New anti-PD-L1 antibody

Abstract

To provide antibodies with significantly better efficacy than current anti-PD-L1 antibodies.SOLUTION: The present disclosure provides an isolated PD-L1 antibody whose binding to PD-L1 at an acidic pH is substantially lower than its binding to PD-L1 at a neutral pH at the same assay setting, and an isolated PD-L1 antibody that is not pH-dependent in binding to PD-L1. Also provided are a pharmaceutical composition of the antibody, an encoding polynucleotide and an expression vector, an isolated host cell thereof, and a kit comprising the PD-L1 antibody. Further provided are methods of treating a PD-L1 associated condition using the PD-L1 antibody.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present disclosure relates generally to novel anti-PD-L1 antibodies that specifically bind to human PD-L1. . [Background technology]

[0002] PD-1, a member of the CD28 family, is a physiological regulator of T cell activation and proliferation. It is an inhibitory receptor expressed on the surface of T cells that functions to restrict the PD-L1 (B7-H1 / CD274) is expressed on antigen-presenting cells and tumor cells. Binding of PD-L1 to its receptor inhibits T cell activation and inhibits B7 binding to CD28. Balances T cell stimulatory signals such as T cell binding.

[0003] PD-L1 is not expressed by normal epithelial tissues but is aberrantly expressed in a wide range of human cancers. In this context, PD-L1 is thought to mediate cancer progression by abrogating host antitumor responses. Its expression in tumor cells is associated with renal cell carcinoma, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and urinary cancer. It has been associated with poor prognosis in urothelial, gastric, esophageal and hepatocellular carcinoma.

[0004] Multiple antibodies targeting PD-1 have been generated and are effective in preventing tumor growth and are randomized controlled trials. It has been shown to improve overall survival in a randomized controlled trial. Antibodies that inhibit the IL-18 receptor have been discovered and have shown activity in inhibiting tumor growth. It is internalized by PD-L1 expressing cells and is secreted into endosomal and lysosomal compartments. These antibodies are rapidly degraded in patients with PD-L1-expressing cells, Thus, the ability of these antibodies to inhibit tumor growth depends on the amount of antibody delivered. Sustained inhibition of tumor growth is limited by the activation of PD-L1. To maintain a concentration of antibody at the tumor site, continuous infusion of large amounts of antibody is required. Therefore, the cost of providing these substances is prohibitively expensive as it requires injecting large amounts of antibodies into patients. More importantly, the ability to achieve a complete response is also limited. Therefore, it is possible to generate antibodies with substantially better efficacy than current anti-PD-L1 antibodies. It is necessary. Summary of the Invention

[0005] The present disclosure relates to novel monoclonal anti-PD-L1 antibodies (particularly chimeric and humanized antibodies); Polynucleotides encoding same, methods of using same and human PD-L1 protein The binding epitope of the antibody is provided qualitatively.

[0006] In certain embodiments, the heavy chain HCDR1, HCDR2 and HCDR3 and the light chain LC An isolated PD-L1 antibody comprising a DR1, LCDR2 and LCDR3 sequence is provided herein. Provided in The HCDR1 sequence is TYWX. 1 H (SEQ ID NO: 1) or at least 80% thereof (e.g. , at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, and 99%) of sequence identity to the corresponding homologous sequences. The HCDR2 sequence is MIQPNSGGTKYNX. 2 X 3 FKX 4 (SEQ ID NO:2) or At least 80% of it (e.g., at least 85%, 88%, 90%, 91%, 92%, Homologues with sequence identity of 93%, 94%, 95%, 96%, 97%, 98%, 99%) is an array, The HCDR3 sequence is GAGTVDYFDY (SEQ ID NO: 3) or at least 80% thereof (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95% %, 96%, 97%, 98%, 99%) of sequence identity to the homologous sequence, The LCDR1 sequence is RASESVDIYGNSFMH (SEQ ID NO: 4) or at least At least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 9 4%, 95%, 96%, 97%, 98%, and 99%) of sequence identity to the homologous sequences. , The LCDR2 sequence may be RASNLES (SEQ ID NO:5) or at least 80% thereof (e.g. If possible, at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, and 99%) of sequence identity to the homologous sequence. LCDR3 array is X 5 QSX 6 X 7 DPYT (SEQ ID NO: 6) or at least 8 0% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) of sequence identity to the homologous sequence, X 1 is I or M, and X 2 is D or E, and X 3 is Q or K, X 4 is N or K, and X 5 is Q or H, and X 6 is N or T, and X 7 is D or E.

[0007] In certain embodiments, X 1 is I or M, and X 2 is D or E, and X 3teeth , Q or K, and X 4 is N or K, and X 5 is Q and X 6 is N or T and X 7 is D or E. In certain embodiments, X 1 is I and X 2 teeth , D and X 3 is Q and X 4 is N and X 5 is Q and X 6 is N X 7 is D. In certain embodiments, X 1 is M and X 2 is E and X 3 is K and X 4 is K and X 5 is Q and X 6 is T and X 7 E In certain embodiments, X 1 is I and X 2 is D and X 3 is Q, X 4 is N and X 5 is H and X 6 is N and X 7 D. In the embodiment, X 1 is M and X 2 is E and X 3 is K and X 4 is K X 5 is H and X 6 is T and X 7 is E.

[0008] In certain embodiments, the PD-L1 antibodies provided herein are selected from the group consisting of SEQ ID NOs: 7-1 2 or at least 80% thereof (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) of sequence identity In certain embodiments, the CDRs include one, two, three, four, five or six CDRs selected from the homologues of Thus, the PD-L1 antibodies provided herein are selected from three sequences selected from SEQ ID NOs: 7 to 9. In certain embodiments, the PD-L1 antibodies provided herein comprise a variety of heavy chain CDRs. In certain embodiments, the antibody comprises three light chain CDRs selected from SEQ ID NOs: 10 to 12. The PD-L1 antibodies provided herein are six types selected from SEQ ID NOs: 7 to 12. Contains the CDRs of.

[0009] In certain embodiments, the PD-L1 antibodies provided herein are The heavy chain framework sequences of HFR1, HFR2, HFR3 and HFR4, and LFR1, LFR2, The heavy chain variable region comprises the light chain framework sequences of LFR3 and LFR4 and has the sequence : Follow HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 The sequence of the light chain variable region is represented by the following formula: LFR1-LCDR1-LFR2-LCDR2-LFR Follow 3-LCDR3-LFR4.

[0010] In certain embodiments, the HFR1 sequence is 1 VQLXa 2 QXa 3 GAEXa 4 Xa 5 KPGASVKXa 6 SCKASGYXa 7 FT (SEQ ID NO: 13), The HFR2 sequence is WVXa 8 QXa 9 PGQGLEWIG (SEQ ID NO: 14), The HFR3 sequence is 10 Xa 11 TLTVDXa 12SXa 13 Xa 14 TAXa 1 5 MXa 16 LSXa 17 LXa 18 SXa 19 DXa 20 AVYYCAR (SEQ ID NO:1 5) and The HFR4 sequence is WGQGXa 21 TLXa 22 Xa 23 SS (SEQ ID NO: 16) , The LFR1 sequence is DIVLTXa 24 SPXa 25 SLXa 26 VSXa 27 GQRA TIXa 28 C (SEQ ID NO: 17), The LFR2 sequence is WYQQKPGQXa 29 PKLLIY (SEQ ID NO: 18), The LFR3 sequence is GIPARFSGSGSRTDFTLTINPVEAXa 30 DXa 31 ATYYC (SEQ ID NO: 19), The LFR4 sequence is FGGGTKLEXa 32 K (SEQ ID NO:20), Xa 1 is Q or L, and Xa 2 is Q or V, and Xa 3 is S or P Xa 4 is L or V, and Xa 5 is V or K, and Xa 6 is L or V and Xa 7 is T, S or I, and Xa 8 is W, K or R, and Xa 9 teeth , R or A, and Xa 10 is R or K, and Xa 11 is V or A, Xa 12 is K or T, and Xa 13 is S or I, and Xa 14 is S or T and Xa 15 is Y or S, and Xa 16 is Q or E, and Xa 17 teeth , S, G or R, and Xa 18 is T or R, and Xa 19 is E or D Xa 20 is S or T, and Xa 21 is T or S, and Xa 22 Is S or T, and Xa 23 is V or I, and Xa 24 is Q or H, and Xa 2 5 is A or V, and Xa 26 is A or T, and Xa 27 is L, A or V and Xa 28 is S or T, and Xa 29 is S, P or A, and Xa 30 is D, N or Q, and Xa 31 is V or T, and Xa 32 is L, T or I and Xa 33 is F or Y, and Xa 34 is T or Q, and Xa 35 teeth , V or L; Xa 36 is D or S, and Xa 37 is M or L, Xa 38 is T or S, and Xa 39 is F or S, and Xa 40 D or Q and Xa 41 is V or A, and Xa 42 is V or I, and Xa 43 teeth , D or A, and Xa 44 is T or S, and Xa 45 is Y or S, Xa 46 is G or R, and Xa 47 is F or L, and Xa 48 is S or N and Xa 49 is T or P, and Xa 50 is Q or E, and Xa 51 teeth , V or T, and Xa 52 is F or Y, and Xa 53 is A or G.

[0011] In certain embodiments, Xa 1 is Q and Xa 2 is V and Xa 3 is S , Xa 4 is V and Xa 5 is K and Xa 6 is L and Xa 7 is I , Xa 8 is K and Xa 9 is R and Xa 10 is R or K, and Xa 11 is A and Xa 12 is K and Xa 13 is I and Xa 14 is S, Xa 15 is Y and Xa 16 is E and Xa 17 is R and Xa 18 is T and Xa 19 is D and Xa 20 is T and Xa 21 is T or S , Xa 22 is T or S, and Xa 23 is I or V, and Xa 24 Is Q Xa 25 is A and Xa 26 is A and Xa 27 is V and Xa 28 teeth , T and Xa 29 is A or P, and Xa 30 is Q and Xa 31 is T Yes, Xa 32 is T or I, and Xa 33 is Y and Xa 34 is Q, Xa 35 is L and Xa 36 is D and Xa 37 is L and Xa 38 is S and Xa 39 is S and Xa 40 is Q and Xa 41 is A and Xa 4 2 is I and Xa 43 is A and Xa 44 is S and Xa 45 is S , Xa 46 is R and Xa 47 is L and Xa 48 is N and Xa 49 teeth, P and Xa 50 is E and Xa 51 is T and Xa 52 is Y and Xa 53 is G.

[0012] In certain embodiments, Xa 1 is Q and Xa 2 is V and Xa 3 is S , Xa 4 is V and Xa 5 is K and Xa 6 is L and Xa 7 is I , Xa8 is K and Xa 9 is R and Xa 10 is R and Xa 11 is A Yes, Xa 12 is K and Xa 13 is I and Xa 14 is S and Xa 15 is Y and Xa 16 is E and Xa 17 is R and Xa 18 is T, Xa 19 is D and Xa 20 is T and Xa 21 is T and Xa 22 is S and Xa 23 is I and Xa 24 is Q and Xa 25 is A and Xa 2 6 is A and Xa 27 is V and Xa 28 is T and Xa 29 is A , Xa 30 is Q and Xa 31 is T and Xa 32 is T and Xa 33 teeth, Y and Xa 34 is Q and Xa 35 is L and Xa 36 is D and Xa 37 is L and Xa 38 is S and Xa 39 is S and Xa 40 Is Q Xa 41 is A and Xa 42 is I and Xa 43 is A and Xa 44 teeth , S and Xa 45 is S and Xa 46 is R and Xa47 is L and X a 48 is N and Xa 49 is P and Xa 50 is E and Xa 51 is T Yes, Xa 52 is Y and Xa 53 is G.

[0013] In certain embodiments, Xa 1 is Q and Xa 2 is V and Xa 3 is S , Xa 4 is V and Xa 5 is K and Xa 6 is L and Xa 7 is I , Xa 8 is K and Xa 9 is R and Xa 10 is K and Xa 11 is A Yes, Xa 12 is K and Xa 13 is I and Xa 14 is S and Xa 15 is Y and Xa 16 is E and Xa 17 is R and Xa 18 is T, Xa 19 is D and Xa 20 is T and Xa 21 is S and Xa 22 is T and Xa 23 is V and Xa 24 is Q and Xa 25 is A and Xa 2 6 is A and Xa 27 is V and Xa 28 is T and Xa 29 is P , Xa 30is Q and Xa 31 is T and Xa 32 is I and Xa 33 teeth, Y and Xa 34 is Q and Xa 35 is L and Xa 36 is D and Xa 37 is L and Xa 38 is S and Xa 39 is S and Xa 40 Is Q Xa 41 is A and Xa 42 is I and Xa 43 is A and Xa 44 teeth , S and Xa 45 is S and Xa 46 is R and Xa 47 is L and X a 48 is N and Xa 49 is P and Xa 50 is E and Xa 51 is T Yes, Xa 52 is Y and Xa 53 is G.

[0014] In certain embodiments, Xa 1 is L or Q, and Xa 2 is V and Xa 3 teeth, S and Xa 4 is V and Xa 5 is K and Xa 6 is L and Xa 7 teeth, I and Xa 8 is K and Xa 9 is R and Xa 10 is R and Xa 11 is A and Xa 12 is K and Xa 13 is I and Xa 14 is S, Xa 15 is Y and Xa 16 is E and Xa 17 is R and Xa 18 is T and Xa 19 is D and Xa 20 is T and Xa 21 is T and Xa 2 2 is S and Xa 23 is I and Xa 24 is Q and Xa 25 is A , Xa 26 is A and Xa 27 is V and Xa 28 is T and Xa 29 teeth, S and Xa 30 is N and Xa 31 is T and Xa 32 is T and Xa 33 is Y and Xa 34 is Q and Xa 35 is L and Xa 36 is D Xa 37 is L and Xa 38 is S and Xa 39 is S and Xa 40 teeth , Q and Xa 41 is A and Xa 42 is I and Xa 43 is A and X a 44 is S and Xa 45 is S and Xa 46 is R and Xa 47 is L Yes, Xa 48 is N and Xa 49 is P and Xa 50 is E and Xa 51 is T and Xa 52 is Y and Xa53 is G.

[0015] In certain embodiments, Xa 1 is L and Xa 2 is V and Xa 3 is S , Xa 4 is V and Xa 5 is K and Xa 6 is V and Xa 7 is I , Xa 8 is R and Xa 9 is A and Xa 10 is R and Xa 11 is V Yes, Xa 12 is T and Xa 13 is I and Xa 14 is S and Xa 15 is Y and Xa 16 is E and Xa 17 is R and Xa 18 is R, Xa 19 is D and Xa 20 is T and Xa 21 is T and Xa 22 is S and Xa 23 is I and Xa 24 is Q and Xa 25 is A and Xa 2 6 is A and Xa 27 is V and Xa 28 is T and Xa 29 is S , Xa 30 is N and Xa 31 is T and Xa 32 is T and Xa 33 teeth, Y and Xa 34 is Q and Xa 35 is L and Xa 36 is D and Xa 37 is L and Xa 38 is S and Xa 39 is S and Xa 40 Is Q Xa 41 is A and Xa 42 is I and Xa 43 is A and Xa 44 teeth , S and Xa 45 is S and Xa 46 is R and Xa 47 is L and X a 48 is N and Xa 49 is P and Xa 50 is E and Xa 51 is T Yes, Xa 52 is Y and Xa 53 is G.

[0016] In certain embodiments, Xa 1 is L and Xa 2 is V and Xa 3 is S , Xa 4 is V and Xa 5 is K and Xa 6 is V and Xa 7 is I , Xa 8 is R and Xa 9 is A and Xa 10 is R and Xa 11 is V Yes, Xa 12 is T and Xa 13 is I and Xa 14 is S and Xa 15 is Y and Xa 16 is E and Xa 17 is R and Xa 18 is R, Xa 19 is D and Xa 20 is T and Xa21 is T and Xa 22 is S and Xa 23 is I and Xa 24 is Q and Xa 25 is A and Xa 2 6 is A and Xa 27 is V and Xa 28 is T and Xa 29 is A , Xa 30 is Q or N, and Xa 31 is T and Xa 32 is T and Xa 33 is Y and Xa 34 is Q and Xa 35 is L and Xa 36 is D Xa 37 is L and Xa 38 is S and Xa 39 is S and Xa 40 teeth , Q and Xa 41 is A and Xa 42 is I and Xa 43 is A and X a 44 is S and Xa 45 is S and Xa 46 is R and Xa 47 is L Yes, Xa 48 is N and Xa 49 is P and Xa 50 is E and Xa 51 is T and Xa 52 is Y and Xa 53 is G.

