometry.

[0228] T cell activation Pre-activated peripheral blood mononuclear cells (PBMCs) were used in several ADCC experiments. (a) Dynabeads (human T cell activator CD3 / CD28) are used for the activation of PBMCs. (b) After washing with buffer, add Dynabeads to PBMCs at a 1:1 ratio together with 30 U / mL of interleukin-2 (IL2). (c) Incubate the cell mixture for a sufficient period of time. (d) At the end of the incubation, remove the beads with a magnet and use the activated PBMCs for the ADCC assay.

[0229] Cell binding assay containing MDA231 cells (a) Culture MDA231 cells in medium at 1x10 6Prepare at a concentration of cells / mL. (b) Dilute the antibody sample to an appropriate concentration. (c) Transfer 50 μL of cells to each well of a 96-well V-bottom plate. (d) Transfer 50 μL of antibody to each well of a 96-well V-bottom plate. (e) Incubate the cell mixture at room temperature for 60 minutes. (f) Pellet the cells by centrifugation and wash twice with FACS buffer. (g) Resuspend the cells (1:500) in 100 μL of FACS buffer containing Cy3-conjugated goat anti-human (GAH) IgG antibody in each well. (h) Incubate at room temperature for 30 minutes and wash twice with FACS buffer. (i) FACS analysis.

[0230] Internalization assay involving MDA231 and SIHA cells (a) Add 50 μL of cell suspension (MDA231 or SIHA cells) to each well of a 96-well plate at 1x10 6 / m. (b) Add 50 μL of Ab to the corresponding wells. (c) Incubate at 37 °C for 30 minutes. (d) Add 100 μL of pHrodo Red-labeled GAH IgG to each well and incubate at 37 °C for 24 hours. (d) Trypsinize and collect the cells, wash twice, and perform FACS.

[0231] Complement-dependent cytotoxicity (CDC) assay involving MDA231 cells (a) Target cells: Wash MDA231 cells twice with PBS and then adjust to a concentration of 0.5x10 6 / mL in PBS. (b) Seed the cells at 300 μL / well in a flat-bottom 24-well plate. (c) Add 300 μL of 20 μg / mL antibody to the corresponding wells to give a final concentration of 10 μg / mL. (d) Incubate the plate at 37 °C for 48 hours. (e) At the end of the incubation, digest the cells with trypsin and wash twice with simple medium. (f) Resuspend the cell pellet in 100 μL of simple medium and transfer the cells to a 96-well plate. (g) Add 100 μL of 10% complement-concentrated serum to each well. (h) Incubate the cells at 37 °C for 4 hours. (i) Wash the cells twice with FACS buffer. (j) Add 100 μL of trypsin and dissociate the cells for 3 minutes. (k) Resuspend the cell pellet in FACS buffer containing 7AAD (1:50 dilution). (l) After incubation at room temperature for 15 minutes, wash the cells twice. (m) Perform FACS analysis.

[0232] Example 2: Bispecific antibody that binds to CD20 and CD3 The bispecific antibody was designed to bind to CD20 and CD3. This bispecific antibody has two common light chains (having the same sequence) and two different heavy chains. The variable regions of the two heavy chains and the sequence of the common light chain are shown below.

[0233] VHa for CD20 (designed from rituximab VH): TIFF2025111661000002.tif19159 VHb for CD3 (designed from MAb12F6 VH): TIFF2025111661000003.tif18160 Common VL (VL of rituximab) TIFF2025111661000004.tif11160

[0234] The 12F6 antibody is described, for example, in Construction and characterization of a humanized anti - human CD3 monoclonal antibody 12F6 with effective immunoregulation functions, Immunology, 116(4),487 - 498(2005), which is hereby incorporated by reference in its entirety. The sequences for the parental antibodies are also shown below for comparison purposes.

[0235] Parental CD20 VH (Rituximab VH): TIFF2025111661000005.tif18160 Parental CD20 VL (Rituximab): TIFF2025111661000006.tif11160 Parental CD3 VH (MAb12F6 VH): TIFF2025111661000007.tif18161 Parental CD3 VL (MAb12F6 VL): TIFF2025111661000008.tif11158

[0236] The CDR sequences of the redesigned VH and VL are also summarized in the following table.

[0237] (Table 1) VHa for CD20 heavy chain TIFF2025111661000009.tif37161

[0238] (Table 2) VHb for CD3 heavy chain TIFF2025111661000010.tif37161

[0239] (Table 3) VL for common light chain TIFF2025111661000011.tif37161

[0240] The 3D (three-dimensional) isoelectric point (PI) of rituximab Fv (VH+VL) is 9.9, and the 3DPI of 12F6Fv is 9.8. After redesigning the sequences, the 3DPI of VHa+common VL was 10.0, and the 3DPI of VHb+common VL was 9.1. The PI change did not affect the binding affinity for CD20, and the second antigen-binding region still maintained a reasonable binding affinity for CD3. The mutations in the two VH chains are shown in the following table.

[0241] (Table 4) Modified amino acids in VH (CD20) TIFF2025111661000012.tif15161

[0242] (Table 5) Modified amino acids in VH (CD3) TIFF2025111661000013.tif35161

[0243] In Figures 1A and 1B, the antigen-binding abilities of the redesigned rituximab (antibody A) and the redesigned 12F6 (antibody B) were tested respectively. Figure 1A shows that the redesigned rituximab (antibody A) binds to CD20-positive Raji cells. Antibody A is a homodimer having two VHa (SEQ ID NO:1), and two common VL (SEQ ID NO:3). Figure 1B shows that the redesigned 12F6 (antibody B) binds to CD3-positive Jurkat cells. Antibody B is also a homodimer having two VHb (SEQ ID NO:2) and two common VL (SEQ ID NO:3). These data suggest that the redesigned rituximab heavy chain, the redesigned 12F6 heavy chain, and the common light chain are conjugated into a functional bispecific antibody, for example, via the "knob-into-hole" technology.

[0244] Therefore, a CD20 / CD3 "unbalanced bispecific antibody" was designed. Knob-and-hole mutations were also introduced into the constant region of the heavy chain to facilitate the formation of bispecific antibodies.

[0245] The full-length sequences of the heavy and light chains are shown below.

[0246] Full length of CD20 heavy chain version 1 (wild-type IgG1 Fc) TIFF2025111661000014.tif54161 Full length of CD20 heavy chain version 2 (IgG1 Fc with Y407T (hole) mutation): TIFF2025111661000015.tif55161 Full length of CD20 heavy chain version 3 (IgG1 Fc with T366Y (knob) mutation): TIFF2025111661000016.tif54161 Full length of CD3 heavy chain version 1 (wild-type IgG1 Fc): TIFF2025111661000017.tif54161 Full length of CD3 heavy chain version 2 (IgG1 Fc with T366Y (knob) mutation): TIFF2025111661000018.tif54161 Full length of CD3 heavy chain version 3 (IgG1 Fc with Y407T (hole) mutation): TIFF2025111661000019.tif54161 Full length of the common light chain: TIFF2025111661000020.tif26160

[0247] The IgG1 heavy chain for CD20 with Y407T (EU numbering) (version 2; SEQ ID NO: 35), and the IgG1 heavy chain for CD3 with the T366Y (EU numbering) mutation (version 2; SEQ ID NO: 38) were selected to generate bispecific antibodies for further experiments. This bispecific antibody also has two common light chains (SEQ ID NO: 40).

[0248] This unbalanced bispecific antibody also includes the following characteristics: (1) the CD3 binding affinity is significantly reduced to increase safety; (2) the ADCC / CDC effector function is maintained to expand clinical implementation; (3) the biochemical and biophysical properties of the CD20 binding arm and the CD3 binding arm are differentiated to enable better separation of the bispecific antibody during downstream purification processes.

[0249] As shown in the following examples, this antibody had better CD20+ Raji cell killing efficacy than CD20 homodimer and rituximab in the presence of human PBMC. On the other hand, under the same conditions, this antibody failed to kill CD3+ Jurkat cells or depleted normal T cells. Therefore, this antibody has shown promise for a broader clinical application than current anti-CD20 cancer therapies: (1) compared with rituximab, this antibody has a T cell mobilization function; (2) compared with CAR-T / other T cell mobilization therapies, this antibody maintains a functional effector function; (3) this antibody shows no safety concerns in vitro. Overall, the CD20 / CD3 bispecific antibody and its platform described in this disclosure can address unmet needs in the field of cancer target therapy.

[0250] The bispecific antibody disclosed herein was purified through two steps: affinity purification using Protein A (round 1) and anion exchange purification using monoQ5 / 50 (round 2). In the second round, a gradient pH buffer (e.g., PBS) was used to elute the antibody. T cell activation assays were evaluated on different fractions after elution. In Figure 20, the numbers indicate different fractions. Only the CD20 / CD3 bispecific antibody can activate T cells, so the T cell activation assay can evaluate the purity and content of the CD20 / CD3 bispecific antibody in each fraction. The results showed that fractions 4-7 had relatively pure CD20 / CD3 bispecific antibody, and demonstrated that the CD20 / CD3 bispecific antibody was purified by the method described herein.

[0251] Furthermore, the pI of the antibodies described herein was also measured. This information may be useful for choosing an appropriate pH for elution.

[0252] (Table 6) TIFF2025111661000021.tif78128

[0253] Example 3: Binding Affinity of Bispecific Antibodies After computer-aided design, the CD20 homodimeric IgG containing the designed VH sequence (SEQ ID NO:1) and the common VL sequence (SEQ ID NO:3) showed a similar binding ability to CD20 as compared to the parental anti-CD20 IgG (parental CD20). A cell-binding affinity assay was performed using Raji cells (expressing CD20). The binding results are shown in Figure 2A.

[0254] The CD3 homodimeric IgG containing the designed VH sequence (SEQ ID NO:2) and the common VL sequence (SEQ ID NO:3) had a decreased binding ability to CD3 compared to the parental anti-CD3 IgG (parental CD3). A cell-binding affinity assay was performed using Jurkat cells (expressing CD3). The binding results are shown in Figure 2B.

[0255] Example 4: Activation of T Cells by Unbalanced CD20 / CD3 Bispecific Antibodies The unbalanced CD20 / CD3 bispecific monoclonal antibody (BsMab) activated only T cells in the presence of target tumor cells. The following experiments were conducted by using Raji cells as CD20+ target tumor cells, 293 cells as CD20 control cells, and Jurkat cells as a T cell model to test whether the CD20 / CD3 BsMab could activate T cells in the presence of target tumor cells due to clusters formed by multiple BsMabs that bind to both T cells and target tumor cells. In contrast, in the presence of CD20 control cells, the CD20 / CD3 BsMab did not activate T cells due to weak binding by one arm to CD3 on T cells.

[0256] In this example, the following experimental procedures were used: (1) Seed Raji&Jurkat and Jurkat&293 at 1x10 5 separately into U-bottom 96-well plates. (2) Add the test antibody and incubate overnight (19 hours). (3) Wash the cells once with PBS + 0.1% BSA. (4) Add anti-human CD69 antibody (labeled with PE) (1.5 μL / well) and incubate at room temperature for 30 minutes. (5) Wash the cells once. (6) Read.

[0257] The results are shown in Figure 3. The T cell activation efficacy by different concentrations of the test antibody in the presence of Raji cells is shown in Figure 4. An isotype antibody (non-specific IgG1 antibody) was used as a control. T cell activation was measured by the expression of CD69 on the surface of Jurkat cells.

[0258] Example 5: Unbalanced CD20 / CD3 BsMab Induces PBMC-Mediated Cell Killing Unbalanced CD20 / CD3 BsMab showed better PBMC-mediated cell killing than rituximab and CD20 homodimeric antibodies before and after T cell activation.

[0259] Before T cell activation: Fresh peripheral blood mononuclear cells (PBMC) from healthy donors were left overnight at 37°C and incubated with calcein-labeled CD20+ Raji cells for 4 hours in the presence of different antibodies shown in the figure. The cell death rate was measured by calcein release. The results are shown in Figure 5.

[0260] After T cell activation (4 days): Fresh PBMCs from healthy donors were incubated with recombinant IL-2 and CD3 / CD28 beads for 4 days to activate T cells, and then incubated with calcein-labeled CD20+ Raji cells in the presence of different antibodies shown in the figure for 2 hours. The cell death rate was measured by calcein release. The results are shown in Figure 6.

[0261] After T cell activation (7 days): Fresh PBMCs from healthy donors were incubated with recombinant IL-2 and CD3 / CD28 for 7 days to activate T cells, and then incubated with calcein-labeled CD20+ Raji cells in the presence of different antibodies shown in the figure for 2 hours. The cell death rate was measured by calcein release. The results are shown in Figure 7.

[0262] Under the same conditions as shown in the PBMC killing assay, to address the safety concern of whether unbalanced CD20 / CD3 BsMab also kills CD3+ T cells, CD3+ Jurkat cells were used as a control in each experiment. Only the highest antibody concentration (10 μg / mL) was tested. The results of T cell activation are shown in Figure 8. The results after T cell activation (4 days) are shown in Figure 9. The results after T cell activation (7 days) are shown in Figure 10. The number of Jurkat cells in the group treated with PBS was set as the baseline. Therefore, when the number of Jurkat cells is equal to that of the PBS-treated group, the percentage of killing is 0. When the number of cells is more than that of the PBS-treated group, the percentage of killing is negative.

[0263] As a result, no killing of Jurkat cells was observed before T cell activation and 4 days after T cell activation. However, killing of Jurkat cells was observed 7 days after T cell activation, and rituximab and CD20 homodimer IgG did not cause killing of Raji cells under the same conditions. This suggests that the killing of Jurkat cells 7 days later was caused by T cell super-activation. To further test whether the natural T cells activated for 7 days were also killed in the presence of unbalanced CD20 / CD3 BsMab, the same PBMC activated for 7 days were incubated overnight with different antibodies shown in the figure, and the T cell depletion status was checked. The results are shown in Figure 11. LALA in the figure is a CD20 / CD3 BsMab with L234A and L235A mutations (EU numbering). Antibodies with L234A and L235A mutations do not have Fc effector functions and were used as negative controls.

[0264] Example 6: T Cell Depletion by Unbalanced Bispecific CD20 / CD3 Antibody Experiments were also conducted to test whether pre-activated T cells could be depleted by unbalanced CD20 / CD3 BsMab.

[0265] Figure 12 shows that non-activated T cells in PBMC were not depleted by overnight incubation with unbalanced CD20 / CD3 BsMab.