[0017] In certain embodiments, Xa 1 is Q and Xa 2 is V and Xa 3 is S , Xa 4is V and Xa 5 is K and Xa 6 is V and Xa 7 is I , Xa 8 is R and Xa 9 is A and Xa 10 is K and Xa 11 is A Yes, Xa 12 is T and Xa 13 is I and Xa 14 is S and Xa 15 is Y and Xa 16 is E and Xa 17 is R and Xa 18 is R, Xa 19 is D and Xa 20 is T and Xa 21 is S and Xa 22 is T and Xa 23 is V and Xa 24 is Q and Xa 25 is A and Xa 2 6 is A and Xa 27 is V and Xa 28 is T and Xa 29 is P , Xa 30 is N and Xa 31 is T and Xa 32 is I and Xa 33 teeth, Y and Xa 34 is Q and Xa 35 is L and Xa 36 is D and Xa 37 is L and Xa 38 is S and Xa 39 is S and Xa 40 Is Q Xa 41 is A and Xa 42 is I and Xa 43is A and Xa 44 teeth , S and Xa 45 is S and Xa 46 is R and Xa 47 is L and X a 48 is N and Xa 49 is P and Xa 50 is E and Xa 51 is T Yes, Xa 52 is Y and Xa 53 is G.

[0018] In certain embodiments, Xa 1 is Q and Xa 2 is V and Xa 3 is S , Xa 4 is V and Xa 5 is K and Xa 6 is V and Xa 7 is I , Xa 8 is R and Xa 9 is A and Xa 10 is K and Xa 11 is A Yes, Xa 12 is T and Xa 13 is I and Xa 14 is S and Xa 15 is Y and Xa 16 is E and Xa 17 is R and Xa 18 is R, Xa 19 is D and Xa 20 is T and Xa 21 is S and Xa 22 is T and Xa 23 is V and Xa 24 is Q and Xa 25 is A and Xa 2 6 is A and Xa27 is V and Xa 28 is T and Xa 29 is P , Xa 30 is N or Q, and Xa 31 is T and Xa 32 is I and Xa 33 is Y and Xa 34 is Q and Xa 35 is L and Xa 36 is D Xa 37 is L and Xa 38 is S and Xa 39 is S and Xa 40 teeth , Q and Xa 41 is A and Xa 42 is I and Xa 43 is A and X a 44 is S and Xa 45 is S and Xa 46 is R and Xa 47 is L Yes, Xa 48 is N and Xa 49 is P and Xa 50 is E and Xa 51 is T and Xa 52 is Y and Xa 53 is G.

[0019] In certain embodiments, Xa 1 is Q and Xa 2 is V and Xa 3 is S , Xa 4 is V and Xa 5 is K and Xa 6 is L and Xa 7 is I , Xa 8 is K and Xa 9 is R and Xa 10 is K and Xa 11 is A Yes, Xa 12 is K and Xa 13 is I and Xa 14 is S and Xa 15 is Y and Xa 16 is E and Xa 17 is R and Xa 18 is T, Xa 19 is D and Xa 20 is T and Xa 21 is S and Xa 22 is T and Xa 23 is V and Xa 24 is Q and Xa 25 is A and Xa 2 6 is A and Xa 27 is V and Xa 28 is T and Xa 29 is P , Xa 30 is N and Xa 31 is T and Xa 32 is I and Xa 33 teeth, Y and Xa 34 is Q and Xa 35 is L and Xa 36 is D and Xa 37 is L and Xa 38 is S and Xa 39 is S and Xa 40 Is Q Xa 41 is A and Xa 42 is I and Xa 43 is A and Xa 44 teeth , S and Xa 45 is S and Xa 46 is R and Xa 47 is L and X a 48 is N and Xa 49 is P and Xa50 is E and Xa 51 is T Yes, Xa 52 is Y and Xa 53 is G.

[0020] In certain embodiments, the PD-L1 antibody comprises a heavy chain variable region set forth in SEQ ID NO:61 and In certain embodiments, the PD-L1 antibody comprises a light chain variable region set forth in SEQ ID NO: 62. Contains the heavy chain variable region set forth in SEQ ID NO: 71 and the light chain variable region set forth in SEQ ID NO: 72 In certain embodiments, the PD-L1 antibody comprises the heavy chain variable region set forth in SEQ ID NO: 71 and In certain embodiments, the PD-L1 antibody comprises a light chain variable region set forth in SEQ ID NO: 70. Contains a heavy chain variable region set forth in SEQ ID NO:69 and a light chain variable region set forth in SEQ ID NO:72 In certain embodiments, the PD-L1 antibody comprises the heavy chain variable region set forth in SEQ ID NO:69 and In certain embodiments, the PD-L1 antibody comprises a light chain variable region set forth in SEQ ID NO: 70. Contains the heavy chain variable region set forth in SEQ ID NO: 73 and the light chain variable region set forth in SEQ ID NO: 74 .

[0021] In certain embodiments, the PD-L1 antibody comprises a heavy chain variable region set forth in SEQ ID NO:63 and In certain embodiments, the PD-L1 antibody comprises a light chain variable region set forth in SEQ ID NO: 64. Comprising a heavy chain variable region set forth in SEQ ID NO: 75 and a light chain variable region set forth in SEQ ID NO: 76 In certain embodiments, the PD-L1 antibody comprises the heavy chain variable region set forth in SEQ ID NO: 77 and In certain embodiments, the PD-L1 antibody comprises a light chain variable region set forth in SEQ ID NO: 78. Comprising a heavy chain variable region set forth in SEQ ID NO: 75 and a light chain variable region set forth in SEQ ID NO: 78 In certain embodiments, the PD-L1 antibody comprises the heavy chain variable region set forth in SEQ ID NO: 77 and In certain embodiments, the PD-L1 antibody comprises a light chain variable region set forth in SEQ ID NO: 76. Comprising a heavy chain variable region set forth in SEQ ID NO: 79 and a light chain variable region set forth in SEQ ID NO: 80 .

[0022] The present disclosure also provides one, two, three, four, five or six CDs selected from SEQ ID NOs: 7 to 12. R. In certain embodiments, the antibody is selected from the group consisting of SEQ ID NOs: 7-9. In certain embodiments, the antibody comprises three selected heavy chain CDRs. In certain embodiments, the antibody comprises three light chain CDRs selected from SEQ ID NO: 21. In certain embodiments, the antibody comprises the heavy chain variable region shown, or a humanized version thereof. , comprising the light chain variable region set forth in SEQ ID NO: 22, or a humanized version thereof. In one embodiment, the antibody comprises a heavy chain variable region as shown in SEQ ID NO:21 and a nucleotide sequence as shown in SEQ ID NO:22. The light chain variable region or a humanized version thereof.

[0023] PD-L1 binding at acidic pH as measured by assay and Binding to PD-L1 at neutral pH as measured by Binding of isolated PD-L1 antibodies to PD-L1 at neutral pH is substantially lower than binding to PD-L1 at neutral pH. In certain embodiments, the binding of a PD-L1 antibody to PD-L1 at acidic pH is consistent with the same antibody. Assay settings showed 80%, 70%, 60%, 50%, and 40% binding to PD-L1 at neutral pH. %, 30%, 20%, 10% or 5% or less. In certain embodiments, the neutral pH is 7.4 and the acidic pH is 6.0, 5.5, 5.0, 4.5 or 4.0. In an embodiment of the present invention, the method of the present invention is carried out at an acidic pH of 6.0, 5.5 or 5.0. The binding of the provided PD-L1 antibodies to PD-L1 was measured at pH 7.4 using the same assay setup. This is substantially lower than the binding of PD-L1 to IgG1.

[0024] In certain embodiments, the assay setup includes ELISA, FACS, surface plasmon resonance, GST pull-down, epitope tag, immunoprecipitation, far-western, fluorescence resonance energy -Transfer, time-resolved fluorescent immunoassay (TR-FIA), radioimmunoassay (RIA) ), enzyme immunoassay, latex agglutination, Western blot and immunohistochemistry The in vitro binding or blocking activity of either the It is measured thus.

[0025] In certain embodiments, the present disclosure also provides antibodies that bind to the same epitope as a PD-L1 antibody, or an isolated PD-L1 antibody that has competitive binding with a PD-L1 antibody. In certain embodiments, the epitope is selected from the following amino acid residues of PD-L1: E58, K62, N At least one of 63, I64, S80, and Y81 (e.g., at least two, In certain embodiments, the epitope comprises one, two or three of the following PD-L1 sequences: Amino acid residues: at least one of R113, M115, Y123, K124 and R125 In certain embodiments, the epitope further comprises at least one (e.g., at least two or three) The topes consist of the following combinations of PD-L1 amino acid residues: 1) E58, R113, and M115. , Y123 and K124; 2) one of E58, S80, R113 and R125 In certain embodiments, the epitope comprises the following amino acid residues of PD-L1: K62, At least one of N63, I64, Y81, and D122 (e.g., at least two In certain embodiments, the epitope further comprises the following PD-L1 antigens: Combination of amino acid residues: 1) E58, N63, I64, S80, Y81, R113 and R 125;2) E58, S80, R113, M115, D122, Y123, K124 and and R125; 3) E58, K62, N63, S80, Y81, R113 and R125; 4) E58, N63, I64, S80, Y81, R113, K124 and R125 Includes one type.

[0026] Binding to PD-L1 at acidic pH was significantly correlated with binding to PD-L1 at neutral pH in the same assay setup. administering to a subject an isolated PD-L1 antibody having substantially less than binding to the subject; and treating a PD-L1-associated condition in a subject in need thereof, including treating a PD-L1-associated condition in a subject in need thereof. Methods for treating a 1-related condition are provided herein.

[0027] In certain embodiments, the antibody is administered at 80%, 70%, 60%, 50%, 45% of the reference dose. , 40%, 35%, 30%, 25%, 20%, 15% or 10% or less The reference dose is determined by the reference antibody to achieve a comparable in vivo therapeutic effect. The reference antibody showed similar binding to PD-L1 at both acidic and neutral pH. As shown, the reference antibody and the antibody have similar binding to PD-L1 at neutral pH.

[0028] In certain embodiments, the antibody is administered at a dosing frequency that is less than that of the reference dosing frequency, The frequency of administration of the reference antibody is determined by the reference antibody to achieve a comparable in vivo therapeutic effect. The reference antibody showed similar binding to PD-L1 at both acidic and neutral pH. The control and antibody have similar binding to PD-L1 at neutral pH.

[0029] In certain embodiments, the antibody has a longer than reference in vivo half-life in a target organ. The reference in vivo half-life is determined by a reference antibody. The reference antibody showed similar binding to PD-L1 at both acidic and neutral pH. The control antibody and the antibody have similar binding to PD-L1 at neutral pH. In certain embodiments, The target organs are serum, kidney, lung, pancreas, liver, gallbladder, urinary bladder, skin, esophagus, ovaries, breasts, This includes one or more of the colon, rectum, stomach, spleen or brain.

[0030] The present disclosure provides heavy chain HCDR1', HCDR2' and HCDR3' and light chain LCDR An isolated PD-L1 antibody comprising an LCDR1', LCDR2' and LCDR3' sequence is provided herein. and HCDR1' sequences include DYYMN (SEQ ID NO:23), SEQ ID NOs:29, 35, 41 and At least 80% of it (e.g., at least 85%, 88%, 90%, 91%, 92%, Homologues with sequence identity of 93%, 94%, 95%, 96%, 97%, 98%, 99%) is selected from the group consisting of sequences The HCDR2' sequence is DINPNNGGTSYNX' 1 KFX' 2 G (SEQ ID NO: 24) , SEQ ID NO: 30, 36, 42 or at least 80% thereof (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) of sequence identity to the sequence of the homologous sequence, The HCDR 3' sequence is VKWGDGPFAY (SEQ ID NO: 25), SEQ ID NO: 31, 37, 43 and at least 80% thereof (e.g., at least 85%, 88%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) of sequence identity. a homologous sequence selected from the group consisting of The LCDR1' sequence is X' 3 ASQNVGAAVA (SEQ ID NO: 26), SEQ ID NO: 32, 38, 44 and at least 80% thereof (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) of sequence identity and The LCDR2' sequence is SASNX' 4 X' 5 T (SEQ ID NO: 27), SEQ ID NO: 33, 39 , 45 and at least 80% thereof (e.g., at least 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity a homologous sequence selected from the group consisting of The LCDR3' sequence is QQYSNYPT (SEQ ID NO: 28), SEQ ID NO: 34, 40, 46 and at least 80% thereof (e.g., at least 85%, 88%, 90%, 91%, 9 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) of sequence identity homologous sequences, X' 1 is H or Q, and X' 2 is K or Q, and X' 3 is K or Q Yes, X' 4 is R or L, and X' 5 is Y or E.

[0031] In certain embodiments, X'1 is Q and X' 2 is Q and X' 3 is Q , X' 4 is R and X' 5 is Y.

[0032] In certain embodiments, the PD-L1 antibodies provided herein comprise a heavy chain framework of HFR1', HFR2', HFR3', and HFR4' and a light chain framework sequence of LFR1', LFR2', LFR3', and LFR4', and the heavy chain variable region sequence is represented by the formula: HFR1'-HCDR1'-HFR2'-HCDR 2 '-HFR3'-HCDR 3 According to '-HFR4', the sequence of the light chain variable region is of the formula LFR1'-LCDR1'-LFR2'-LCDR 2 '-LFR3'-LCDR 3 Follow '-LFR4'.

[0033] In certain embodiments, the HFR1′ sequence is 1 VQLX'a 2 QSGX'a 3 EX 'a 4 X'a 5 KPGASVKX'a 6 SCKASGYVFT (SEQ ID NO: 47), The HFR2' sequence is 7 QX'a 8 X'a 9 GX'a 10 X'a 11 LEWI G (SEQ ID NO: 48); The HFR3' sequence is 12 X'a 13 TVTVDX'a 14 SX'a 15 X'a 1 6 TAYMELX'a 17 X'a 18 LX'a 19 SX'a 20 DX'a 21AVYYC (SEQ ID NO: 49), The HFR4' sequence is WGQGTLVTVSX'a 22 (SEQ ID NO: 50), The LFR1' sequence is DIX'a 23 MTQSX'a 24 X'a 25 X'a 26 X'a 2 7 SX'a 28 SVGDRVX'a 29 ITC (SEQ ID NO:51), The LFR2' sequence is WYQQKPGX'a 30 X'a 31 PKLLIY (SEQ ID NO:52 ) and The LFR3' sequence is GVPX'a 32 RFX'a 33 GSGSGTDFTLTISX' a 34 X'a 35 QX'a 36 EDX'a 37 AX'a 38 YFC (SEQ ID NO:53) the law of nature, The LFR4' sequence is FGSGTKLGIK (SEQ ID NO:54), Here, X'a 1 is Q or E, and X'a 2 is Q or V, and X'a 3 teeth , A or P, and X'a 4 is L or V, and X'a 5 is V or K, X'a 6 is I or V, and X'a 7 is K or R, and X'a 8 is S or A and X'a 9 is H or P, and X'a 10 is Q or K, and X'a 1 1 is S or G, and X'a12 is K or R, and X'a 13 is A or V and X'a 14 is K or T, and X'a 15 is S, T or I, and X' a 16 is S or R, and X'a 17 is L or S, and X'a 18 , S or is R, and X'a 19 is R or T, and X'a 20 is E or D, and X' a 21 is S or T, and X'a 22 is A or S, and X'a 23 Q or is V, and X'a 24 is Q or P, and X'a 25 is K or S, and X' a 26 is F or S, and X'a 27 is M or L, and X'a 28 T or is A and X'a 29 is S or T, and X'a 30 is Q or K, and X' a 31 is S or A, and X'a 32 is S or D, and X'a 33 , S or is T and X'a 34 is S or N, and X'a 35 is M or L, and X' a 36 is S or P, and X'a 37 is L or I, and X'a 38 D or is T.

[0034] In certain embodiments, X'a 1 is Q and X'a 2 is V and X'a 3 A and X'a 4 is V and X'a 5 is V and X'a 6 is I and X'a 7 is K and X'a 8 is A and X'a 9 is P, and X'a 10 Is Q , X'a 11 is G and X'a 12 is R, and X'a 13 is A and X' a 14 is K and X'a 15 is T and X'a 16 is R, and X'a 17 teeth , S, and X'a 18 is R, and X'a 19 is R, and X'a 20 is D , X'a 21 is T and X'a 22 is S and X'a 23 is Q and X' a 24 is Q and X'a 25 is S and X'a 26 is S and X'a 27 teeth , L, and X'a 28 is A and X'a 29 is T and X'a 30 is K , X'a 31 is A and X'a 32 is S and X'a 33 is S and X' a 34 is S and X'a 35 is M and X'a 36 is P, and X'a 37 teeth , I and X'a 38 is T.

[0035] In certain embodiments, X'a1 is Q and X'a 2 is V and X'a 3 A and X'a 4 is V and X'a 5 is K and X'a 6 is V and X'a 7 is R, and X'a 8 is A and X'a 9 is P, and X'a 10 Is Q , X'a 11 is G and X'a 12 is R, and X'a 13 is V and X' a 14 is T and X'a 15 is I and X'a 16 is R, and X'a 17 teeth , S, and X'a 18 is R, and X'a 19 is R, and X'a 20 is D , X'a 21 is T and X'a 22 is S and X'a 23 is Q and X' a 24 is Q and X'a 25 is S and X'a 26 is S and X'a 27 teeth , L, and X'a 28 is A and X'a 29 is T and X'a 30 is K , X'a 31 is A and X'a 32 is S and X'a 33 is S and X' a 34 is S and X'a 35 is M and X'a 36 is P, and X'a 37 teeth , I and X'a38 is T.

[0036] In certain embodiments, X'a 1 is Q and X'a 2 is V and X'a 3 A and X'a 4 is V and X'a 5 is V and X'a 6 is I and X'a 7 is K and X'a 8 is A and X'a 9 is P, and X'a 10 Is Q , X'a 11 is G and X'a 12 is R, and X'a 13 is A and X' a 14 is K and X'a 15 is T and X'a 16 is R, and X'a 17 teeth , S, and X'a 18 is R, and X'a 19 is R, and X'a 20 is D , X'a 21 is T and X'a 22 is S and X'a 23 is Q and X' a 24 is P, and X'a 25 is S and X'a 26 is S and X'a 27 teeth , L, and X'a 28 is A and X'a 29 is T and X'a 30 is K , X'a 31 is A and X'a 32 is S and X'a 33 is S and X' a 34 is S and X'a 35is L, and X'a 36 is P, and X'a 37 teeth , I and X'a 38 is T.

[0037] In certain embodiments, X'a 1 is Q and X'a 2 is V and X'a 3 A and X'a 4 is V and X'a 5 is K and X'a 6 is V and X'a 7 is R, and X'a 8 is A and X'a 9 is P, and X'a 10 Is Q , X'a 11 is G and X'a 12 is R, and X'a 13 is V and X' a 14 is T and X'a 15 is I and X'a 16 is R, and X'a 17 teeth , S, and X'a 18 is R, and X'a 19 is R, and X'a 20 is D , X'a 21 is T and X'a 22 is S and X'a 23 is Q and X' a 24 is P, and X'a 25 is S and X'a 26 is S and X'a 27 teeth , L, and X'a 28 is A and X'a 29 is T and X'a 30 is K , X'a 31 is A and X'a 32 is A and X'a33 is S and X' a 34 is S and X'a 35 is L, and X'a 36 is P, and X'a 37 teeth , I and X'a 38 is T.

[0038] In certain embodiments, the PD-L1 antibodies provided herein have the sequence set forth in SEQ ID NO:65. and a light chain variable region as shown in SEQ ID NO: 66. In some embodiments, the PD-L1 antibodies provided herein have a heavy chain homologue as set forth in SEQ ID NO:67. In certain embodiments, the antibody of the present invention comprises a light chain variable region as set forth in SEQ ID NO: 68 and a light chain variable region as set forth in SEQ ID NO: 69. The PD-L1 antibodies provided herein have a heavy chain variable region and a sequence as set forth in SEQ ID NO:65. In certain embodiments, the light chain variable region comprises the light chain variable region set forth in sequence number 68. The PD-L1 antibody of interest has a heavy chain variable region set forth in SEQ ID NO:67 and a PD-L1 antibody of interest has a heavy chain variable region set forth in SEQ ID NO:66. In certain embodiments, the PD-L 1 antibody comprises a heavy chain variable region shown in SEQ ID NO: 85 and a light chain variable region shown in SEQ ID NO: 86. Includes the area.

[0039] The present disclosure relates to at least 1, 2, 3, 4, 5 or 6 selected from SEQ ID NOs: 29 to 46. Further provided herein is another isolated PD-L1 antibody comprising two CDRs. In certain embodiments, 1) a heavy chain HCDR1' that is SEQ ID NO:29, a HCDR2' that is SEQ ID NO:30, DR2' and HCDR3' which is SEQ ID NO:31; 2) heavy chain HCDR which is SEQ ID NO:35 1', an HCDR2' which is SEQ ID NO:36 and an HCDR3' which is SEQ ID NO:37; or 3) a heavy chain HCDR1' having SEQ ID NO: 41, an HCDR2' having SEQ ID NO: 42, and An isolated PD-L1 antibody comprising an HCDR3' which is sequence number 43. In certain embodiments, The PD-L1 antibody comprises 1) a light chain LCDR1' having a sequence of SEQ ID NO: 32, a light chain LCDR2' having a sequence of SEQ ID NO: 33, CDR2' and LCDR3' of SEQ ID NO: 34; 2) light chain LCDR3' of SEQ ID NO: 38 R1', LCDR2' which is SEQ ID NO:39 and LCDR3' which is SEQ ID NO:40; 3) a light chain LCDR1' having SEQ ID NO: 44, an LCDR2' having SEQ ID NO: 45, and In certain embodiments, the PD-L1 antibody comprises 1) an LCDR3' having sequence number 46. Heavy chain HCDR1' is SEQ ID NO: 29, HCDR2' is SEQ ID NO: 30 and SEQ ID NO: 3 1, light chain LCDR1' is SEQ ID NO: 32, LC 1) a heavy chain HCDR2' and an LCDR3' that is SEQ ID NO: 34; 2) a heavy chain HCDR that is SEQ ID NO: 35 1', HCDR2' which is SEQ ID NO: 36 and HCDR3' which is SEQ ID NO: 37, light chain LCDR1' having SEQ ID NO: 38, LCDR2' having SEQ ID NO: 39 and LCDR3 having SEQ ID NO: 40 or 3) a heavy chain HCDR1' that is SEQ ID NO:42; and HCDR2' which is SEQ ID NO: 43, and HCDR3' which is SEQ ID NO: 44. R1', LCDR2' which is SEQ ID NO:45 and LCDR3' which is SEQ ID NO:46 .

[0040] In certain embodiments, the PD-L1 antibody is selected from SEQ ID NOs: 55, 57, and 59. In certain embodiments, the PD-L1 antibody comprises a heavy chain variable region comprising SEQ ID NOs: 56, 58 and 59. and a light chain variable region selected from 60.

[0041] In certain embodiments, the PD-L1 antibody comprises the heavy chain variable region of SEQ ID NO: 55 or a human and the light chain variable region of SEQ ID NO:56 or a humanized version thereof. In certain embodiments, the PD-L1 antibody comprises the heavy chain variable region of SEQ ID NO: 57, or a humanized version thereof. and the light chain variable region of SEQ ID NO:58 or a humanized version thereof. In embodiments, the PD-L1 antibody comprises the heavy chain variable region of SEQ ID NO:59, or a humanized version thereof. and the light chain variable region of SEQ ID NO: 60, or a humanized version thereof.

[0042] The present disclosure also relates to antibodies that bind to the same epitope as the PD-L1 antibodies provided herein. or another isolated antibody that has competitive binding with the PD-L1 antibodies provided herein. In certain embodiments, the epitope is at least E5 In a particular embodiment, the epitope comprises the amino acid residues S8 and S80 of the following PD -L1 amino acid residues: Y56, R113, D122, Y123 and R125 At least one (e.g., at least two, three, or four) of the In this embodiment, the epitope is a combination of the following amino acid residues of PD-L1: 1) E58, R11 3) D122, Y123 and R125; 2) Y56, E58 and R113; 3) E5 In certain embodiments, the epitope comprises one of the following: At least one (or at least two) of the following PD-L1 amino acid residues: S80 and D122 In certain embodiments, the epitope further comprises at least one of the following epitopes of PD-L1: Combination of amino acid residues: 1) E58, S80, R113, D122, Y123 and R1 25;2) Contains one of E58, S80, R113 and R125.

[0043] The present disclosure also provides a method for producing a thermostable ferroelectric material having a temperature above 76° C. (e.g., 80° C.) as measured by differential scanning calorimetry. An isolated PD-L1 antibody with a thermal transition midpoint (Tm) of 100°C or higher (>85°C, >90°C, or >85°C) In certain embodiments, the antibodies provided herein are bispecific. Heterologous antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, recombinant antibodies, labeled antibodies, bispecific antibodies In certain embodiments, the antibody is a camelized monovalent antibody or an anti-idiotypic antibody. Domain antibodies, diabodies, scFv, scFv dimers, BsFv, dsFv, (ds Fv) 2 , dsFv-dsFv', Fv fragment, Fab, Fab', F(ab') 2 , ds Diabodies, nanobodies, domain antibodies, isolated CDRs and bivalent domain antibodies The antigen-binding fragment is selected from the group consisting of:

[0044] In certain embodiments, the PD-L1 antibodies provided herein (4B6, 26F5 , 21F11, 23A11, 23F11 and 22C9) are used in humans and non-human primates. In certain embodiments, the present invention The PD-L1 antibodies provided in the specification (such as 18G4) are directed against human and mouse PD-L1 1.

[0045] The present disclosure also relates to pharmaceutical compositions comprising the antibodies provided herein.

[0046] The present disclosure also relates to polynucleotides encoding the antibodies provided herein. A vector comprising the polynucleotide is provided herein. Also provided herein are isolated host cells. In certain embodiments, the host cells are Antibodies encoded by the polynucleotide are produced.

[0047] The present disclosure also relates to kits that include the antibodies provided herein.

[0048] The present disclosure also provides a method for producing an antibody provided herein, comprising: and culturing under conditions such that the polynucleotide is expressed.

[0049] The present disclosure also relates to a method of treating a PD-L1 associated condition in a subject in need thereof. administering to a subject a therapeutically effective amount of an antibody provided herein. In certain embodiments, the second therapeutic agent is selected from the group consisting of radiation therapy, chemotherapy, targeted therapy, Gene therapy, immunotherapy, hormone therapy, anti-angiogenesis, palliative care, surgery or a combination thereof In certain embodiments, the second therapeutic agent is an agent used in combination with a VEGFR 2 antibody.

[0050] Pharmaceutical compositions comprising a PD-L1 antibody provided herein and a second therapeutic agent also include Provided in this disclosure.

[0051] 1. A method of treating a PD-L1 associated condition in a subject in need thereof, comprising administering to the subject: Further provided in the present disclosure are methods comprising administering a therapeutically effective amount of the pharmaceutical composition. .

[0052] The present disclosure also provides an isolated nucleic acid sequence comprising a polynucleotide encoding a human PD-L1 protein. In certain embodiments, the non-human tumor cells are derived from rodents (e.g., For example, mouse, rat or hamster tumor cells.

[0053] Also provided herein are polypeptides comprising a polynucleotide encoding the human PD-L1 protein. A method for producing a non-human tumor cell comprising a polynucleotide encoding human PD-L1. Also provided are methods comprising introducing an endogenous non-human In a particular embodiment, the inactivation further comprises inactivating the PD-L1 gene. Gene disruption, mutation, addition, gene silencing or deletion of protein coding regions Gene deletions, thereby eliminating or minimizing expression of a target gene or inhibiting its function In certain embodiments, human PD-L is produced as an inactive / truncated protein. The gene segment is operably inserted into the endogenous non-human PD-L1 locus. In certain embodiments, the human PD-L1 gene segment is an endogenous non-human PD-L1 gene. In certain embodiments, the human PD-L1 gene segment is inserted at a site other than the locus. It is in the form of an episomal DNA segment.

[0054] Screening or evaluating the in vivo efficacy of antibodies against human PD-L1 The method comprises the steps of: detecting a non-human tumor comprising a polynucleotide encoding the human PD-L1 protein; inoculating a non-human animal with tumor cells and contacting the antibody with the non-human tumor cells in the non-human animal. and determining the tumor burden of the non-human tumor cells. The non-human cells may be tumor cells or non-solid tumor cells. In certain embodiments, the non-human cells are isolated from syngeneic tissue. Syngeneic non-human animals are inoculated to generate genetic tumor models. In some embodiments, the non-human animal is a rodent (e.g., a mouse, a rat, or a hamster). do.

[0055] In one aspect, the disclosure provides a combination of the following PD-L1 amino acid residues: 1) E58 and S80; 2) E58 and R113; 3) E58 and D122; 4) E58 and R1 25 Provided herein are PD-L1 antibodies that have been modified to target PD-L1. In certain embodiments, the epitope is Additionally, at least one of the following PD-L1 amino acid residues: Y56 and Y123 In certain embodiments, the epitope comprises the following combination of amino acid residues of PD-L1: 1) E58, R113, M115, Y123 and K124; 2) E58, S80, R1 13 and R125; 3) E58, R113, D122, Y123 and R125; 4) Y56, E58 and R113, 5) Contains one of E58, R113 and R125 In certain embodiments, the epitope is located at the following amino acid residues of PD-L1: K62, N6 In certain embodiments, the nucleic acid further comprises at least one of epitaxial growth factor 1 (EGF), epitaxial growth factor 2 (EGF), epitaxial growth factor 3 (EGF), epitaxial growth factor 4 (EGF), epitaxial growth factor 5 (EGF), epitaxial growth factor 6 (EGF), epitaxial growth factor 7 (EGF), epitaxial growth factor 8 (EGF), epitaxial growth factor 9 (EGF), epitaxial growth factor 10 (EGF), epitaxial The topes consist of the following combinations of PD-L1 amino acid residues: 1) E58, N63, I64, S 80, Y81, R113 and R125; 2) E58, S80, R113, M115, D 122, Y123, K124 and R125; 3) E58, K62, N63, S80, Y 81, R113 and R125; 4) E58, N63, I64, S80, Y81, R11 3) K124 and R125; 5) E58, S80, R113, D122, Y123 and and R125.

[0056] In one embodiment, it binds to an epitope consisting of amino acid residue S80 of PD-L1; or Also provided herein are isolated PD-L1 antibodies that have competitive binding therewith.

[0057] In one aspect, the present disclosure provides a method for the treatment of a cancer, comprising administering to a patient a therapeutically effective amount of a cancer cell for at least 7 days (e.g., at least 8, 9, 10, 11, 12, 14, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 2, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 30, 35 or 40 days) of sustained PD-L1 receptor occupancy. Provides PD-L1 antibodies.