[0266] Example 7: Induction of Complement-Dependent Cytotoxicity Since CD20 / CD3 BsMab has an arm that binds to CD20 with high affinity, experiments were conducted to test whether CD20-arm binding was sufficient to induce complement-dependent cytotoxicity. This antibody was incubated with human complement-concentrated serum and CD20+ Raji cells. Unbalanced CD20 / CD3 BsMab decreased the CDC efficacy compared to rituximab and CD20 homodimer antibodies. The detection results by FACS (7AAD) are shown in Figure 13. The detection results by calcein release are shown in Figure 14.

[0267] Example 8: Safety Evaluation It was also tested whether CD3+ Jurkat cells and normal T cells were killed by unbalanced CD20 / CD3 BsMab. Figure 15 shows that a high dose of unbalanced CD20 / CD3 BsMab did not induce CDC against Jurkat cells.

[0268] Figure 16 shows that unbalanced CD20 / CD3 BsMab did not induce T cell death after co-incubation with human serum containing PBMC and human complement concentrated serum.

[0269] Example 9: Unbalanced CD20 / CD3 BsMab can kill rituximab-resistant Raji cells To test whether unbalanced CD20 / CD3 BsMab could kill rituximab-resistant Raji cells (RRCL), RRCL was incubated with activated PBMC from three different donors for 7 days in the presence of the antibodies shown in the figure. Significant killing of RRCL was observed in the presence of unbalanced CD20 / CD3 BsMab (Figures 17-19).

[0270] Example 10: Animal studies on unbalanced CD20 / CD3 BsMab Experiments were conducted to evaluate the effect of CD20 / CD3 BsMab in animals.

[0271] Raji cells, human PBMC, and unbalanced CD20 / CD3 BsMab were mixed and injected into mice by intravenous administration. These Raji cells were labeled with luciferase. Each mouse in the treatment group (B-NDG, Biocytogen, Beijing, Cat#201811808) was administered 5x10 5 Raji cells, 2.5x10 6 human PBMC cells, and 60 μg of the antibody. On day 0, day 2, day 3, and every 3 days after day 3, the mice were imaged to track Raji cell depletion.

[0272] On day 0, luciferase-labeled Raji cells and human PBMC cells were mixed with either phosphate-buffered saline PBS (Group G1; control; n = 4), CD20 / CD3 BsMab (Group G2; n = 4), or rituximab (anti-CD20 antibody; Group G3; n = 4). These mice were imaged for the first time 15 minutes after intravenous (i.v.) injection and then on days 2, 3, and every 3 days after day 3.

[0273] Figure 21A shows that CD20 / CD3 BsMab and rituximab had no obvious toxic effects. Figure 21B shows that both CD20 / CD3 BsMab and rituximab had a tumor inhibitory effect, and rituximab was less effective than CD20 / CD3 BsMab. The difference in the tumor inhibitory effect was observed after injection on day 16.

[0274] Example 11: Characterization of unbalanced bispecific antibodies Experiments were conducted to characterize the purified CD20 / CD3 bispecific antibody sample.

[0275] First, reducing capillary electrophoresis sodium dodecyl sulfate (Re-CE-SDS) was performed on the purified CD20 / CD3 bispecific antibody sample. The results showed that there were three main peaks. Based on the molecular weight, peak #1 was the common light chain (LC), and peaks #2 and #3 were two different heavy chains (HC) (Figure 22A).

[0276] Non-reducing CE (non-Re-CE-SDS) was also performed. The results showed that there was one main peak of CD20 / CD3 bispecific IgG (Figure 22B). The results in Figures 22A and 22B suggest that the 1 CD20 / CD3 bispecific antibody sample has good purity.

[0277] Second, differential scanning fluorimetry (DSF) was performed for the measurement of protein melting temperature (Tm), and static light scattering (SLS) was performed for the measurement of the aggregation temperature at 266 nm (Tagg266) and the aggregation temperature at 473 nm (Tagg473). These samples were submitted to the UNcle system for analysis. A temperature gradient of 1 °C / min was performed for DSF and SLS with monitoring from 20 °C to 95 °C. UNcle measured SLS at 266 nm and 473 nm. Tm and Tagg were calculated and analyzed using the UNcle analysis software.

[0278] Some of the tested antibodies had two Tm values, and some had three Tm values. This is because IgG has a multi-domain structure, the CH2 domain usually has a Tm of about 70 °C in PBS, and the CH3 is more stable with a Tm of about 80 °C. Fab has a wide range of Tm values from about 50 to 85 °C due to its large sequence variation. Therefore, the Tm values measured by various analytical methods are usually "apparent" transition temperatures rather than the formal melting temperature. When the antibody is the whole IgG, often two to three Tm values are present in the DSF measurement. It is not easy to determine which Tm represents which domain.

[0279] In the case of this bispecific antibody, the Tm of 86.7 °C may represent only the CH3 domain. The other one or two lower Tm values represent Fab, CH2, or Fab + CH2.

[0280] Tagg is the temperature at which SLS begins to detect aggregation. Tagg266 measures SLS at 266 nm, which is more sensitive and suitable for detecting smaller particles. Tagg473 is measured at 473 nm and is suitable for measuring larger particles.

[0281] Both the DSF and SLS data indicate that the CD20 / CD3 bispecific antibody has good thermal stability.

[0282] (Table 7) TIFF2025111661000022.tif31161

[0283] Thirdly, dynamic light scattering (DLS) detected only molecular particles of one size (10.15 nm). This result indicated that there was no aggregation in the sample.

[0284] (Table 8) TIFF2025111661000023.tif25161

[0285] These characterization data suggest that the CD20 / CD3 bispecific antibody has good development potential as a therapeutic antibody.

[0286] Example 12: Bispecific Antibody that Binds to PD-L1 and CD55 Two versions of bispecific antibodies were designed to bind to PD-L1 and CD55 (PD-L1 / CD55 BsMab v1 and PD-L1 / CD55 BsMab v2). These bispecific antibodies have two common light chains and two different heavy chains.

[0287] The sequences of the variable regions of the two heavy chains and the common light chain of the first version of the bispecific antibody (PD-L1 / CD55 BsMab v1) are shown below.

[0288] VHa for PD-L1 (designed from abelumab): TIFF2025111661000024.tif19161 VHb for CD55 (designed from CD55 ScFV): TIFF2025111661000025.tif19160 Common VL (designed from CD55 ScFV): TIFF2025111661000026.tif18159

[0289] The CD55 ScFV is described, for example, in Identification of a human anti-CD55 single-chain Fv by subtractive panning of a phage library using tumor and nontumor cell lines, Cancer Res. 59(11), 2718-2723(1999), which is hereby incorporated by reference in its entirety. The sequence of this parental antibody is also shown below for comparison purposes.

[0290] Parental PD-L1 VH: TIFF2025111661000027.tif18161 Parental PD-L1 VL: TIFF2025111661000028.tif19159 Parental CD55 VH: TIFF2025111661000029.tif18160 Parental CD55 VL: TIFF2025111661000030.tif18159

[0291] The 3DPI of abelumab Fv (VH+VL) is 9.4, and the 3DPI of anti-CD55 Fv is 9.8. After redesign of the sequences, the 3DPI of VHa+common VL was 9.9, and the 3DPI of VHb+common VL was 9.3. The mutations to the two VH chains are shown in the table below.

[0292] (Table 9) Modified amino acids in VH (PD-L1) TIFF2M25111661000031.tif20161

[0293] (Table 10) Modified amino acids in VH (CD55) TIFF2025111661000032.tif20161

[0294] Example 13: Binding affinities of newly designed PD-L1 and CD55 antibodies Experiments were conducted to determine the binding affinity of the newly designed PD-L1 and CD55 antibodies.