[0058] Approximately 0.03 μg / ml as measured by ELISA (e.g. Measured 0.001μg / ml to 1μg / ml (e.g., 0.001μg / ml to 0. 5μg / ml, 0.001μg / ml~0.2μg / ml, 0.001μg / ml~0. 1μg / ml, 0.01μg / ml~0.2μg / ml, 0.01μg / ml~0.1μ g / ml, 0.01μg / ml~0.05μg / ml, 0.01μg / ml~0.03μ g / ml or 0.001 μg / ml to 0.01 μg / ml) EC50 value, or FA 0.01 μg / ml to 1 μg / ml (e.g., 0.01 μg / ml) measured by CS l~0.5μg / ml, 0.01μg / ml~0.2μg / ml, 0.02μg / ml~ 1μg / ml, 0.02μg / ml~0.5μg / ml, 0.02μg / ml~0.2μ Activated β-lactamase with an EC50 of 0.02μg / ml or 0.1μg / ml An isolated PD-L1 antibody that binds to selected human T cells. [Brief description of the drawings]

[0059] [Figure 1] Figure 1A is a bar graph presenting the ability of mouse anti-PD-L1 antibodies in hybridoma supernatants to block the binding of hPD-L1-Fc to hPD-1-his coated on a plate as measured by ELISA analysis. Figure 1B is a bar graph presenting the ability of mouse anti-PD-L1 antibodies in hybridoma supernatants to block the binding of hPD-L1-Fc to hPD-1-his coated on a plate. Figure 1C is also a bar graph presenting the ability of mouse anti-PD-L1 antibodies in hybridoma supernatants to block the binding of hPD-L1-Fc to hPD-1-his coated on a plate. [Diagram 2] Figure 2A is a graphical representation showing the pH dependent binding of murine anti-PD-L1 antibody 23A11 to hPD-L1-his in hybridoma supernatants, with higher binding at pH 7.4 (triangles) and lower binding at pH 6.0 (diamonds) as measured by ELISA analysis. Figure 2B shows the pH dependent binding of murine antibody clone 23F11 to hPD-L1 in hybridoma supernatants, with higher binding at pH 7.4 (triangles) and lower binding at pH 6.0 (diamonds) as measured by ELISA analysis. [Diagram 3] Figure 3 shows the binding of serially diluted different clones of purified mouse monoclonal PD-L1 antibodies (21F11 (x), 23F11 (diamond), 26F5 (open circle), 22C9 (open triangle), 4B6 (closed square), 23A11 (closed triangle)) to hPD-L1 as measured by ELISA analysis. [Figure 4]Figure 4A shows the activity of serially diluted purified mouse monoclonal PD-L1 antibodies (23A11 (light grey filled squares), 23F11 (filled triangles), 4B6 (filled circles), 22C9 (filled diamonds), 26F5 (dark filled squares), 21F11 (inverted triangles), and BM-GT (open circles)) in blocking the binding of hPD-1-Fc N297A to hPD-L1 expressed on HCC827 as measured by ELISA. Figure 4B shows the activity of PD-L1 antibodies at 10μg / ml in blocking the binding of hPD-1-Fc N297A to hPD-L1 expressed on HCC827 as measured by FACS analysis. [Diagram 5] Figure 5A shows the dose-dependent binding of mouse purified PD-L1 antibodies to hPD-L1 expressed on HCC827. Figure 5B shows the dose-dependent binding of mouse purified PD-L1 antibodies in blocking the binding of hPD-1-N297A to hPD-L1 expressed on HCC827. 21F11 (circle), 23A11 (triangle), 23F11 (inverted triangle), 22C9 (diamond) and 4B6 (large circle). [Figure 6-1] [Figures 6A and 6B] Figure 6A (4B6-C), Figure 6B (23A11), Figure 6C (23F11), Figure 6D (22C9) and Figure 6E (21F11) show that mouse and / or chimeric PD-L1 antibodies bind similarly to human and cynomolgus PD-L1-his as measured by ELISA, while those that specifically bind to human and cynomolgus PD-L1 do not bind to mouse PD-L1-his, except for 18G4 which binds to human and mouse PD-L1 (Figure 6F). [Figure 6-2] [Figures 6C and 6D] Ibid. [Figure 6-3] Figure 6G shows that all antibodies were capable of binding to hPD-L1, but not to hPD-L2, as determined by ELISA. [Figure 7] Figure 7 illustrates the binding of chimeric and / or humanized PD-L1 antibodies to hPD-L1 expressed on HCC827 as measured by FACS. [Figure 8]Figure 8 shows the activity of chimeric and / or humanized PD-L1 antibodies in blocking the binding of PD-1 to hPD-L1 expressed on HCC827 as measured by FACS. [Figure 9] Figure 9A (18G4), Figure 9B (4B6) and Figure 9C (23A11) are bar graphs showing the results of epitope binding of PD-L1 antibodies to hPD-L1-Fc in the presence of competing antibodies 4B6, 23A11, 23F11, 22C9 and 21F11, respectively, as measured by competitive ELISA. [Figure 10] Figure 10A (23F11-C), Figure 10B (23A11-C), Figure 10C (22C9-C), Figure 10D (21F11-C), Figure 10E (4B6-C), and Figure 10F (26F5-C) are graphs showing the pH dependent binding of chimeric PD-L1 antibodies to hPD-L1-his at pH 7.4 and pH 5.5, respectively, as measured by ELISA analysis. [Figure 11] Figure 11A shows the averaged tumor volumes measured over time in the MC38 / human-PD-L1 knock-in tumor model demonstrating the in vivo anti-tumor activity of purified murine anti-PD-L1 antibodies (4B6 (filled diamonds), 23A11 (open inverted triangles), 23F11 (filled striped diamonds) and 26F5 (open diamonds)) and vehicle (filled circles) in inhibiting tumor growth. Figure 11B shows the averaged tumor volumes at day 29 for the same antibodies. Figure 11C shows the averaged body weights measured over time for the same antibodies. Mice (n=8 for each group) were injected IP with 10 mg / kg (i.e. mpk) of antibody 3 times per week for 3 weeks and the results are expressed as the mean ± SEM. [Figure 12-1]Figure 12A shows the averaged tumor volumes measured over time in an MC38 / human-PD-L1 knock-in tumor model injected with purified murine / chimeric PD-L1 antibodies (4B6-C (light grey filled triangle), 23F11 (dark filled inverted triangle)) and benchmark antibodies BM-GT (open circle) and BM-ME (open square) or control PBS (filled circle) in inhibiting tumor growth, demonstrating the in vivo anti-tumor activity of the antibodies. Figure 12B shows the averaged tumor volumes at day 20 in mice (n=8 for each group) injected IP with 1 mpk (mg / kg) of the antibodies, 3 times / week for 3 weeks. [Figure 12-2] Figure 12C shows the averaged tumor volumes measured over time, and Figure 12D shows the averaged tumor volumes at day 25 for mice (n=8 for each group) injected IV with 1 mpk (light grey filled inverted triangles) or 3 mpk (filled diamonds) mouse antibody 18G4. [Figure 12-3] Figure 12E shows the averaged tumor volume measured over time. Figure 12F shows the averaged tumor volume on day 25 for mice (n=8 for each group) IV-injected with 1 mpk (open circle) or 3 mpk (open square) mouse 22C9, respectively. Results are expressed as mean±SEM. [Figure 13] Figure 13A shows the averaged tumor volumes measured over time in the MC38 / human-PD-L1 knock-in tumor model, demonstrating the in vivo anti-tumor activity of purified pH-dependent chimeric PD-L1 antibodies (23A11-C in open circles, 23F11-C in open triangles) in inhibiting tumor growth. Figure 13B shows the averaged tumor volumes at day 29. Mice (n=8 for each group) were injected IP with 1mpk antibody 3 times / week for 3 weeks, and results are expressed as mean±SEM, P<0.05. Vehicle is represented by closed circles and benchmark GT is closed inverted triangles. [Figure 14]Figure 14A shows the amino acid sequences of the germline and humanized heavy chain variable regions of 4B6 (4B6-germline, 4B6-Hzd-HC-V3 and 4B6-HC-V4). Figure 14B shows the amino acid sequences of the germline and humanized light chain variable regions of 4B6 (4B6LC germline, 4B6-Hzd-LC-V3 and 4B6-LC-V4). [Figure 15] Figure 15A shows the amino acid sequences of the germline and humanized heavy chain variable regions of the 23A11 antibody (23A11-HC-germline, 23A11-HC-V3 and 23A11-HC-V5). Figure 15B shows the amino acid sequences of the germline and humanized light chain variable regions of the 23A11 antibody (23A11-LC-germline, 23A11-LC-V3 and 23A11-LC-V5). [Figure 16] Figure 16A shows the amino acid sequences of the germline and humanized heavy chain variable regions of the 23F11 antibody (23F11 germline HC, 23F11-HC-V4 and 23F11-HC-V6). Figure 16B shows the amino acid sequences of the germline and humanized light chain variable regions of the 23F11 antibody (23F11 germline LC, 23F11-LC-V4 and 23F11-LC-V6). [Figure 17] Figure 17A is a bar graph showing binding of 23F11 antibodies (H4L4, H4L6, H6L4, H6L6 and chimera).Figure 17B shows binding of humanized 23A11 antibodies (H3L3, H3L5, H5L3 and H5L5) to hPD-L1-his as measured by ELISA. [Figure 18-1][Figure 18A and Figure 18B] Figures 18A-18D are bar graphs showing the activity of humanized PD-L1 antibodies in blocking the binding of hPD-L1-Fc to hPD-1-N297A as measured by ELISA. The results for humanized 4B6 (H3L3, H3L4, H4L3, and H4L4, Figure 18A), 23F11 (H4L4, H4L6, H6L4, H6L6, and chimeric (C), Figure 18B), humanized 23A11 (H3L3, H3L5, H5L3, and H5L5, Figure 18C), humanized 23A11 (H3L3 and H3L5), and humanized 23F11 (H4L4) and benchmark antibodies (BM-GT and BM-ME, see Figure 18D), are shown, respectively. [Figure 18-2] [Figures 18C and 18D] Ibid. [Figure 19] Figure 19 shows the binding of humanized PD-L1 antibodies (BM-GT, BM-ME, 4B6-H3L4, 4B6-H4L3, 23F11-H4L4, 23F11-H4L6, 23F11-H6L4, 23F11-H6L6, 23A11-H3L3, 23A11-H3L5, 23A11-H5L3, and 23A11-H5L5) to hPD-L1 expressed on HCC827 as measured by FACS. [Figure 20] Figure 20A is a bar graph showing the activity of humanized 4B6 (H3L4 and H4L3) and benchmark antibodies (BM-GT and BM-ME) in blocking the binding of hPD-1-N297A to hPD-L1 expressed on HCC827 as measured by FACS. Figure 20B shows the blocking activity of humanized 23A11 H3L5 (produced in CHO and 293, respectively), humanized 23F11 H4L4 (produced in CHO and 293, respectively), and benchmark antibodies (BM-GT and BM-ME) on the binding of hPD-1-N297A to hPD-L1 expressed on HCC827 as measured by FACS. [Figure 21-1]Figure 21A is a bar graph illustrating IL-2 produced in activated donor 5 CD4 T cells stimulated with humanized 4B6-H3L3 antibody, hIgG1, BM-GT and BM-ME in a mixed lymphocyte reaction (MLR). Figure 21B shows IFN-γ produced in activated donor 5 CD4 T cells stimulated with humanized 4B6-H3L3 antibody, hIgG1, BM-GT and BM-ME in the same study. [Figure 21-2] Figure 21C shows IL-2 produced in activated donor 6 CD4 T cells stimulated with humanized 23A11-H3L3, 23A11-H3L5, 23F11-H4L4 and 23F11-H6L4, hIgG1, BM-GT and BM-ME in the same study. Figure 21D shows IFN-γ produced in activated donor 6 CD4 T cells stimulated with humanized 23A11-H3L3, 23A11-H3L5, 23F11-H4L4 and 23F11-H6L4 antibodies, hIgG1, BM-GT and BM-ME in the same study. [Figure 22] Figures 22A and 22C show the pH dependent binding of humanized 23A11-H3L5 produced in CHO cells and 293 cells to hPD-L1-his as measured by ELISA analysis at pH 7.4 (triangles) and pH 5.5 (diamonds) respectively. Figures 22B and 22D show the binding of humanized 23F11-H4L4 to hPD-L1-his as measured by ELISA analysis at pH 7.4 (triangles) and pH 5.5 (diamonds) respectively. Figure 22E shows the binding of benchmark antibody BM-GT to hPD-L1-his as measured by ELISA analysis at pH 7.4 (triangles) and pH 5.5 (diamonds) respectively. [Figure 23-1]Figure 23A shows the pH-dependent binding of 23A11-H3L5 to hPD-L1-his at neutral (i.e., 7.4) and acidic pH (i.e., 6.0, 5.5, 5.0, 4.5, 4.0) as measured by ELISA analysis. Figure 23B shows the same results expressed as the percentage of the signal normalized to that at pH 7.4. [Figure 23-2] Figure 23C shows the pH dependent binding of 23F11-H4L4 to hPD-L1-his at neutral (i.e. 7.4) and acidic pH (i.e. 6.0, 5.5, 5.0, 4.5, 4.0) as measured by ELISA analysis. Figure 23D shows the same results expressed as percentage of the signal normalized to that at pH 7.4. Figure 23E shows the pH dependent binding of 22C9-C to hPD-L1-his at neutral (i.e. 7.4) and acidic pH (i.e. 6.0, 5.5, 5.0, 4.5, 4.0) as measured by ELISA analysis. [Figure 23-3] Figure 23F shows the same results expressed as percentage of signal normalized to that at pH 7.4. In all figures in Figure 23, pH 7.4 (grey circles), pH 6.0 (dark circles), pH 5.5 (dark squares), pH 5.0 (grey inverted triangles), pH 4.5 (grey diamonds), pH 4.0 (grey triangles). [Figure 24-1] [Figure 24A, Figure 24B, and Figure 24C] Figures 24A-I are bar graphs showing the binding of PD-L1 antibodies (23F11 (Figure 24A), 23A11 (Figure 24B), 26F5 (Figure 24C), 18G4 (Figure 24D), 4B6 (Figure 24E), 21F11 (Figure 24F), 22C9 (Figure 24G), 23F11-H4L4 (Figure 24H), and 23A11-H3L5 (Figure 24I)) to alanine mutant hPD-L1-Fcs (prepared in Example 18) as measured by ELISA. [Figure 24-2] [Figures 24D, 24E and 24F] Ibid. [Figure 24-3] [Figures 24G, 24H and 24I] Ibid. [Diagram 25]Figure 25 shows the in vivo anti-tumor activity of humanized PD-L1 antibodies (vehicle, open square, BT-GM, and open circle, 4B6-H3L3-N297A) in inhibiting tumor growth in the MC38 / human-PD-L1 knock-in tumor model as indicated by averaged tumor volume over time. Mice (n=8 per group) were injected IP with 3mpk antibody 3 times / week for 3 weeks and the results are presented as mean±SEM. [Figure 26-1] Figure 26A shows the averaged tumor volumes measured over time in the MC38 / human-PD-L1 knock-in tumor model administered vehicle (filled circle), BM-ME (open square), BM-GT (open diamond), 23A11-H3L5 (open triangle) and 23F11-H4L4 (open inverted triangle), demonstrating the in vivo anti-tumor activity of humanized PD-L1 antibodies in inhibiting tumor growth. Figure 26B shows the averaged tumor volumes at day 28 using mice injected with 1 mpk of antibody. [Figure 26-2] Figure 26C shows the same test as Figure 26A, but using 10 mpk antibody, respectively. Figure 26D shows the same test as Figure 26B, but using 10 mpk antibody, respectively. [Figure 26-3] Figure 26E compares tumor volumes in mice injected with various antibodies and doses (vehicle, diamond, BM-GT with 1 mpk, triangle, BM-GT with 3 mpk, square, BM-GT with 10 mpk, and circle, 23F11-H4L4 with 1 mpk). Mice (n=8 for each group) were injected IP 3 times / week for 3 weeks and the results are presented as mean±SEM. [Figure 27]Figure 27A shows the averaged tumor volumes measured over time in the MC38 / human-PD-L1 knock-in tumor model, demonstrating the in vivo anti-tumor activity of antibodies (vehicle, open triangle, BM-GT, 23A11-H3L5, and dotted circle, 23F11-H4L4) in inhibiting tumor growth. Figure 27B shows the averaged tumor volumes on day 26 with mice dosed with antibodies (vehicle, open triangle, BM-GT, 23A11-H3L5, inverted triangle, and 23F11-H4L4, open circle). Mice (n=12 for each group) were injected IV with 1mpk antibody on days 0 and 15 and the results are presented as the mean±SEM. [Figure 28] Figures 28A-D are waterfall graphs showing the change in tumor size normalized to the starting point in mice treated with IV injection of 1 mg / kg of PD-L1 antibodies (vehicle (Figure 28A), BM-GT (Figure 28B), 23A11-H3L5 (Figure 28C), and 23F11-H4L4 (Figure 28D). [Figure 29] Figure 29A shows the average tumor volume measured over time in inhibiting tumor growth in the MC38 / human-PD-L1 knock-in tumor model IV dosed with various doses of PD-L1 antibodies (closed circle, vehicle; open square, BM-GT 1mpk; closed square, BM-GT 10mpk; closed diamond, 23F11-H4L4 1mpk; closed inverted triangle, 23F11-H4L4 10mpk; open circle, 23A11-H3L5 1mpk; closed triangle, 23A11-H3L5 10mpk), demonstrating in vivo anti-tumor activity. Figure 29B shows the averaged tumor volumes on day 15 with mice dosed with various antibodies (vehicle in filled circles, BM-GT 1mpk in filled open squares, BM-GT 10mpk in filled open squares, 23A11-H3L5 1mpk in filled open circles, 23A11-H3L5 10mpk in filled open triangles, 23F11-H4L4 1mpk in filled open circles, and 23F11-H4L4 10mpk in filled inverted open triangles). Mice (n=12 for each group) were injected IV with antibodies on day 0 and results are presented as mean±SEM. [Diagram 30] Figure 30 shows the in vivo anti-tumor activity of humanized PD-L1 antibodies 23A11-H3L5 (in triangles) and 23F11-H4L4 (in circles) in an MC38 / human-PD-L1 knock-in tumor model re-challenged with tumor cells on day 35. [Figure 31-1] Figure 31A shows exemplary images of the emission signal of labeled pH-dependent PD-L1 antibody 23F11-H4L4 and BM-GT penetrating into MC38 / hPD-L1 tumor cells at various time points after dosing at 1 mg / kg intravenously. Figure 31B shows the ratio of emission / tumor volume measured over several hours in each mouse (2-1 (grey inverted triangle), 2-2 (black grey diamond), 2-3 (white grey circle) for BM-GT and 4-1 (white solid diamond), 4-2 (black solid diamond), 4-3 (solid star) for 23F11-H4L4). [Figure 31-2] FIG. 31C shows the tumor volumes measured over several days in each mouse dosed with BM-GT (indicated by squares) or 23F11-H4L4 (inverted triangles); the lines represent the averaged values. [Figure 32-1]Figure 32A shows the averaged tumor volumes measured over time in mice in the hPD-L1 / MC38 tumor model dosed with vehicle (filled circles), DC101 alone (anti-VEGFR2 antibody with open squares), 23A11-C alone (open inverted triangles), and the combination of 23A11-C and DC101 (filled circles), respectively, demonstrating the in vivo anti-tumor activity of the combination. Figure 32B shows the averaged tumor volumes at day 29 with mice injected with vehicle (filled circles), DC101 alone (anti-VEGFR2 antibody with open squares), 23A11-C alone (filled inverted triangles), and the combination of 23A11-C and DC101 (filled diamonds), respectively. FIG. 32C shows averaged mouse body weights measured over time in the same study dosed with vehicle (closed circles), DC101 alone (anti-VEGFR2 antibody in closed squares), 23A11-C alone (closed inverted triangles) and a combination of 23A11-C and DC101 (closed diamonds); mice (n=8 for each group) were injected IV with 0.3 mpk of 23A11-C and / or 20 mpk of DC101 three times a week for three weeks. [Figure 32-2] Figure 32D shows the averaged tumor volumes measured over time using mice dosed with 10mpk control IgG (filled circle), 1mpk DC101 alone (filled square), 1mpk 23A11-H3L5 alone (filled diamond), and a combination of 1mpk 23A11-H3L5 and 1mpk DC101 (filled triangle), respectively. Figure 32E shows the averaged tumor volumes measured over time using mice dosed with 10mpk control IgG (filled circle), 3mpk DC101 alone (filled triangle), 3mpk 23A11-H3L5 alone (open circle), and a combination of 3mpk 23A11- H3L5 and 3mpk DC101 (open inverted triangle), respectively. Mice (n=8 for each group) were injected IV with the antibodies once a week for 2-3 weeks. Results are expressed as mean±SEM. [Diagram 33] FIG. 33A and FIG. 33B show graphs illustrating the thermal transition midpoints (Tm) of 23F11-H4L4 and 23A11-H3L5, respectively, as measured by differential scanning calorimetry (DSC). [Figure 34-1] FIG. 34 shows a list of the sequences described in this disclosure. [Figure 34-2] [List continued]Ibid. [Figure 34-3] [List continued]Ibid. [Figure 34-4] [List continued]Ibid. [Figure 34-5] [List continued]Ibid. [Diagram 35] Figure 35 shows a graph of internalization of 23F11-H4L4 into MC38 / hPD-L1 cells. The Y-axis shows the fluorescent signal of 23F11-H4L4 in cells at various antibody concentrations as measured by Varioskan Flash. Small grey circles represent 23F11-H4L4 tested after 24 hours, and large black circles represent 23F11-H4L4 tested after 6 hours. [Diagram 36] Figures 36A and 36B illustrate the binding of 23F11-H4L4 to activated human T cells. Figure 36A shows the binding of 23F11-H4L4 to CD3 and CD28 positive monocytes, whereas very little binding was detected to CD3 and CD28 negative monocytes. [Figure 37] Figures 37A-37C illustrate an in vivo efficacy study of PD-L1 antibodies in a hPD-1 knock-in mouse model with MC38 / hPD-L1 cells. Figure 37A shows tumor volume over time in human PD-1 knock-in C57BL / 6 mice dosed with PBS and 23F11-H4L4, respectively. Figure 37B shows the change in tumor size in individual mice normalized to the starting point treated with PBS or IV injection of 3 mg / kg PD-L1 antibody, respectively. Figure 37C shows the averaged mouse body weight over time measured in the same study dosed with PBS (filled circle) and 23F11-H4L4 (filled square), respectively. [Figure 38-1]Figures 38A-38C illustrate in vivo efficacy studies of PD-L1 antibodies in a hPD-1 knock-in mouse model with MC38 / hPD-L1 cells. Figures 38A and 38B show tumor volume over time in human PD-1 knock-in C57BL / 6 mice treated with 23F11-H4L4 (1 mg / kg, 3 mg / kg, and 10 mg / kg), control hIgG (AB160160, 10 mg / kg), and BM-GT (10 mg / kg), respectively. [Figure 38-2] Figure 37C shows the change in tumor size in individual mice normalized to the starting point treated with IP injection of PD-L1 antibody, control hIgG, or BM-GT, respectively. [Figure 39] Figure 39 shows PD-L1 receptor occupancy of 23F11-H4L4 and BM-GT measured 2, 7, and 14 days after IV dosing in MC38 / hPD-L1 tumor-bearing mice. [Diagram 40] Figure 40 shows representative images of 23F11-H4L4 infiltration and TILs in frozen MC38 / hPD-L1 CDx tumor sections at 7 days after dosing at 10mpk. Panel A: 23F11-H4L4 stained with Alexa Fluor 488 goat anti-human IgG (H+L) secondary antibody (green); Panel B: TILs stained with Alexa Fluor® 594 anti-mouse CD8b.2 (red); Panel C: Blood vessels stained with Alexa Fluor® 594 anti-mouse CD31 (red); Panel D: Negative control. Nuclei stained with DAPI (blue). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] The following description of the disclosure is intended merely to illustrate various embodiments of the disclosure. As such, the specific modifications discussed are intended to be considered limitations on the scope of the disclosure. As will be apparent to those skilled in the art, various equivalents, modifications and alterations may be made. , may be made without departing from the scope of the present disclosure, and such equivalent embodiments It is understood that all references herein are incorporated by reference in their entirety. All references cited herein, including the applications therefor, are hereby incorporated by reference in their entirety. Be absorbed.

[0061] definition As used herein, "PD-L1" (also known as B7-H1 / CD274) refers to , programmed cell death ligand 1 (PD-L1, e.g., Freeman et al. (2000) J. Exp. Med. 192:1027). It is expressed in the pelvis, spleen, lymph nodes, thymus, heart, and fetal liver, and in many tumor or cancer cells. "PD-L2" stands for programmed cell death ligand 2 (PD-L2, For example, Latchman et al. (2001) Nat. Immunol. 2:261). "PD-1" (also known as CD279) PDCD1 gene is a member of the CD28 family of cells that are involved in the regulation of cell death. It is encoded by IL-1, is expressed on the surface of T cells, and acts as a physiological limiting factor for T cell activation and proliferation. The representative amino acid sequence of human PD-1 is NCB I Accession No. NP_005009.2, which encodes human PD-1 A representative nucleic acid sequence is shown under NCBI accession number: NM_005018.2. "PD-1 ligand" refers to either or both of PD-L1 and PD-L2 and to the Any variant or isoform and / or full length naturally expressed by the cell. The polypeptide includes a fragment thereof having at least one biological activity of the polypeptide. The columns are NCBI accession numbers: NP_054862.1 (representative amino acid sequence) and NC Disclosed under BI Accession Number: NM_014143.3 (representative nucleic acid sequence).

[0062] As used herein, the term "antibody" refers to any immunoglobulin that binds to a specific antigen. Monoclonal, polyclonal, multivalent, multispecific or bispecific antibodies A naturally occurring, intact antibody is a disulfide bonded (bivalent) antibody, or a functional portion thereof. It contains two heavy chains (H) and two light chains (L) interconnected by cleavage. Each heavy chain comprises The variable region (VH) and the first, second and third constant regions (CH1, CH2, and CH3), and each light chain consists of a variable region (VL) and a constant region (CL) Mammalian heavy chains are classified as α, δ, ε, γ, and μ, while mammalian light chains are classified as λ or The variable regions of the light and heavy chains are involved in antigen binding. The variable region is generally divided into three regions of hypervariability called complementarity determining regions (CDRs). (Light chain (L) CDRs, including LCDR1, LCDR2 and LCDR3, HC Heavy chain (H) CDRs including DR1, HCDR2, HCDR3. The CDR boundaries of antibodies and antigen-binding fragments are determined according to the methods described in Kabat, Chothia or Al-L It can be defined or identified by the practice of azikani (Al-Lazikani, B ., Chothia, C., Lesk, AM, J. Mol. Bio l., 273(4), 927 (1997); Chothia, C. et a l., J Mol Biol. Dec 5;186(3):651-63 (198 5);Chothia, C. and Lesk, AM, J.Mol.Bio l., 196,901 (1987); Chothia, C. et al., Nature. Dec 21-28;342(6252):877-83 (1989 );Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991). 3 CDRs are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The stretches at each end, known as framework regions (FR), hold Thus, each VH and VL is inserted between the following amino acid residues: to C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, 3 The constant regions of the heavy and light chains are involved in antigen binding. Although the antibody has no specific amino acid sequence, it exhibits various effector functions. Based on the sequence, they are assigned to five main classes: α, δ, ε, γ, and μ. Characterized by the presence of heavy chains, IgA, IgD, IgE, IgG and IgM. Several subclasses of the major antibody classes are IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or I gA2 (α2 heavy chain), etc.

[0063] As used herein, the term "antigen-binding fragment" refers to an antibody that contains one or more CDRs. Antibody fragments formed from fragments or antigen-binding fragments that do not contain an intact native antibody structure In certain embodiments, the antibody provided herein is Examples of antigen-binding fragments include, but are not limited to, diabodies. Di, Fab, Fab', F(ab') 2 , Fv fragment, disulfide-stabilized Fv fragment (d (sFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulfide Dodecyl stabilized diabody (ds diabody), single chain antibody molecule (scFv), scFv bilayer (bivalent diabodies), multispecific antibodies, camelized single domain antibodies, nanobodies, Antigen-binding fragments include domain antibodies, isolated CDRs and bivalent domain antibodies. , capable of binding to the same antigen that the parent antibody binds. In certain embodiments, the antigen-binding fragment is It may include one or more CDRs derived from a particular human antibody.

[0064] "Fab" with respect to an antibody refers to a single heavy chain variable region and a second heavy chain bound together by disulfide bonds. Monovalent antibody consisting of a single light chain (both variable and constant regions) bound to one constant region Fab refers to an antigen-binding fragment of a heavy chain that is N-terminal to the disulfide bond in the hinge region. It can be obtained by papain digestion of the antibody at proximal residues.

[0065] "Fab'" refers to the residues proximal to the C-terminus of the disulfide bond between the heavy chains in the hinge region. Fab can be obtained by pepsin digestion of an antibody, and thus, Fab is a fragment of the hinge region. A single nucleotide polymorphism in the hinge region differs in a small number of residues (including one or more cysteines) in the This refers to a Fab fragment that contains the Fab portion.

[0066] "F(ab') 2 " refers to a dimer of Fab' containing portions of two light chains and two heavy chains. Refers to...

[0067] "Fc" with respect to an antibody refers to the second and third heavy chains bound together by disulfide bonds. A portion of an antibody consisting of the second and third constant regions of a first heavy chain combined with a constant region of The IgG and IgM Fc regions consist of three heavy chain constant regions (the second, third and fourth constant regions in each chain). and the fourth heavy chain constant region). It can be obtained by papain digestion of an antibody. The Fc portion of an antibody is involved in various effector functions, such as ADCC and CDC. However, it does not function in antigen binding.

[0068] "Fv" with respect to an antibody refers to the smallest fragment of an antibody that retains an intact antigen-binding site. An Fv fragment consists of the variable region of a single light chain associated with the variable region of a single heavy chain. "dsFv" refers to a Fv in which the linkage between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. The term "disulfide-stabilized Fv fragment" refers to a disulfide-stabilized Fv fragment that is a disulfide bond.

[0069] A "single chain Fv antibody" or "scFv" refers to an Fv antibody that is synthesized either directly or via a peptide linker sequence. An engineered antibody comprising a light chain variable region and a heavy chain variable region connected to each other via a Refers to the body (Huston JS et al. Proc Natl Acad Sci USA, 85:5879(1988)). An "scFv dimer" is a dimer of two heavy chain variable fragments. In certain embodiments, the term "light chain variable region" refers to a single chain comprising a light chain variable region and two light chain variable regions together with a linker. An "scFv dimer" is a dimer of one part of the V H But the other part of the V L In cooperation with Two that can target one antigen (or epitope) or different antigens (or epitopes) Another V H -VL The V dimerized with the moiety H -V L Includes ( Bivalent diabodies or bivalent ScFvs (BsFvs linked by a peptide linker) In other embodiments, the "scFv dimer" is H1 and V L1 In cooperation , V H2 and V L2 and each coordinated pair has a different antigen specificity. , V L1 -V H2 (also linked by a peptide linker) H 1 -V L2 (linked by a peptide linker). do.

[0070] A "single chain Fv-Fc antibody" or "scFv-Fc" refers to a single chain Fv-Fc antibody that is linked to the Fc region of an antibody. The term "antibody" refers to an engineered antibody consisting of an scFv.

[0071] "Camelized single domain antibodies", "heavy chain antibodies", "nanobodies" or "HCAbs" There are two V H Refers to antibodies that contain the IgG domain and do not contain light chains (Riechmann L. and Muyldermans S., J Immunol Methods . Dec 10;231(1-2):25-38 (1999); Muylderm ans S., J Biotechnol. Jun;74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005, Heavy chain antibodies were originally derived from the Camelidae family (camels, dromedaries and Despite the lack of light chains, camelized antibodies have reliable antigen-binding receptors. Partry (Hamers-Casterman C. et al., Na ture. Jun 3;363(6428):446-8 (1993); Nguy en VK. et al. “Heavy-chain antibodies in Camelidae; a case of evolutionary innov ation,” Immunogenetics. Apr;54(1):39-47 (2002); Nguyen VK. et al. Immunology. Ma 109(1):93-101 (2003)). The main component corresponds to the smallest known antigen-binding unit generated by the adaptive immune response (Ko ch-Nolte F. et al., FASEB J. Nov;21(13): 3490-8. Epub 2007 Jun 15 (2007). "Diabody " includes small antibody fragments having two antigen binding sites, the fragments being composed of a single polypeptide chain. V in L V connected to the domain H Domain Included (V H -V L or V L -V H ). For example, Holliger P. et al., Proc Natl Acad S ci US A. Jul 15;90(14):6444-8 (1993);EP 404097; see WO93 / 11161). The linker is too short to The two domains cannot pair, and therefore the domains cannot pair with the complementary domains on another strand. The antigen-binding site is forced to pair with the ribosome, thereby generating two antigen-binding sites. Whether targeting the same antigen (or epitope) or different antigens (or epitopes) may also be targeted.

[0072] A "domain antibody" is an antibody fragment that contains only the variable region of a heavy chain or the variable region of a light chain. In certain embodiments, two or more V H The domains are shared with a peptide linker The two domains are joined by binding to form bivalent or multivalent domain antibodies. The V H The domains may target the same antigen or different antigens. do.

[0073] In certain embodiments, "(dsFv) 2 " is composed of three peptide chains: a peptide linker and two V L Two V's joined together H Part include.

[0074] In certain embodiments, a "bispecific ds diabody" is H1 and V L1 Between V by disulfide bridge L1 -V H2 (also linked by a peptide linker) V H1 -V L2 (linked by a peptide linker).