[0295] The anti-PD-L1 homodimer IgG (PD-L1v1) containing the designed VH sequence (SEQ ID NO:4) and the common VL sequence (SEQ ID NO:6) had a weaker binding affinity than the parental anti-PD-L1 antibody (PD-L1wt) (Figure 23A). The anti-CD55 homodimer IgG (CD55v1) containing the designed VH sequence (SEQ ID NO:5) and the common VL sequence (SEQ ID NO:6) had a similar binding affinity compared to the parental anti-CD55 antibody (CD55wt) (Figure 23B).

[0296] Since the bispecific antibody had to bind to the cancer-specific antigen (PD-L1) with high affinity and the other arm of the bispecific antibody had to bind to the cancer-related antigen (CD55) with low affinity, the antibodies (CD55v1 and PD-L1v1) did not meet this requirement.

[0297] Therefore, a second version of the bispecific antibody was designed to bind to PD-L1 and CD55 (PD-L1 / CD55 BsMab v2). VHa and VHb for the second version of the bispecific antibody were the same as VHa and VHb of the first version of the bispecific antibody. However, the common light chain was redesigned based on the method described herein. The sequence of the redesigned common light chain is shown below.

[0298] Common VL2 (redesigned from SEQ ID NO:6): TIFF2025111661000033.tif19159

[0299] An alignment of common VL (SEQ ID NO:6) and common VL2 (SEQ ID NO:7) is shown in Figure 24. The underlined sequences are the sequences of the light chain constant region.

[0300] The redesigned CDR sequences of VH and VL are shown below.

[0301] (Table 11) VHa for the PD-L1 heavy chain TIFF2025111661000034.tif37161

[0302] (Table 12) VHb for the CD55 heavy chain TIFF2025111661000035.tif37161

[0303] (Table 13) VL version 1 for the common VL TIFF2025111661000036.tif37161

[0304] (Table 14) VL version 2 for the common VL TIFF2025111661000037.tif37161

[0305] Furthermore, since the λ light chain has less commonality compared to the κ light chain in human serum, in the examples, the constant region of the λ light chain was replaced with the constant region of the κ light chain.

[0306] Experiments were conducted to determine the binding affinity of the second version of the antibody. The anti-PD-L1 homodimer IgG (PD-L1v2) containing the designed VH sequence (SEQ ID NO:4) and the common VL2 sequence (SEQ ID NO:7) had a similar binding affinity compared to the parental anti-PD-L1 antibody (PD-L1wt) (Figure 25A). The anti-CD55 homodimer IgG (CD55v2) containing the designed VH sequence (SEQ ID NO:5) and the common VL2 sequence (SEQ ID NO:7) had a weaker binding affinity compared to the parental anti-CD55 antibody (CD55wt) (Figure 25B). Therefore, the binding affinity of the antibody with the redesigned sequence met the requirements, and PD-L1 / CD55 BsMab v2 was selected for further experiments. PD-L1 / CD55 BsMab v2 has two common light chains (κ chains) containing SEQ ID NO:7, one IgG1 heavy chain containing SEQ ID NO:4, and one IgG1 heavy chain containing SEQ ID NO:5. Furthermore, the heavy chain against PD-L1 has a Y407T mutation (EU numbering), and the IgG1 heavy chain against CD55 has a T366Y mutation (EU numbering).

[0307] The full-length sequences of these heavy and light chains are shown below.

[0308] Full length of PD-L1 heavy chain: TIFF2025111661000038.tif54161 Full length of CD55 heavy chain: TIFF2025111661000039.tif54161 Full length of CD55 common light chain version 1: TIFF2025111661000040.tif26160 Full length of CD55 common light chain version 2: TIFF2025111661000041.tif26160

[0309] Furthermore, the pI of the antibodies described herein was also measured. This information is important for selecting the appropriate pH for elution.

[0310] (Table 15) TIFF2025111661000042.tif60132

[0311] Example 14: Complement-dependent cytotoxicity (CDC) for the PD-L1 / CD55 bispecific antibody An experiment was conducted to test the complement-dependent cytotoxicity of the PD-L1 / CD55 bispecific antibody. These parental antibodies were included for comparison purposes. The assay was performed on MDA231 cells based on the protocol described herein, and the concentration of each antibody was 10 μg / mL. The results are shown in Figures 26A - 26B.

[0312] As shown in the figure, both the anti-PD-L1 (PD-L1wt) and anti-CD55 (CD55wt) parental antibodies can induce CDC. The PD-L1 / CD55 bispecific antibody v1 had a much weaker CDC compared to the parental anti-PD-L1 antibody (PD-L1wt) and the parental anti-CD55 antibody (CD55wt). In contrast, the PD-L1 / CD55 bispecific antibody v2 had a much higher CDC compared to the first version, the parental anti-PD-L1 antibody (PD-L1wt), and the parental anti-CD55 antibody (CD55wt). The CDC effect of the PD-L1 / CD55 bispecific antibody v2 was approximately 4.5-fold higher compared to the CDC effectiveness of the first version of the bispecific antibody.

[0313] Example 15: Internalization induced by the PD-L1 / CD55 bispecific antibody An experiment was conducted to evaluate the internalization induced by the PD-L1 / CD55 bispecific antibody.

[0314] Internalization assays were performed on two versions of the PD-L1 / CD55 bispecific antibodies and their parental antibodies. MDA231 cells were used in the first internalization experiment (Figure 27A), and SIHA cells were used in the second internalization experiment (Figure 27B). The cells were mixed with 20 μg / mL antibody and incubated at 37 °C for 30 minutes. Then the pHrodo-labeled secondary antibody was added and the cells were incubated at 37 °C for 24 hours. The cells were then harvested and analyzed by FACS.

[0315] CD55 is a receptor for echovirus and coxsackievirus B infection and is known as a receptor with internalization ability. Thus, the anti-CD55 parental monoclonal antibody (CD55wt) causes rapid internalization of CD55. As shown in Figure 27A, in MDA231 cells with similar PD-L1 and CD55 expression levels, the internalization induced by the anti-PD-L1 antibody was much slower than that of CD55. However, both the PD-L1 / CD55 bispecific antibodies v1 and v2 were able to cause internalization, and this internalization rate was comparable to that of the anti-CD55 parental monoclonal antibody.

[0316] In Figure 27B, the expression of CD55 in SIHA cells is higher than that of PD-L1. The PD-L1 / CD55 bispecific antibodies v1 and v2 can induce better internalization than the parental anti-PD-L1 antibody and the parental anti-CD55 antibody. Nevertheless, considering the decreased binding to CD55 of PD-L1 / CD55 BsMab v2 compared to v1, PD-L1 / CD55 BsMab v2 should have a better efficacy / safety balance in vivo and should be safer than PD-L1 / CD55 BsMab v1. Therefore, it is expected that the PD-L1 / CD55 BsMab can induce the death of target cancer cells at three different levels: (1) blocking the PD1 / PD-L1 interaction; (2) inducing PD-L1 internalization; 3) when complexed with a drug, the antibody-drug conjugate can kill cancer cells.

[0317] Other embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is illustrative of the scope of the invention as defined by the appended claims and is not limiting. Other aspects, advantages, and modifications are within the scope of the appended claims.