[0075] In certain embodiments, a "bispecific dsFv" or "dsFv-dsFv'" is Two peptide chains: the heavy chain is connected by a peptide linker (e.g., a long flexible linker). are bonded to each other via disulfide bridges, L1and V L2 It is paired with RuV H1 -V H2 The heavy and light chains each contain a disulfide pair. , with different antigenic specificities.

[0076] As used herein, the term "humanized" or "humanized form" refers to an antibody or antigen-binding fragment. In connection with non-human animals (e.g., rodents, rabbits, dogs, goats, horses or chickens), ), CDRs derived from human, FR regions derived from human, and, where applicable, constant regions derived from human. In certain embodiments, the constant region from a human antibody is The humanized antibody or antigen-binding fragment can be used as a human therapeutic. In certain embodiments, the antibodies have reduced immunogenicity as compared to non-human species antibodies. Some of these are either not approved for use in humans or are unlikely to induce an immune response in humans. In one embodiment, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, or human. Rhinoceros monkeys, guinea pigs, hamsters or non-human primates (e.g., monkeys (e.g., cynomolgus monkeys) monkeys or rhesus monkeys) or apes (e.g., chimpanzees, gorillas, simians, In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or an affen. is composed of substantially all human sequences, except for the CDR sequences, which are non-human. In some embodiments, the humanized antibody or antigen-binding fragment has the same or similar properties as the antibody, such as binding or binding affinity. For example, one or more non-human CDRs may be modified to improve performance. In this study, multiple amino acid residues were altered to reduce potential immunogenicity in humans. So, the amino acid residues to be changed are not critical for immunospecific binding, or The changes are conservative changes, so that the binding of the humanized antibody to the antigen is not significantly affected. In some embodiments, the human-derived FR region is identical to the human antibody from which it is derived. It may contain one amino acid sequence or several amino acid changes, e.g., 10 of the amino acids, Some embodiments may include up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 permutation. In this case, such changes in amino acids are limited to the heavy chain FR region and not to the light chain FR region. In some preferred embodiments, the humanized antibody comprises a human Contains FR1-3 and human JH and Jκ.

[0077] The term "chimeric" as used herein refers to a chimeric antibody that is a mixture of heavy and / or light chains derived from a given species. Antibodies or antigens having portions and remainders of heavy and / or light chains derived from different species In an exemplary embodiment, a chimeric antibody comprises a human-derived constant region and a mouse-derived constant region. The antibody may contain variable regions derived from a non-human species, such as from a human.

[0078] "Anti-PD-L1 antibodies" as used herein refer to antibodies having diagnostic and / or therapeutic uses. PD-L1 (e.g., human or non-human primate PD-L1) with sufficient affinity to provide 1) refers to an antibody that can specifically bind to

[0079] "Substantially" and "substantially the same" are used herein to refer to a high degree of similarity between two values. The term "activity" refers to the activity of a compound, and one of skill in the art would understand the statistical and / or biological activity represented by the value. Does not recognize or think there is a significant difference between the two values, or perceives there to be little difference In contrast, "substantially lower" means that the numerical value is less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10%. This means:

[0080] The terms "specific binding" or "specifically binding" as used herein refer to the binding of two molecules It refers to a non-random binding reaction between, for example, an antibody and an antigen. The antibodies or antigen-binding fragments provided herein can be used to treat human and / or non-human spirits. PD-1 and PD-2 at pH 7.4, about 0.01 nM to about 100 nM, about 0.1 nM to about 10 0nM, 0.01nM to approx. 10nM, approx. 0.1nM to approx. 10nM, 0.01nM to approx. 1n M, about 0.1 nM to about 1 nM or about 0.01 nM to about 0.1 nM) D ) specifically binds to D As used herein, the ratio of the dissociation rate to the association rate (k off / k on ), for example, surface plasma using instruments such as Biacore. This can be determined using the Montgomery resonance method.

[0081] The ability to "block binding" or "compete for the same epitope" is defined herein as In the method, two molecules of an antibody or antigen-binding fragment (e.g., human PD-L1 and anti-PD This refers to the ability to inhibit, to any detectable extent, the binding interaction between a specific In embodiments, an antibody or antigen-binding fragment that blocks binding between two molecules In certain embodiments, the inhibition is at least 50%. %, greater than 70%, greater than 80% or greater than 90%.

[0082] The term "epitope" as used herein refers to a region of interest that is bound to an antigen by an antigen-binding protein, such as an antibody. A particular group of atoms on the antigen to be bound (e.g., sugar side chains, phosphoryl groups, sulfonyl groups) or amino acids. Epitopes can be conformational or linear. Conformational epitopes are non-contiguous but are related to the three-dimensional tertiary folding of a protein. The amino acid residues may be spatially juxtaposed for interaction, where the residues It directly contributes to the affinity of the β-lactamase to the β-lactamase and loses its ability to interact when exposed to denaturing solvents. Generally, all of the interaction points of a linear epitope are aligned along primary amino acid residues on the protein. The small segments of consecutive amino acids are linearly arranged in the major histocompatibility complex ( can be digested from antigen binding to MHC molecules or retained upon exposure to denaturing solvents (Salmeron A et al., J Immunol. 1991 N ov 1;147(9):3047-52; Goldsby et al., Imm unology(Fifth ed.). New York: WH Free man and Company. pp. 57-75.ISBN0-7167-49 In one embodiment of the present disclosure, the PD-L1 antibodies provided herein In another embodiment of the present disclosure, the epitope bound by the The epitopes bound by the PD-L1 antibodies provided in are linear. Antibodies bind to the same epitope in an antigen if they exhibit competitive binding with the antigen. For example, the antibody or antigen-binding fragment may be 21F11, 18G4, 4B6, 26F5, 23A11, 23F11, 22C9, their chimeric antibodies, humanized 4B6, humanized 23A Blocks the binding of exemplary antibodies of the disclosure, such as human PD-1, 11 and humanized 23F11 In this case, the antibody or antigen-binding fragment binds to the same epitope as the exemplary antibodies. It can be considered to be combined.

[0083] Specific amino acid residues within the epitope can be modified, for example, by alanine scanning mutagenesis. and mutating the polypeptide to identify mutations that reduce or prevent protein binding. "Alanine scanning mutagenesis" is a method for the identification of an epitope by mutating it with another compound or tag that binds to it. To identify specific residues or regions of a protein that affect its interaction with a protein The method can be carried out in a simple manner. (most preferably alanine or polyalanine or a conservative amino acid Reduces protein binding below a threshold or displaces the protein or the amino acid residue that encodes the one that reduces binding of Any mutation in a codon is likely to be within the epitope bound by the protein In certain embodiments of the disclosure, the epitopes that are critical for pH-dependent PD-L1 antibodies The fragment contains at least one of the amino acid residues E58, N63, S80, Y81, and I64. Includes one.

[0084] The sequences set forth below can be seen in FIG.

[0085] As used herein, "18G4" refers to the heavy chain variable region of SEQ ID NO: 55 (SEQ ID NO: 93 the light chain variable region of SEQ ID NO: 56 (corresponding to the coding DNA sequence of SEQ ID NO: 94); It refers to a mouse monoclonal antibody that has a chimeric DNA sequence corresponding to the chimeric DNA sequence. The 18G4-C / 18G4-chimera antibody is fused to a mouse variable region. It contains a human constant region of the IgG1 isotype.

[0086] As used herein, "4B6" refers to a heavy chain variable region of SEQ ID NO: 85, a light chain variable region of SEQ ID NO: 86, It refers to a mouse monoclonal antibody having a chain variable region. Its chimeric antibody (i.e., 4B 6-C / 4B6-chimera) is a human IgG1 isotype fused to mouse variable regions. The heavy and light chains of the antibody comprise a humanized heavy chain (H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, H19, H20, H21, H22, H23, H24, H25, H36, H37, H48, H49, H50, H1 corresponds to the sequence of SEQ ID NO: 83, and H2 corresponds to the sequence of SEQ ID NO: 65 (sequence H1 has the coding DNA sequence of SEQ ID NO: 67, and H2 has the coding DNA sequence of SEQ ID NO: 68. ) and light chain (L1 corresponds to the sequence of SEQ ID NO: 82 and L2 corresponds to the sequence of SEQ ID NO: 84 L3 corresponds to the sequence of SEQ ID NO:66 (the coding DNA sequence of SEQ ID NO:100) and L2 corresponds to the sequence of SEQ ID NO: 68). Humanized, thereby 4B6-H3L3, 4B6-H3L4, 4B6-H4L3 and Humanized antibodies such as 4B6-H4L4 and 4B6-H4L4 have also been produced using humanized antibodies with positions in the Fc region. Produced in CHO cells for small / large scale production using the N to A mutation at 297 It is possible.

[0087] As used herein, "26F5" refers to the heavy chain variable region of SEQ ID NO: 59 (SEQ ID NO: 97 a light chain variable region of SEQ ID NO:60 (having a coding DNA sequence of SEQ ID NO:98); It refers to a mouse monoclonal antibody having a DNA sequence similar to that of the chimeric antibody. The chimera (i.e., 26F5-C / 26F5-chimera) is a chimera in which the I It contains the human constant region of the gG1 isotype.

[0088] "21F11" refers herein to the heavy chain variable region of SEQ ID NO: 57 (SEQ ID NO: 95 a light chain variable region of SEQ ID NO: 58 (having a coding DNA sequence of SEQ ID NO: 96); It refers to a mouse monoclonal antibody having a chimeric The antibody (i.e., 21F11-C / 21F11-chimera) is fused to mouse variable regions. It contains a human constant region of the IgG1 isotype.

[0089] As used herein, "23A11" refers to the heavy chain variable region of SEQ ID NO: 73, the heavy chain variable region of SEQ ID NO: 74 It refers to a mouse monoclonal antibody having a light chain variable region of 23A11-C / 23A11-chimera) is an IgG1 antibody fused to a mouse variable region. The antibody heavy and light chains contain a human constant region of the same type. The humanized heavy chain (H3 is SEQ ID NO: No. 71 (having a coding DNA sequence of SEQ ID NO: 103), and H5 corresponds to L1 corresponds to the sequence of SEQ ID NO: 69) and the light chain (L2 corresponds to the sequence of SEQ ID NO: 72, L3 corresponds to the sequence of SEQ ID NO: 73, 5 corresponds to the sequence of SEQ ID NO: 70 (having the coding DNA sequence of SEQ ID NO: 104) 23A11- H3L5, 23A11-H5L3, 23A11-H3L3 and 23A11-H5L5 The resulting humanized antibody is 23A11-H3L5 (the corresponding sequences of H3 and L5 are Antibodies such as SEQ ID NO: 61 and SEQ ID NO: 62, respectively, also include those having positions in the Fc region Produced in CHO cells for small / large scale production using the N to A mutation at 297 It is possible.

[0090] As used herein, "23F11" refers to the heavy chain variable region of SEQ ID NO: 79, the heavy chain variable region of SEQ ID NO: 80 It refers to a mouse monoclonal antibody having a light chain variable region of 23F11-C / 23F11-chimera) is an IgG1 antibody fused to a mouse variable region. The antibody heavy and light chains contain a human constant region of the same type. The humanized heavy chain (H4 is SEQ ID NO: No. 75 (having the coding DNA sequence of SEQ ID NO: 105), and H6 corresponds to 77) and the light chain (L4 corresponds to the sequence of SEQ ID NO: 76 (SEQ ID NO: 1 06) and L6 corresponds to the sequence in SEQ ID NO: 78 23F11- H4L4, 23F11-H6L4, 23F11-H4L6, 23F11-H6L6, etc. A humanized antibody is generated. 23F11-H4L4 (the corresponding sequences of H4 and L4 are Antibodies such as SEQ ID NO: 63 and SEQ ID NO: 64 also contain a nucleotide sequence at position 29 in the Fc region. 7 and produced in CHO cells for small / large scale production using the N to A mutation It is possible.

[0091] "22C9" refers herein to the heavy chain variable region of SEQ ID NO: 21 (coding NA sequence SEQ ID NO: 101), the light chain variable region of SEQ ID NO: 22 (coding DNA It refers to a mouse monoclonal antibody having the sequence SEQ ID NO: 102. The chimera (i.e., 22C9-C / 22C9-chimera) is a chimera in which the I It contains the human constant region of the gG1 isotype.

[0092] "Conservative substitution", in the context of amino acid sequences, refers to replacing amino acid residues with those that have similar physicochemical properties. or by replacing the amino acid residue with a different amino acid residue having a side chain having For example, conservative substitutions are amino acids with non-polar side chains. Among amino acid residues (e.g., Met, Ala, Val, Leu, and Ile, Pro, P He, Trp), among residues with uncharged polar side chains (e.g., Cys, Ser, Thr, Asn, Gly and Gln), among residues with acidic side chains (e.g., Asp, Glu) Among amino acids with basic side chains (e.g., His, Lys, and Arg), Among amino acids with sulfur-containing side chains (e.g., Thr, Val, and Ile), in amino acids with aromatic side chains (e.g., Cys and Met) or in residues with aromatic side chains (e.g., For example, Trp, Tyr, His and Phe. An insertion or deletion may also be considered as a "conservative substitution." The number of amino acids that can be conserved can range from about 1 to 5. Conservative substitutions usually do not cause significant changes in the protein conformational structure. , and thus may retain the biological activity of the protein.

[0093] "Percent sequence identity" with respect to an amino acid sequence (or a nucleic acid sequence) refers to the degree to which a sequence After aligning and introducing gaps, if necessary, to achieve maximum correspondence, the reference Amino acid (or nucleic acid) residues in the candidate sequence that are identical to amino acid (or nucleic acid) residues in the sequence Conservative substitutions of amino acid residues are considered to be identical residues. Percent amino acid (or nucleic acid) sequence identity may or may not be possible. Alignment for the purpose of determining the target sequence can be performed using, for example, BLASTN, BLASTp, etc. (US National Center for Biotechnology Information on the National Center for Biological Diversity (NCBI) website, Altschul SF et al. , J. Mol. Biol., 215:403-410 (1990); Ste. phen F. et al, Nucleic Acids Res., 25:33 89-3402 (1997)), ClustalW2 (European Bioinformatics European Bioinformatics Institute Available on the website of the Met. hods in Enzymology, 266:383-402 (1996); Larkin MA et al, Bioinformatics (Oxfor d, England), 23(21): 2947-8 (2007). ) and publicly available software such as ALIGN or Megalign (DNASTAR) This can be accomplished using publicly available tools. Those skilled in the art will appreciate that the defaults provided by the tools are Default parameters may be used, or a suitable algorithm may be selected, for example. etc., may be customized as appropriate for the alignment.

[0094] As used herein, "homolog sequence" and "homologous sequence" are used interchangeably and are any In the selection, the sequence is aligned to another sequence by at least 80% (e.g., at least 8 5%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%) sequence identity (or its complementary strand) refers to an amino acid sequence.

[0095] "T cells," as used herein, include helper T cells (e.g., CD4 + T cell, T helper T helper type 1 T cells, T helper type 2 T cells, T helper type 3 T cells, T helper type 17 T cells cells), cytotoxic T cells (e.g. CD8 + T cells), memory T cells (e.g., central Memory T cells (TCM cells), effector memory T cells (TEM cells and TEMRA cells) and resident memory T cells (TR cells), which are either CD8+ or CD4+. M) in cell-mediated immunity, including natural killer T (NKT) cells and inhibitory T cells. This refers to lymphocytes that play a vital role in

[0096] "Effector function" or "antibody effector function" as used herein refers to an antibody that is capable of acting as an effector for a C1 complex and Fc receptors, resulting from the binding of the Fc region of an antibody to its effector. Exemplary effector functions include the effector function of antibodies and C1q on the C1 complex. Complement-dependent cytotoxicity (CDC) induced by the interaction of the Fc region of an antibody with Antibody-dependent cell-mediated cytotoxicity (ADC) induced by binding to Fc receptors on human donor cells ( These include ADCC (adaptive cytotoxicity complex) and phagocytosis. is characterized in that the antibody effector function is at least 50% (e.g., 60%, 70%, This refers to a reduction of 80%, 90%, 95%, 96%, 97%, 98%, or 99%. In certain embodiments, the effector function is a cleavage site in the Fc region to eliminate glycosylation. Mutations that cause leukemia, such as N297A or D265A (Shields et al., J. See Biol. Chem. 276 (9): 6591-6604 (2001). (See references), K322A, L234A / L235A, and K322A are eliminated. As used herein, refers to the C-terminal region of an immunoglobulin heavy chain.

[0097] "Cancer" or "cancerous condition," as used herein, refers to malignant cell growth or neoplasm, abnormal refers to any medical condition characterized by rapid proliferation, invasion, or metastasis, including solid tumor cancers and Solid tumors include both solid tumors (hematological malignancies) such as sarcomas and carcinomas. Sarcomas are non-epithelial tumors in blood vessels, bone, fatty tissue, ligaments, lymphatic vessels, muscles, or tendons. A "tumor" is a tumor of the skin, a carcinoma is an epithelial tumor in the lining of the skin, glands, and organs. As used herein, refers to a solid mass of neoplastic and / or malignant cells.

[0098] As used herein, "treating," "treatment," or "therapy" of a condition are used interchangeably. It may be used to prevent or reduce a condition, reduce the rate of onset or occurrence of a condition, to accelerate the onset of the condition, reduce the risk of the condition occurring, or prevent the onset of symptoms associated with the condition to slow or delay the onset of a condition, to reduce or terminate symptoms associated with the condition, producing complete or partial regression of the condition, curing the condition or any of its In the context of cancer, "treating" includes therapeutic treatment, preventative or prophylactic measures, such as combinations. "and," "treatment," or "therapy" refers to the prevention or treatment of neoplastic or malignant cell growth (the increase or decrease in tumor reducing the volume of the tumor), inhibiting proliferation, invasion or metastasis to other organs, or slowing or preventing the occurrence of neoplastic or malignant cell growth, proliferation or metastasis; It may refer to the progression, slowing or halting of a tumor, or some combination thereof. With respect to tumors, "treating" or "treatment" refers to eradicating all or part of the tumor. inhibiting or slowing tumor growth and metastasis; preventing tumor initiation "Isolated" includes the initiation or delay of a given stage of a disease or some combination thereof. A material has been altered by the hand of man from its natural state. An "isolated" composition or material When a substance occurs in nature, it has been modified or removed from its original environment. For example, an "isolated" polynucleotide or polynucleotides A polypeptide is a natural product that does not contain other polynucleotides or polypeptides, respectively. Not associated with naturally occurring components that accompany the polynucleotide or polypeptide in the natural state A polynucleotide or a polypeptide. In certain embodiments, an "isolated" antibody The method is carried out by at least one step electrophoretic method (Coomassie blue or silver staining) (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis, etc.) Chromatographic methods (such as ion exchange chromatography or reverse phase HPLC) or Lowry At least 90%, 91%, 92%, 93%, 94%, 95% or more as determined by law %, 96%, 97%, 98% and 99% purity.

[0099] The term "vector" as used herein refers to a vector that is capable of expressing a protein in a host cell. A polynucleotide encoding a protein is operably inserted and delivered to the A vector is a vehicle designed to cause the expression of the genetic elements it carries in a host cell. In addition, they can be used to transform, transduce or transfect host cells. Exemplary types of vectors include, but are not limited to, plasmids (e.g., phage cosmid, yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived human vectors (such as λ phage or M13 phage) vectors, bacterial vectors or non-episomal mammalian vectors. A category of animal viruses used as vectors is the retrovirus. Viruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, Vectors include romanviruses and papovaviruses (e.g., SV40). , promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements and reporters In addition, vectors (e.g., Vectors (e.g., bacterial or episomal mammalian vectors) may contain an origin of replication. The vector may also be, but is not limited to, a viral particle, a liposome or a protein coater. The antibody may contain materials that aid in its entry into the cell, including stents.

[0100] A "host cell," as used herein, is a cell that expresses one or more exogenous proteins. Thus, the term refers to a cell into which an exogenous polynucleotide and / or vector has been introduced. It is intended to refer to both the particular subject cell and its progeny. Host cells include any of a variety of organisms, including prokaryotic, eukaryotic, and The regulatory agent may be introduced into a cell, a plant cell, an animal cell, or a hybridoma. Unless otherwise specified, or the regulatory sequence is introduced into the host cell so as to be operably linked to the nucleic acid. It may be a cell that contains the nucleic acid but does not express the protein at the desired level unless directed to do so.

[0101] "PD-L1-associated condition," as used herein, refers to a condition that is associated with PD-L1 (e.g., human PD-L 1) caused by or exacerbated by an increase or decrease in the expression or activity of "Condition" refers to any condition occurring or otherwise associated with

[0102] The term "therapeutically effective amount" or "effective dosage" as used herein refers to a therapeutically effective amount of human PD-L1 and It refers to the dosage or concentration of a drug effective to treat the relevant disease or condition. For example, Regarding the use of the antibodies or antigen-binding fragments disclosed herein to treat cancer A therapeutically effective amount is a therapeutically effective amount that is capable of reducing tumor volume, eradicating all or part of a tumor, The present invention relates to a method for treating cancer, comprising administering to a patient a therapeutically effective amount of ... cellular component that mediates a cancerous condition and that can inhibit or slow tumor growth or cancer cell invasion into other organs. Inhibit or slow tumor cell metastasis; inhibit tumor or or any symptom or marker associated with the cancerous condition. Antibody or antigen binding that can prevent or delay the onset of a disease, or some combination thereof. The dosage or concentration of the combined fragment.

[0103] The term "pharmaceutical acceptable" refers to a carrier, vehicle, diluent, excipient, and / or salt that is Generally chemically and / or physically compatible with other ingredients, including those listed above, and compatible with the recipient Demonstrate physiological compatibility.

[0104] The term "receptor occupancy" refers to the receptors occupied by ligands and available ligands at equilibrium. It refers to the proportion of the total number of receptors that are capable of binding to a given receptor and is usually expressed as a percentage of the total number of receptors. Duration of receptor occupancy was assessed by measuring and comparing receptor occupancy at various time points. FACS, positron emission tomography (PET), autoradiography Measurements of receptor occupancy, such as raffinates, are known in the art.

[0105] Anti-PD-L1 antibody The present disclosure provides anti-PD-L1 antibodies and antigen-binding fragments thereof. In certain embodiments, The present disclosure relates to exemplary monoclonal antibodies 21F11, 18G4, 4B6, 26F5, 23 A11, 23F11, 22C9, their chimeric antibodies, humanized 4B6, humanized 23A11 and and humanized 23F11.

[0106] In certain embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof bind to an antigen (e.g., human PD-L1) is pH-dependent. H7.4) The antigen binding exhibited by pH-dependent antibodies is observed at acidic pHs of 5.5 or 6. 0 (e.g., the pH of the endosome). Such pH-dependent antibodies are The pH-dependent antibodies preferentially dissociate from the antigen in endosomes. In the lysosome, by dissociating from the antigen and recycling from the cell This is because the antigen can escape antigen-mediated degradation if the antigen undergoes antigen-mediated clearance. The advantage of pH-dependent antibodies is that they can be used for therapeutic pH-dependent antibodies. Lower doses (relative to the reference PD-L1 antibody to achieve comparable in vivo therapeutic efficacy) 80%, 70%, 60%, 50%, 40%, 30%, or even less of the dose that would normally be needed. 20% or less than 10%) or for improved reusability and enhanced serum half-life include less frequent dosing compared to the reference PD-L1 antibody, both of which have comparable i The reference antibody is pH-independent and therefore achieves a therapeutic effect in vivo. L1, and binds similarly at both acidic and neutral pH, and exhibits better binding at neutral pH compared to pH-dependent antibodies. In certain embodiments, the pH dependent antibody has similar binding to PD-L1. It has the above advantages when compared to its non-pH dependent counterpart having the same bond.

[0107] In certain embodiments, the pH dependent anti-PD-L1 antibodies and antigen-binding fragments thereof are Specific binding to D-L1 was observed at acidic pH (e.g., pH 6.0, 5.5, 5.0, 4. At neutral pH (e.g., pH 7.4) it is more effective than at neutral pH (e.g., pH 7.5 or 4.0). In the acidic pH state, the binding of the antibody to PD-L1 was significantly higher than that of the control in the same assay setup. Its binding to PD-L1 is substantially lower than that of H7.4. In certain embodiments, the acidic p The binding of the PD-L1 antibodies and antigen-binding fragments thereof provided herein with H is Up to 2% at its neutral pH (i.e., 7.4) as measured by LISA; 5%, 10%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 6 5%, 70%, 75%, 80% or 85%. In certain embodiments, the acidic pH / medium K of pH-dependent anti-PD-L1 antibodies against human PD-L1 at neutral pH D Ratio and / or The Koff ratio is 2, 3, 4, 5, 8, 10, 15, 20, 30, 40 or 100 or In certain embodiments, the pH dependent anti-PD-L1 antibody binds to human PD-L1. The dissociation half-lives (t1 / 2) at 25°C or 37°C and acidic pH are 5, 4, 3, 2, and In certain embodiments, the method described herein is The described pH-dependent anti-PD-L1 antibodies and antigen-binding fragments thereof bind to human PD-L1. and at least about 2 at acidic pH compared to that of a reference PD-L1 antibody at neutral pH. 3, 5, 8, 10, 15, 20, 30, 50, or 100 times the dissociation half-life (t 1 / 2 ) In certain embodiments, the reduction in the in vivo amount of PD-1 protein is , pH-dependent binding, compared to a reference PD-L1 antibody that has similar binding to PD-L1 at neutral pH In certain embodiments, the expression of human PD-L1 is prolonged when the patient is exposed to a human PD-L1-dependent anti-PD-L1 antibody. pH-dependent anti-PD-L1 antibodies and antigen-binding fragments thereof that bind to PD-L1 at a specific dose Upon administration, increased target organ half-life compared to that of a reference PD-L1 antibody at the same dose In certain embodiments, the antibodies and antigen-binding fragments thereof have an affinity for acidic pH and neutral pH. H showed similar binding to PD-L1, In certain embodiments, the target organ is Organs include, but are not limited to, blood or serum, kidneys, lungs, pancreas, liver, gallbladder, and urinary These include the bladder, skin, esophagus, ovaries, breasts, colon, rectum, stomach, spleen or brain.

[0108] As will be appreciated by those of skill in the art, the CDR sequences provided herein are The resulting antibody may have one or more properties, such as improved binding or binding affinity, increased pharmacokinetics, or increased pharmacokinetics. Reduced CDR residues due to their half-life, pH sensitivity, and suitability for conjugation (e.g., risk of glycosylation and / or deamination at amino acid residues and reduced immunogenicity) and is otherwise identical to the parent antibody (i.e., except for the modifications or alterations described above). otherwise identical set of CDR sequences) or the antigen of the parent antibody. One or more substitutions of amino acid residues (e.g., For example, antibody variants (Fa b or scFv variants) and then use phage display technology to generate libraries of and then screened for binding or binding affinity to human PD-L1. For another example, computer software can be used to The antibody is designed to form a binding interface that substantially simulates the binding of the antibody to human PD-L1. Amino acid residues can be identified. Such residues can be used to prevent a decrease in binding or binding affinity. can be avoided in substitution or targeted for substitution to provide stronger binding. In certain embodiments, at least one (or all) of the substitutions in the CDR sequences are This is a conservative substitution.

[0109] In certain embodiments, the antibodies and antigen-binding fragments thereof are those provided herein. At least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and wherein the CDR sequence has sequence identity to human PD-L1. The binding activity or affinity for the agonist or antagonists can be measured using the methods provided herein. ) substantially identical sequences, except for the corresponding CDR sequences, which have 100% sequence identity to The antibody retains its binding affinity at levels similar to or even higher than its parent antibody.

[0110] In certain embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof are chimeric. The mer antibody may be a polypeptide that is a hybrid of one or more regions derived from an antibody as well as other antibodies or species. In certain embodiments, at least one of the chimeric anti-PD-L1 antibodies At least one CDR is derived from one species. In certain embodiments, all of the CDRs are In certain embodiments, the variable regions of the chimeric anti-PD-L1 antibodies are derived from one Chimeric antibodies are antibodies derived from one species and linked to the constant regions of an antibody from another species. The antibody retains its specificity or binding affinity.

[0111] In certain embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof are humanized. In certain embodiments, a humanized antibody originates from a non-human species and has some amino acids in the framework. The amino acid residues and constant regions of the heavy and light chains reduce or avoid immunogenicity in humans. In certain embodiments, the variable regions of the non-human species are mutated to conform to the definition of a human antibody. In certain embodiments, humanized antibodies are CDR-grafted, i.e. That is, by replacing the CDRs of a human antibody with the corresponding CDRs of a non-human antibody. Thus, the immunogenicity of humanized antibodies in humans is low. In certain embodiments, The human framework regions, for example, improve or retain binding activity or binding affinity. To this end, the CDR sequences may be derived from a non-human antibody (e.g., a murine framework region). In certain embodiments, a humanized antibody is a humanized antibody that is a humanized antibody having one or more amino acid residues substituted relative to the parent antibody. The binding activity or affinity of the antibody or antibody complex is maintained or increased.

[0112] In some embodiments, chimeric or humanized anti-PD-L1 antibodies and antigen-binding fragments thereof. The pieces are sequence numbers 21, 47, 55, 57, 59, 61, 63, 65, 67, 69, 71, A heavy chain variable region selected from the group consisting of 73, 75, 77, 79, 81, 83 and 85 and at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92% %, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) of sequence identity and / or SEQ ID NOs: 22, 48, 56, 58, 60, 62, 64, 6 From the group consisting of 6, 68, 70, 72, 74, 76, 78, 80, 82, 84 and 86 The light chain variable region selected and at least 80% (e.g., at least 85%, 88% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% These humanized antibodies contain homologous sequences that have sequence identity to human PD-L1. The binding activity or binding affinity to the exemplary antibodies: 4B6, 18G4, 26 F5, 21F11, 23A11, 23F11, 22C9 and their chimeric and humanized antibodies 4B6, humanized 23F11, and humanized 23A11 at a similar level. do.

[0113] In certain embodiments, 4B6, 26F5, 21F11, 23A11, 23F11, 22 C9 PD-L1 antibody and its chimeric antibodies, humanized 4B6, humanized 23F11, and Humanized 23A11 binds to human and non-human primate PD-L1 but not to mouse PD-L In certain embodiments, the PD-L1 antibody (e.g., 18G4), its kinase inhibitor, The Mela antibody and its antigen-binding fragments can bind to both human and mouse PD-L1. Binding activity or binding affinity can be measured using ELISA assays, radioligand competitive binding assays, etc. The antibody titer is determined based on competitive assays and FACS analysis.

[0114] In some embodiments, the anti-PD-L1 antibodies provided herein and their anti-PD-L1 The original binding fragment binds human PD-L1 with approximately 1% affinity as measured by plasmon resonance binding assay. 0 -7 M or less (e.g., 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 - 12 The binding affinity is expressed as K D Depending on the value This can be expressed as the association rate when the binding between the antigen and the antigen-binding molecule reaches equilibrium. The ratio of dissociation rates to (k off / k on ) is calculated as the antigen binding affinity (e.g. , K D ) includes plasmon resonance binding assays using instruments such as Biacore, This can be conveniently determined using suitable methods known in the art (see, e.g., Murphy, M. et al, Current protocols in protein s science, Chapter 19, unit 19.14, 2006).

[0115] In certain embodiments, the PD-L1 antibodies and antigen-binding fragments thereof are 0.001μg / ml to 1μg / ml (e.g., 0.001 μg / ml~0.5μg / ml, 0.001μg / ml~0.2μg / ml, 0.001 μg / ml~0.1μg / ml, 0.01μg / ml~0.2μg / ml, 0.01μg / ml~0.1μg / ml, 0.01μg / ml~0.05μg / ml, 0.01μg / EC5 of 0.01μg / ml to 0.03μg / ml or 0.001μg / ml to 0.01μg / ml at 0 (i.e., 50% binding concentration) or 0.01 μg / m as measured by FACS l~1μg / ml (e.g. 0.01μg / ml~0.5μg / ml, 0.01μg / m l~0.2μg / ml, 0.05μg / ml~1μg / ml, 0.05μg / ml~0. It can bind with an EC50 of 5μg / ml or 0.05μg / ml to 0.2μg / ml. Binding of the antibody to human PD-L1 can be determined by methods known in the art, for example, ELISA, FACS, surface plasmon resonance, GST pull-down, epitope tag, immunoprecipitation, phas -Western, Fluorescence Resonance Energy Transfer, Time-Resolved Fluorescence Immunoassay (TR-FIA) , radioimmunoassay (RIA), enzyme immunoassay, latex agglutination, This can be measured by stain blot and immunohistochemistry or other binding assays. In an illustrative example, the test antibody (i.e., the first antibody) is an antibody that binds immobilized human PD-L1. L1 or human PD-L1 expressing cells and then washed to remove unbound antibody. After removal, a labeled antibody is allowed to bind and allow detection of the bound first antibody. A secondary antibody is introduced. Detection is by microscopy if immobilized PD-L1 is used. When cells expressing human PD-L1 are used, FA This can be done by using CS analysis.