[0318] Sequence information SEQUENCE LISTING <110> AB THERAPEUTICS, INC. <120> BISPECIFIC ANTIBODIES AND USES THEREOF <150> US 62 / 654,112 <151> 2018-04-06 <150> US 62 / 539,970 <151> 2017-08-01 <160> 68 <170> PatentIn version 3.5 <210> 1 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 1 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ala 115 120 <210> 2 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 2 Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Glu Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gln Asp Tyr Asp Val Tyr Phe Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 3 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 3 Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 4 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 4 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Glu Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Glu 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 5 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 5 Gln Val Lys Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Gly Met Ser Trp Ile Arg Gln Thr Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Ser Gly Gly Ser Tyr Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Asn Gly Thr Leu Tyr Tyr Tyr Leu Met Asp Tyr Trp Gly 100 105 110 Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 6 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 6 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ala 85 90 95 Ser Thr Arg Ile Phe Gly Gly Gly Thr Lys Val Thr Val Leu Arg 100 105 110 <210> 7 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 7 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Arg Ile Phe Gly Gly Gly Thr Lys Val Thr Val Leu Arg 100 105 110 <210> 8 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 8 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ala 115 120 <210> 9 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 9 Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 10 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 10 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gln Asp Tyr Asp Val Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 11 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 11 Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Thr Lys Arg 100 105 <210> 12 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 12 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 13 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 13 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 14 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 14 Gln Val Lys Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Gly Met Ser Trp Ile Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Ser Gly Gly Ser Tyr Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Asn Gly Thr Leu Tyr Tyr Tyr Leu Met Asp Tyr Trp Gly 100 105 110 Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 15 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 15 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Phe 35 40 45 Met Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Val 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ala 85 90 95 Ser Thr Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 16 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 16 Ser Tyr Asn Met His 1 5 <210> 17 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 17 Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 18 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 18 Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val 1 5 10 <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 19 Gly Tyr Thr Phe Thr Ser Tyr 1 5 <210> 20 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 20 Tyr Pro Gly Asn Gly Asp 1 5 <210> 21 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 21 Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val 1 5 10 <210> 22 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 22 Ser Tyr Thr Met His 1 5 <210> 23 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 23 Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 24 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 24 Trp Gln Asp Tyr Asp Val Tyr Phe Asp Tyr 1 5 10 <210> 25 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 25 Gly Tyr Thr Phe Thr Ser Tyr 1 5 <210> 26 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 26 Asn Pro Ser Ser Gly Tyr 1 5 <210> 27 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 27 Trp Gln Asp Tyr Asp Val Tyr Phe Asp Tyr 1 5 10 <210> 28 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 28 Arg Ala Ser Ser Ser Val Ser Tyr Ile His 1 5 10 <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 29 Ala Thr Ser Asn Leu Ala Ser 1 5 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 30 Gln Gln Trp Thr Ser Asn Pro Pro Thr 1 5 <210> 31 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 31 Arg Ala Ser Ser Ser Val Ser Tyr Ile His 1 5 10 <210> 32 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 32 Ala Thr Ser Asn Leu Ala Ser 1 5 <210> 33 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 33 Gln Gln Trp Thr Ser Asn Pro Pro Thr 1 5 <210> 34 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 34 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 35 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 35 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 36 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 36 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Tyr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 37 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 37 Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Glu Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gln Asp Tyr Asp Val Tyr Phe Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 38 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 38 Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Glu Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gln Asp Tyr Asp Val Tyr Phe Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Tyr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 39 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 39 Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Glu Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gln Asp Tyr Asp Val Tyr Phe Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 40 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 40 Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 41 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 41 Ser Tyr Ile Met Met 1 5 <210> 42 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 42 Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val Lys 1 5 10 15 Gly <210> 43 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 43 Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr 1 5 10 <210> 44 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 44 Gly Phe Thr Phe Ser Ser Tyr 1 5 <210> 45 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 45 Tyr Pro Ser Gly Gly Ile 1 5 <210> 46 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 46 Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr 1 5 10 <210> 47 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 47 Gly Tyr Gly Met Ser 1 5 <210> 48 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 48 Thr Ile Asn Ser Gly Gly Ser Tyr Thr Tyr Tyr Ser Asp Ser Val Lys 1 5 10 15 Gly <210> 49 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 49 Arg Asn Gly Thr Leu Tyr Tyr Tyr Leu Met Asp Tyr 1 5 10 <210> 50 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 50 Gly Phe Thr Phe Ser Gly Tyr 1 5 <210> 51 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 51 Asn Ser Gly Gly Ser Tyr 1 5 <210> 52 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 52 Arg Asn Gly Thr Leu Tyr Tyr Tyr Leu Met Asp Tyr 1 5 10 <210> 53 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 53 Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser 1 5 10 <210> 54 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 54 Asp Val Ser Lys Arg Pro Ser 1 5 <210> 55 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 55 Ser Ser Tyr Thr Ser Ala Ser Thr Arg Ile 1 5 10 <210> 56 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 56 Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser 1 5 10 <210> 57 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 57 Asp Val Ser Lys Arg Pro Ser 1 5 <210> 58 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 58 Ser Ser Tyr Thr Ser Ala Ser Thr Arg Ile 1 5 10 <210> 59 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 59 Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser 1 5 10 <210> 60 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 60 Asp Val Ser Asn Arg Pro Ser 1 5 <210> 61 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 61 Ser Ser Tyr Thr Ser Ser Ser Thr Arg Ile 1 5 10 <210> 62 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 62 Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser 1 5 10 <210> 63 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 63 Asp Val Ser Asn Arg Pro Ser 1 5 <210> 64 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 64 Ser Ser Tyr Thr Ser Ser Ser Thr Arg Ile 1 5 10 <210> 65 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 65 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Glu Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Glu 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Tyr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 66 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 66 Gln Val Lys Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Gly Met Ser Trp Ile Arg Gln Thr Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Ser Gly Gly Ser Tyr Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Asn Gly Thr Leu Tyr Tyr Tyr Leu Met Asp Tyr Trp Gly 100 105 110 Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 67 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 67 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ala 85 90 95 Ser Thr Arg Ile Phe Gly Gly Gly Thr Lys Val Thr Val Leu Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 68 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 68 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Arg Ile Phe Gly Gly Gly Thr Lys Val Thr Val Leu Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

Claims

1. A first heavy chain variable region, a second heavy chain variable region, a first light chain variable region, and a second light chain variable region comprising a bispecific antibody or an antigen-binding fragment thereof, wherein The first heavy chain variable region and the first light chain variable region bind to each other to form a first antigen-binding region that specifically binds to a first antigen with a binding affinity higher than 10 7 M -1 、10 8 M -1 、10 9 M -1 、10 10 M -1 、10 11 M -1 、or 10 12 M -1 and that forms a first antigen-binding region that specifically binds to the first antigen with a binding affinity higher than 10 The second heavy chain variable region and the second light chain variable region bind to each other to form a second antigen-binding region that specifically binds to the second antigen with a binding affinity lower than 10 9 M -1 、10 8 M -1 、10 7 M -1 、10 6 M -1 、10 5 M -1 、or 10 4 M -1 than that specifically binds to the second antigen with a lower binding affinity said bispecific antibody or an antigen-binding fragment thereof.

2. The second antigen-binding region binds specifically to the second antigen with a binding affinity higher than 10 7 M -1 、10 6 M -1 、10 5 M -1 、or 10 4 M -1 The bispecific antibody or antigen-binding fragment thereof according to claim 1.

3. The bispecific antibody or an antigen-binding fragment thereof according to claim 1, wherein the binding affinity of the first antigen-binding region when binding to a first antigen is at least 100-fold, 1000-fold, or 10000-fold higher than the binding affinity of the second antigen-binding region when binding to a second antigen.

4. The bispecific antibody or an antigen-binding fragment thereof according to claim 1, wherein the first light chain variable region and the second light chain variable region are at least 90%, 95%, 99%, or 100% identical.

5. A first arm comprising a first heavy chain variable region and a first light chain variable region; and a second arm comprising a second heavy chain variable region and a second light chain variable region comprising a bispecific antibody or an antigen-binding fragment thereof, wherein The first arm binds specifically to the first antigen with a binding affinity higher than 10 7 M -1 、10 8 M -1 、10 9 M -1 、10 10 M -1 、10 11 M -1 、10 12 M -1 and the second arm binds specifically to the second antigen with a binding affinity lower than 10 9 M -1 、10 8 M -1 、10 7 M -1 、10 6 M -1 、10 5 M -1 、 or 10 4 M -1 。 said bispecific antibody or an antigen-binding fragment thereof.

6. The second arm binds specifically to the second antigen with a binding affinity higher than 10 7 M -1 、10 6 M -1 、10 5 M -1 、or 10 4 M -1 The bispecific antibody or antigen-binding fragment thereof according to claim 5, which binds specifically to the second antigen with a binding affinity higher than

7. The bispecific antibody or an antigen-binding fragment thereof according to claim 5, wherein the binding affinity of said first arm when binding to a first antigen is at least 100-fold, 1000-fold, or 10000-fold higher than the binding affinity of said second arm when binding to a second antigen.

8. The bispecific antibody or an antigen-binding fragment thereof according to claim 5, wherein the first light chain variable region and the second light chain variable region are at least 90%, 95%, 99%, or 100% identical.

9. A first heavy chain comprising a first heavy chain variable region, a second heavy chain comprising a second heavy chain variable region, a first light chain comprising a first light chain variable region, and a second light chain comprising a second light chain variable region comprising a bispecific antibody or an antigen-binding fragment thereof, wherein The first heavy chain variable region and the first light chain variable region bind to each other to form a first antigen-binding region that specifically binds to the first antigen with a binding affinity higher than 10 7 M -1 、10 8 M -1 、10 9 M -1 、10 10 M -1 、10 11 M -1 、10 12 M -1 and forms a first antigen-binding region that specifically binds to the first antigen with a binding affinity higher than 10 The second heavy chain variable region and the second light chain variable region bind to each other to form a second antigen-binding region that specifically binds to a second antigen with a binding affinity lower than 10 9 M -1 、 10 8 M -1 、 10 7 M -1 、 10 6 M -1 、 10 5 M -1 、 or 10 4 M -1 and forms a second antigen-binding region that specifically binds to a second antigen with a lower binding affinity than said bispecific antibody or an antigen-binding fragment thereof.

10. The second antigen-binding region specifically binds to the second antigen with a binding affinity higher than 10 7 M -1 、10 6 M -1 、10 5 M -1 、or 10 4 M -1 The bispecific antibody or antigen-binding fragment thereof according to claim 9.

11. The bispecific antibody or an antigen-binding fragment thereof according to claim 9, wherein the binding affinity of the first antigen-binding region when binding to a first antigen is at least 100-fold, 1000-fold, or 100000-fold higher than the binding affinity of the second antigen-binding region when binding to a second antigen.

12. The bispecific antibody or an antigen-binding fragment thereof according to claim 9, wherein the first light chain and the second light chain are at least 90%, 95%, 99%, or 100% identical.

13. The bispecific antibody or antigen-binding fragment thereof according to claim 9, wherein the first heavy chain and the second heavy chain are bound to each other by the knobs into holes method.

14. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the first antigen is a cancer-specific antigen and the second antigen is CD3.

15. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the first antigen is CD20 and the second antigen is CD3.

16. The bispecific antibody or antigen-binding fragment thereof according to claim 15, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2, and the first and second light chain variable regions comprise a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:

3.

17. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the first antigen is a cancer-specific antigen and the second antigen is a cancer-related antigen.

18. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the first antigen is PD-L1 and the second antigen is CD55.

19. The bispecific antibody or antigen-binding fragment thereof according to claim 18, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 5, and the first and second light chain variable regions comprise a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 6 or SEQ ID NO:

7.

20. A method for producing a bispecific antibody or antigen-binding fragment thereof, the method comprising: (a) Select a first antigen and a second antigen, and identify a first antibody or an antigen-binding fragment thereof that binds to the first antigen and a second antibody or an antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or the antigen-binding fragment thereof comprises a first heavy-chain variable region (VHa) and a first light-chain variable region (VLa), and the second antibody or the antigen-binding fragment thereof comprises a second heavy-chain variable region (VHb) and a second light-chain variable region (VLb); (b) Determine the amino acid sequences of VHa, VLa, VHb, and VLb; (c) Align the amino acid sequences of VLa and VLb and determine that the sequence homology between VLa and VLb is higher than 80%; (d) Design a common light-chain variable region (VLc), wherein VLc maintains the affinity for the first antigen when binding to VHa; (e) Redesign the sequences of VHa and VHb, thereby obtaining VHa' and VHb' that increase the difference in biochemical or biophysical properties between a first protein comprising two polypeptides each containing VHa' and two polypeptides each containing VLc, and a second protein comprising two polypeptides each containing VHb' and two polypeptides each containing VLc; and (f) Produce a bispecific antibody or an antigen-binding fragment thereof having two light-chain variable regions and two heavy-chain variable regions, wherein each of the two light-chain variable regions contains VLc and the two heavy-chain variable regions contain VHa' and VHb', respectively. Claim 21 The method according to claim 20, wherein in step (d), the binding affinity of VLc-VHb for the second antigen may decrease. Claim 22 The method according to claim 20, further comprising: (g) Create a buffer system for purifying the bispecific antibody or an antigen-binding fragment thereof. Claim 23 A method for producing a bispecific antibody or an antigen-binding fragment thereof, the method comprising: (a) Select a first antigen and a second antigen, and identify a first antibody or an antigen-binding fragment thereof that binds to the first antigen and a second antibody or an antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or the antigen-binding fragment thereof comprises a first heavy-chain variable region (VHa) and a first light-chain variable region (VLa), and the second antibody or the antigen-binding fragment thereof comprises a second heavy-chain variable region (VHb) and a second light-chain variable region (VLb); (b) Determine the amino acid sequences of VHa, VLa, and VLb; (c) Align the amino acid sequences of VLa and VLb and determine that the sequence homology between VLa and VLb is less than 80%; (d) Replace all light-chain variable regions in a phage display antibody library with VLa and pan against the second antigen to obtain a third heavy-chain variable region (VHc); (e) Redesign the sequences of VHa and VHc, thereby obtaining VHa' and VHc' that increase the difference in biochemical or biophysical properties between a first protein comprising two polypeptides each comprising VHa' and two polypeptides each comprising VLa and a second protein comprising two polypeptides each comprising VHc' and two polypeptides each comprising VLa; and (f) Produce a bispecific antibody or an antigen-binding fragment thereof having two light-chain variable regions and two heavy-chain variable regions, wherein each of the two light-chain variable regions comprises VLa and the two heavy-chain variable regions each comprise VHa' and VHc'. Claim 24 The method according to claim 23, further comprising the following: (g) Create a buffer system for purifying the bispecific antibody or an antigen-binding fragment thereof. Claim 25 A method for producing a bispecific antibody or an antigen-binding fragment thereof, the method comprising the following: (a) Selecting a first antigen and a second antigen, and identifying a first antibody or an antigen-binding fragment thereof that binds to the first antigen and a second antibody or an antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or the antigen-binding fragment thereof comprises a first heavy-chain variable region (VHa) and a first light-chain variable region (VLa), and the second antibody or the antigen-binding fragment thereof comprises a second heavy-chain variable region (VHb) and a second light-chain variable region (VLb); (b) Determining the amino acid sequences of VHa, VLa, VHb, and VLb; (c) Aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%; (d) Replacing all light-chain variable regions in a phage display antibody library with a plurality of light-chain variable regions that are at least 80%, 85%, 90%, 95%, or 99% identical to VLa or VLb; (e) Panning against the second antigen; (f) Selecting a common light-chain variable region (VLc) and a third heavy-chain variable region (VHc), wherein VHa-VLc binds to the first antigen with a desired affinity and VHc-VLc binds to the second antigen with a desired affinity; (g) Redesigning the sequences of VHa and VHc to obtain VHa' and VHc' that increase the difference in biochemical or biophysical properties between a first protein comprising two polypeptides each comprising VHa' and two polypeptides each comprising VLc and a second protein comprising two polypeptides each comprising VHc' and two polypeptides each comprising VLc; and (h) Producing a bispecific antibody or an antigen-binding fragment thereof having two light-chain variable regions and two heavy-chain variable regions, wherein each of the two light-chain variable regions comprises VLc and the two heavy-chain variable regions comprise VHa' and VHc', respectively. **Claim 26** The method according to claim 25, wherein in step (d), the plurality of light-chain variable regions are produced by error-prone PCR. **Claim 27** The method according to claim 25, further comprising: (i) Making a buffer system for purifying the bispecific antibody or an antigen-binding fragment thereof. **Claim 28** A method for producing a bispecific antibody or an antigen-binding fragment thereof, the method comprising: (a) selecting a first antigen and a second antigen and identifying a first antibody or an antigen-binding fragment thereof that binds to the first antigen and a second antibody or an antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or an antigen-binding fragment thereof comprises a first heavy-chain variable region (VHa) and a first light-chain variable region (VLa), and the second antibody or an antigen-binding fragment thereof comprises a second heavy-chain variable region (VHb) and a second light-chain variable region (VLb); (b) determining the amino acid sequences of VHa, VLa, VHb, and VLb; (c) aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%; (d) replacing all light-chain variable regions in a phage display antibody library with a plurality of light-chain variable regions that are at least 80%, 85%, 90%, 95%, or 99% identical to VLa or VLb; (e) panning against the second antigen; (f) selecting a common light-chain variable region (VLc) and a third heavy-chain variable region (VHc), wherein VHc-VLc binds to the second antigen with a desired affinity; (g) determining that the homology between VLa and VLc is higher than 80%; (h) designing a common light-chain variable region (VLd) that maintains the affinity for the first antigen when binding to VHa and has the desired affinity for the second antigen when binding to VHc; (i) optionally, redesigning the sequences of VHa and VHc to obtain VHa' and VHc' that increase the difference in biochemical or biophysical properties between a first protein comprising two polypeptides each comprising VHa' and two polypeptides each comprising VLd and a second protein comprising two polypeptides each comprising VHc' and two polypeptides each comprising VLd; and (j) Optionally, a step of preparing a bispecific antibody having two light chain variable regions and two heavy chain variable regions or an antigen-binding fragment thereof, wherein each of the two light chain variable regions contains VLd, and the two heavy chain variable regions contain VHa’ and VHc’, respectively, said step. **Claim 29** A method for preparing a bispecific antibody or an antigen-binding fragment thereof, the method comprising: (a) Selecting a first antigen and a second antigen and identifying a first antibody or an antigen-binding fragment thereof that binds to the first antigen and a second antibody or an antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or an antigen-binding fragment thereof contains a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or an antigen-binding fragment thereof contains a second heavy chain variable region (VHb) and a second light chain variable region (VLb), said step; (b) Determining the amino acid sequences of VHa, VLa, VHb, and VLb; (c) Aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is higher than 80%; (d) Designing a common light chain variable region (VLc), wherein VLc maintains the affinity for the first antigen when binding to VHa, said step; and (e) Optionally, a step of preparing a bispecific antibody having two light chain variable regions and two heavy chain variable regions or an antigen-binding fragment thereof, wherein each of the two light chain variable regions contains VLc, and the two heavy chain variable regions contain VHa and VHb, respectively, said step. **Claim 30** A method for preparing a bispecific antibody or an antigen-binding fragment thereof, the method comprising: (a) Selecting a first antigen and a second antigen and identifying a first antibody or an antigen-binding fragment thereof that binds to the first antigen and a second antibody or an antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or an antigen-binding fragment thereof contains a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or an antigen-binding fragment thereof contains a second heavy chain variable region (VHb) and a second light chain variable region (VLb), said step; (b) Determining the amino acid sequences of VHa, VLa, and VLb; (c) Aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%; (d) Replacing all light chain variable regions in the phage display antibody library with VLa and panning against the second antigen to obtain a third heavy chain variable region (VHc); and (e) Optionally, producing a bispecific antibody or an antigen-binding fragment thereof having two light chain variable regions and two heavy chain variable regions, each of the two light chain variable regions containing VLa, and the two heavy chain variable regions containing VHa and VHc, respectively, said step.

31. A method for producing a bispecific antibody or an antigen-binding fragment thereof, the method comprising: (a) Selecting a first antigen and a second antigen and identifying a first antibody or an antigen-binding fragment thereof that binds to the first antigen and a second antibody or an antigen-binding fragment thereof that binds to the second antigen, the first antibody or an antigen-binding fragment thereof containing a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or an antigen-binding fragment thereof containing a second heavy chain variable region (VHb) and a second light chain variable region (VLb), said step; (b) Determining the amino acid sequences of VHa, VLa, VHb, and / or VLb; (c) Aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%; (d) Replacing all light chain variable regions in the phage display antibody library with a plurality of light chain variable regions, said light chain variable regions being at least 80%, 85%, 90%, 95%, or 99% identical to VLa or VLb; (e) Panning against the first and / or second antigen; (f) Selecting a common light chain variable region (VLc) and a third heavy chain variable region (VHc), wherein VHa-VLc binds to the first antigen with a desired affinity and VHc-VLc binds to the second antigen with a desired affinity, said step; and (g) Optionally, producing a bispecific antibody or an antigen-binding fragment thereof having two light chain variable regions and two heavy chain variable regions, each of the two light chain variable regions containing VLc, and the two heavy chain variable regions containing VHa and VHc, respectively, said step.

32. A heavy chain variable region (VH) comprising complementarity determining regions (CDR) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and A light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence An antibody or antigen-binding fragment thereof that binds to CD3, comprising The amino acid sequences of the selected VH CDR1, 2, 3 are set forth in SEQ ID NO: 22-24 respectively, and the amino acid sequences of the selected VL CDR1, 2, 3 are set forth in SEQ ID NO: 28-30 respectively, The antibody or antigen-binding fragment thereof.

33. The antibody or antigen-binding fragment thereof according to claim 32, wherein VH comprises CDR1, 2, 3 having amino acid sequences set forth in SEQ ID NO: 22, 23, 24 respectively, and VL comprises CDR1, 2, 3 having amino acid sequences set forth in SEQ ID NO: 28, 29, 30 respectively.

34. The antibody or antigen-binding fragment thereof according to any one of claims 32 to 33, which specifically binds to human CD3.

35. The antibody or antigen-binding fragment thereof according to any one of claims 32 to 34, which is a bispecific antibody.

36. A heavy chain variable region (VH) comprising complementarity-determining regions (CDR) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and VH CDR3 comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and A light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence, said light chain variable region (VL) An antibody or antigen-binding fragment thereof that binds to PD-L1, comprising The amino acid sequences of the selected VH CDR1, 2, and 3 and the amino acid sequences of the selected VL CDR1, 2, and 3 are (1) The amino acid sequences of the selected VH CDR1, 2, 3 are set forth in SEQ ID NOs: 41-43, respectively, and the amino acid sequences of the selected VL CDR1, 2, 3 are set forth in SEQ ID NOs: 53-55, respectively; (2) The amino acid sequences of the selected VH CDR1, 2, 3 are set forth in SEQ ID NOs: 41-43, respectively, and the amino acid sequences of the selected VL CDR1, 2, 3 are set forth in SEQ ID NOs: 59-61, respectively One of The antibody or antigen-binding fragment thereof.

37. The antibody or antigen-binding fragment thereof according to claim 36, wherein VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 41-43, respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 59-61, respectively.

38. The antibody or antigen-binding fragment thereof according to any one of claims 36-37, which specifically binds to human CD3.

39. The antibody or antigen-binding fragment thereof according to any one of claims 36-38, which is a bispecific antibody.

40. A heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and A light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence comprising an antibody or antigen-binding fragment thereof that binds to CD55, wherein the amino acid sequences of the selected VH CDRs 1, 2, and 3 and the amino acid sequences of the selected VL CDRs 1, 2, and 3 are (1) the amino acid sequences of the selected VH CDRs 1, 2, 3 are set forth in SEQ ID NOs: 47-49, respectively, and the amino acid sequences of the selected VL CDRs 1, 2, 3 are set forth in SEQ ID NOs: 53-55, respectively; (2) the amino acid sequences of the selected VH CDRs 1, 2, 3 are set forth in SEQ ID NOs: 47-49, respectively, and the amino acid sequences of the selected VL CDRs 1, 2, 3 are set forth in SEQ ID NOs: 59-61, respectively is one of the antibody or antigen-binding fragment thereof. **Claim 41** The antibody or antigen-binding fragment thereof according to claim 40, wherein VH comprises CDRs 1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 47-49, respectively, and VL comprises CDRs 1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 59-61, respectively. **Claim 42** The antibody or antigen-binding fragment thereof according to any one of claims 40-41, which specifically binds to human CD3. **Claim 43** The antibody or antigen-binding fragment thereof according to any one of claims 40-42, which is a bispecific antibody. **Claim 44** An immunoglobulin light chain or a fragment thereof comprising VLs containing CDR1, 2, and 3, each containing the amino acid sequences set forth in SEQ ID NOs: 53 to 55 A nucleic acid comprising a polynucleotide encoding a polypeptide comprising When the VL is paired with a VH containing the amino acid sequence set forth in SEQ ID NO: 4, it binds to PD-L1, and / or when paired with a VH containing the amino acid sequence set forth in SEQ ID NO: 5, it binds to CD55 Said nucleic acid

45. An immunoglobulin light chain or a fragment thereof comprising VLs containing CDR1, 2, and 3, each containing the amino acid sequences set forth in SEQ ID NOs: 59 to 61 A nucleic acid comprising a polynucleotide encoding a polypeptide comprising When the VL is paired with a VH containing the amino acid sequence set forth in SEQ ID NO: 4, it binds to PD-L1, and / or when paired with a VH containing the amino acid sequence set forth in SEQ ID NO: 5, it binds to CD55 Said nucleic acid

46. The nucleic acid according to any one of claims 44 to 45, encoding a bispecific antibody

47. The nucleic acid according to any one of claims 44 to 45, which is cDNA

48. A vector comprising one or more of the nucleic acids according to any one of claims 44 to 47

49. A cell comprising the vector according to claim 48

50. The cell according to claim 49, which is a CHO cell

51. A cell comprising one or more of the nucleic acids according to any one of claims 44 to 47

52. A first polypeptide comprising a first heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1; A second polypeptide comprising a second heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2; A third polypeptide comprising a first light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3; A fourth polypeptide comprising a second light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 A bispecific antibody or antigen-binding fragment thereof that binds to CD20 and CD3,

53. wherein the first heavy chain variable region (VH) comprises SEQ ID NO: 1; the second heavy chain variable region (VH) comprises SEQ ID NO: 2; the first light chain variable region (VL) comprises SEQ ID NO: 3; and the second light chain variable region (VL) comprises SEQ ID NO: 3, the bispecific antibody or antigen-binding fragment thereof according to claim 52.

54. wherein the first polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 34, 35, or 36; the second polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37, 38, or 39; the third polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 40; and the fourth polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 40, the bispecific antibody or antigen-binding fragment thereof according to claim 52.

55. wherein the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35; and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 38, the bispecific antibody or antigen-binding fragment thereof according to claim 52.

56. a first polypeptide comprising a first heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4; a second polypeptide comprising a second heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5; a third polypeptide comprising a first light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6 or 7; a fourth polypeptide comprising a second light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6 or 7 A bispecific antibody or antigen-binding fragment thereof that binds to PD-L1 and CD55, comprising

57. wherein the first heavy chain variable region (VH) comprises SEQ ID NO: 4; the second heavy chain variable region (VH) comprises SEQ ID NO: 5; the first light chain variable region (VL) comprises SEQ ID NO: 7; and the second light chain variable region (VL) comprises SEQ ID NO: 7, the bispecific antibody or antigen-binding fragment thereof according to claim 56.

58. wherein the first polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 65; the second polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 66; the third polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 67 or 68; and the fourth polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 67 or 68, the bispecific antibody or antigen-binding fragment thereof according to claim 56.

59. wherein the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 65; the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 66; the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 68; and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 68, the bispecific antibody or antigen-binding fragment thereof according to claim 56.

60. An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, 32 to 43, and 52 to 59 covalently linked to a therapeutic agent.

61. The antibody-drug conjugate according to claim 60, wherein the therapeutic agent is a cytotoxic agent or a cytostatic agent.

62. A method of treating a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, 32 to 43, and 52 to 59, or an antibody-drug conjugate according to claim 60 or 61.

63. The method according to claim 62, wherein the subject has a solid tumor. **Claim 64** The method according to claim 62, wherein the cancer is melanoma, pancreatic cancer, or a hematological malignancy. **Claim 65** The method according to claim 62, wherein the cancer is non-Hodgkin lymphoma, lymphoma, or chronic lymphocytic leukemia. **Claim 66** A method of reducing the rate of tumor growth, comprising contacting tumor cells of a subject with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19, 32 to 43, and 52 to 59, or an antibody-drug conjugate according to claim 60 or 61. **Claim 67** A method of killing tumor cells, comprising contacting tumor cells of a subject with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19, 32 to 43, and 52 to 59, or an antibody-drug conjugate according to claim 60 or 61. **Claim 68** A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19, 32 to 43, and 52 to 59 and a pharmaceutically acceptable carrier. **Claim 69** A pharmaceutical composition comprising an antibody-drug conjugate according to claim 60 or 61 and a pharmaceutically acceptable carrier.

Images