[0116] In certain embodiments, the antibodies and fragments thereof provided herein are directed to human PD- Binding of 1 to human PD-L1 was measured using FACS at 0.05 μg / ml to 1 μg / ml. g / ml (e.g. 0.05μg / ml~0.8μg / ml, 0.05μg / ml~0. IC of 5μg / ml or 0.05μg / ml to 0.3μg / ml 50 In or EL 0.001μg / ml to 0.5μg / ml (e.g., 0.00 1μg / ml~0.2μg / ml, 0.001μg / ml~0.1μg / ml, 0.00 1μg / ml~0.05μg / ml, 0.001μg / ml~0.02μg / ml, 0. 005μg / ml~0.05μg / ml, 0.005μg / ml~0.02μg / ml or IC of 0.005μg / ml to 0.01μg / ml 50 and inhibits.

[0117] In certain embodiments, the antibodies and fragments thereof provided herein are directed to human PD- L1 to its receptor (i.e., human PD-1), thereby e.g. , activated T cells (CD4 + T cells and CD8 + T cells) Inducing the production of activated T cells (CD4 + T cells and CD8 + T cells, etc.) These antibodies provide biological activities including inducing proliferation of T cells and reversing the suppressive function of T regs. Exemplary cytokines include IL-2 and IFNγ. -2" refers to interleukin 2, white blood cells (e.g., leukocytes It refers to a type of cytokine signaling molecule in the immune system that regulates the activity of immune cells (kocytes). The term "interferon gamma (IFNγ)" refers to the natural killer (NK) cells, CD4 + and CD8 + A cytokine produced by T cells, macrophages It is a critical activator of phages and an indicator of major histocompatibility complex (MHC) molecule expression. Cytokine production can be measured using methods known in the art, for example, by measuring the activity of E. This can be determined by LISA. 3 [H]thymidine incorporation assay and luminescent cell viability assay Methods including assays can also be used to detect T cell proliferation.

[0118] The anti-PD-L antibodies and antigen-binding fragments thereof are directed against human PD-L1 and / or non-human primate PD-L1. In certain embodiments, the antibodies and antigen-binding fragments thereof are specific for PD- L2 (e.g., human PD-L2). For example, the binding affinity to PD-L2 is 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, and 3% with human PD-L1 , less than 2% or 1%.

[0119] In certain embodiments, the antibodies and antigen-binding fragments thereof bind to non-human primate PD-L1 and E 0.001 μg / ml to 0.5 μg / ml (e.g., 0.0 01μg / ml~0.2μg / ml, 0.001μg / ml~0.1μg / ml, 0.0 01μg / ml~0.05μg / ml, 0.001μg / ml~0.02μg / ml, 0 .005μg / ml~0.05μg / ml, 0.005μg / ml~0.02μg / ml or 0.005μg / ml to 0.01μg / ml).

[0120] The anti-PD-L1 antibodies and antigen-binding fragments thereof can be used to bind human PD-L1 and / or non-human PD-L1. In certain embodiments, the antibodies and antigen-binding fragments thereof are specific for protozoan PD-L1. The fragment does not bind to mouse PD-L1. For example, the binding affinity to mouse PD-L1 is 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, and 3% with human PD-L1 , less than 2% or 1%.

[0121] In certain embodiments, the antibodies and antigen-binding fragments thereof do not bind to mouse PD-L1. However, non-human primate PD-L1 has similar binding activity or affinity to that of human PD-L1. For example, exemplary antibodies 4B6, 26F5, 21F11, 23A11, 23F 11, 22C9 and its chimeric antibodies, humanized 4B6, humanized 23F11 and humanized 2 Binding of 3A11 to mouse PD-L1 was measured using conventional methods such as ELISA or FACS analysis. Although very low in binding assays, the binding of these antibodies to non-human primate PD-L1 was Similar to that of human PD-L1 as measured by ELISA or FACS In certain embodiments, the antibodies and antigen-binding fragments thereof (such as 18G4) are directed against mouse PD -L1 and non-human primate PD-L1 have similar binding activity to that of human PD-L1 Or bind with binding affinity.

[0122] In certain embodiments, the anti-PD-L1 antibodies and their antigen-binding domains provided herein are The combined fragments were generated using genes encoding tumor cells, purified tumor antigens, and immune-stimulating cytokines. Can be used in combination with immunogenic agents such as transfected cells and tumor vaccines In addition, anti-PD-L1 antibodies and their antigen-binding fragments have been shown to be effective in treating patients with advanced PD-L1 disease after standard chemotherapy and radiation therapy (e.g., radiation therapy, X-ray therapy), target-based small molecule therapy (e.g., tyrosine kinase inhibitors), anti-cancer drugs imatinib, gefitinib; monoclonal antibodies, photodynamic therapy), immunotherapy (e.g. For example, carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, Tag-72, HMFG, sialyl Lewis antigen, MucA, Muc B, PLAP, estrogen receptor, laminin receptor, erb B and p155, DL L4, Notch1, Notch2 / 3, Fzd7 or Wnt, r-spondin (RS Antibodies against tumor markers such as RSPO1, RSPO2, RSPO3 or RSPO4 , other emerging immune checkpoint modulator therapies (e.g., vaccines), hormonal therapy, and Month therapy, angiogenesis inhibition (angiogenesis inhibitors), gene therapy (inducers of cell proliferation, inhibitors of cell proliferation or regulators of programmed cell death), palliative care (i.e. pain (e.g. morphine and oxycodone), nausea, vomiting (e.g., ondansetron and amphetamines), Prepitant), directed at improving quality of care to reduce diarrhea and bleeding In certain embodiments, the antibody can be included in a combination therapy, including chemotherapy, chemotherapy alone or in combination with chemotherapy alone. and antigen-binding fragments thereof, as vectors for antibody-drug conjugates, bispecific or multivalent antibodies. It can be used as a source.

[0123] The anti-PD-L1 antibodies and antigen-binding fragments thereof provided herein are Clonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, bispecific antibodies The recombinant antibody may be an antibody, a labeled antibody, a bivalent antibody or an anti-idiotypic antibody. These are antibodies prepared in vitro using recombinant techniques rather than in vitro. Bispecific or bivalent antibodies are artificial antibodies that contain fragments of two different monoclonal antibodies. and can bind to two different antigens. An antibody or antigen-binding fragment thereof that is "bivalent" is An antibody or antigen-binding fragment is characterized as "bispecific." In the case of a single antigen-binding site, the two antigen-binding sites may bind to the same antigen, or they may each They can bind to different antigens.

[0124] In some embodiments, the anti-PD-L1 antibodies provided herein and their anti-PD-L1 The original binding fragment is a humanized or chimeric antibody. In certain embodiments, the humanized or chimeric Antibodies are prepared using recombinant methods. For example, non-human animals can express human PD-L1 proteins. The subject can be immunized with an appropriate antigen, such as a protein. The gene fragment that encodes the antibody is excised from the mouse monoclonal antibody gene, and this part is , operably linked to a gene for the constant region of an antibody derived from human IgG1. For production of the recombinant gene fragment, the recombinant gene fragment is incorporated into an expression vector and then transformed into a host cell. (U.S. Patent Nos. 4,816,397, 4,816,567, and 5,816,570) (See issue 07,715).

[0125] A "humanized antibody" refers to an antibody in which the variable region framework and constant regions, if present, are The human antibody CDR genes of non-human origin may be cloned into human antibody CDRs derived completely or substantially from human antibody sequences. The antibody is obtained by grafting the human antibody gene. Methods for this purpose are known in the art (e.g., U.S. Pat. No. 5,225,539 ... ,530,101, 6,407,213, 5,859,205, 6,881 ,557, EP239400, EP125023, WO90 / 07861 and WO9 In certain embodiments, the humanized antibody comprises a humanized heavy chain and In certain embodiments, the grafted CD in the humanized PD-L1 antibody comprises a humanized light chain. The sequence of R is at least 60%, 70%, 80%, 82%, 85%, or 100% identical to the corresponding CDR. %, 88%, 90%, 95% or 100% identical. In certain embodiments, the humanized P No more than three conservative amino acid substitutions are made in the CDRs of the D-L1 antibody. For sequence optimization, we first used the amino acid sequence of the variable region framework of the humanized PD-L1 antibody. In certain embodiments, the variable region framework of the humanized PD-L1 antibody chain is The framework sequences are at least 65%, 70%, and 80% identical to the corresponding human variable region framework sequences. %, 75%, 80%, 85%, 90%, 95% or 100% identical.

[0126] In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof are Domain antibodies, diabodies, scFv, scFv dimers, BsFv, dsFv, (ds Fv) 2 , dsFv-dsFv', Fv fragment, Fab, Fab', F(ab') 2 , ds Diabodies, nanobodies, domain antibodies, isolated CDRs or bivalent domain antibodies be.

[0127] In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof are In some embodiments, the immunoglobulin constant region further comprises a heavy chain and The heavy chain constant region comprises a CH1, CH1-CH2 or CH 1-CH3 region. In some embodiments, the constant region confers desirable properties. For example, the constant region may further comprise one or more modifications such as to improve FcRn receptor binding so as to reduce or deplete the effector function of or to introduce one or more cysteine ​​residues.

[0128] In certain embodiments, the anti-PD-L antibodies and antigen-binding fragments thereof are thermostable. The terms "thermal stability" or "thermal resistance" are used herein to refer to thermodynamic properties. This refers to the functional stability of the anti-PD-L1 antibody, including but not limited to, the effects of heating, cooling, freezing, Freeze-thaw cycles, vibration, vortexing, sonication, chemical denaturants, pH, surfactants Thermal and / or physical / chemical manipulations, including agents, salts, additives, proteases or temperature This refers to the resistance of an antibody to irreversible denaturation caused by the action of Irreversible unfolding of the functional conformation of the antibody, resulting in loss of biological activity and Denatured proteins lead to aggregation. Increased thermal stability is a measure of ligand binding. or at increasing temperatures, It is determined by using spectroscopic methods such as light, circular dichroism (CD) or light scattering. The PD-L1 antibodies provided herein can be used to treat at least 2°C. At least 5°C, at least 8°C, at least 10°C, at least 15°C, at least 2 Thermal stability in functional conformational states using 0°C or at least 30°C The stability of the antibodies provided herein can be increased as measured by increasing the thermal stability. Stability can be assessed, for example, by differential scanning fluorimetry (DSF) or differential scanning calorimetry (DSC). (He F et al., J Pharm Sci. 2011 Apr 100(4):1330-40), where the thermal transition midpoint (Tm) is measured. and indicates the relative stability of the protein in liquid. The Tm of is greater than 74°C, greater than 76°C, greater than 78°C, greater than 80°C, greater than 82°C, greater than 84°C, greater than 86°C, Above 88°C, above 90°C or above 92°C, above 94°C, above 96°C or above 98°C. In embodiments, the PD-L1 antibody with improved thermostability is 23F11 (e.g., 23F1 1-H4L4, 23A11-H6L4, 23A11-H4L6 or 23A11-H6L 6) and has a thermal transition midpoint (Tm) of greater than 90°C.

[0129] In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof are A variety of payloads can be used to form the ADCs provided herein. It is contemplated that the antibody or antigen-binding fragment may be linked to the target antibody or antigen-binding fragment (e.g., njugate Vaccines”, Contributions to Micr obiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, (See New York, (1989)). "Payload (singular or plural)" The term is used interchangeably with "drug(s)" and these payloads include The antibodies or antigen-binding fragments may be bound by, among other methods, covalent binding, affinity binding, intercalation, coordination, etc. The linkage may be by covalent bonding, complexation, association, blending or addition. In one embodiment, the antibodies and antigen-binding fragments disclosed herein comprise an epitope-binding portion on the outside of the one or more payloads, e.g., peptides, nucleic acid molecules, drugs, cytotoxins, Polypeptides, proteins, fusion proteins, antibodies, haptens, small molecules, mimetics, synthetic Can be used to bind drugs, inorganic molecules, organic molecules, radioisotopes and reporter groups For example, such sites may be engineered to contain specific sites. To facilitate covalent linkage to the base, one or more reactive amino acid residues, e.g., In certain embodiments, the antibody may include a linker. A payload may be indirectly linked to the payload via a payload-link or by another payload. For example, an antibody or antigen-binding fragment may be conjugated to biotin and then coupled to avidin. The payload may be indirectly conjugated to a second payload that is also conjugated to the payload. The moiety may be a reporter group or a detectable label, a pharmacokinetic-modifying moiety, a purification moiety or a cytotoxic moiety. Examples of detectable labels include fluorescent labels (e.g., fluorescein, -damine, dansyl, phycoerythrin or Texas Red), enzyme substrate label (e.g. , horseradish peroxidase, alkaline phosphatase, Luciferases, glucoamylase, lysozyme, saccharide oxide β-D-galactosidase or β-D-galactosidase), radioisotopes (e.g., 123I, 12 4I, 125I, 131I, 35S, 3H, 111In, 112In, 14C, 64Cu , 67Cu, 86Y, 88Y, 90Y, 177Lu, 211At, 186Re, 188R e, 153Sm, 212Bi and 32P, other lanthanides, luminescent labels), chromophore part Molecular weights include digoxigenin, biotin / avidin, DNA molecules or gold for detection. In certain embodiments, the payload comprises a drug, such as PEG, which serves to increase the half-life of the antibody. Other suitable polymers include, for example, carboxymethyl Cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol In certain embodiments, the copolymers include glycerol / propylene glycol copolymers. The payload can be a purification moiety such as a magnetic bead. is any substance that is harmful to cells or that can damage or kill cells. Examples of cytotoxic moieties include, but are not limited to, chemotherapeutic agents, Antitumor agents, growth inhibitors, drugs, toxins, e.g., Taxol, Cytochalasin B, Gramicidal Zin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine Lystine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydrochloride Dihydroxy anthracin dione, mitoxantrone, mithramycin Synthin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine tetracaine, lidocaine, propranolol, puromycin and similar body, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethene, amine, thioepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, and busulfan. mannitol, dibromomannitol, streptozotocin, mitomycin C and cis-dichloro diamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., Dow norubicin (formerly daunomycin and doxorubicin), antibiotics (e.g., daunomycin Cutinomycin (formerly actinomycin), bleomycin, mithramycin, platinum (e.g., cisplatin and oxaliplatin), plant alkaloids (e.g., topoisomers, somerase inhibitors, vinca alkaloids, taxanes and epipodophyllotoxins) and and anthramycin (AMC)) and mitotic inhibitors (e.g., vincristine and The terms "loading" or "drug loading" or "payload-loaded" are used interchangeably. "Drug loading" as used herein refers to the average number of drugs / payload per antibody. The present invention relates to a method for the determination of chromatographic characteristics of a sample by using techniques in the art such as mass spectrometry, UV / visible spectroscopy, ELISA assays and HPLC. 1-20 (e.g., 1-15) per antibody, as determined by a suitable method in , 1-10, 2-10, 1-8, 2-8, 2-6, 2-5 or 2-4) drug (drug countermeasure In certain embodiments, the drug loading can be in the range of 1, 2, 3, 4 , 5, 6, 7, 8, 9 or 10.

[0130] Polynucleotides and Recombinant Methods The present disclosure provides isolated polynucleotides encoding anti-PD-L1 antibodies and antigen-binding fragments thereof. In certain embodiments, the isolated polynucleotide is The present invention includes one or more nucleotide sequences encoding the CDR sequences provided above.

[0131] The method for producing a monoclonal anti-PD-L1 antibody or antigen-binding fragment thereof comprises administering to a subject a subject, comprising administering to the subject a therapeutically effective amount of the monoclonal anti-PD-L1 antibody or antigen-binding fragment thereof, Immunizing a suitable animal with hPD-L1 protein or with hPD-L1 producing cells. The animal may be a mouse, rat, sheep, goat, rabbit or guinea pig. Using the spleen or lymph nodes, or by collecting B cells from immunized animals, PD-L1 Antibody titers are measured to generate hybridomas. Hybridomas obtained from immunized animals PD-L1 antibodies or their antigen-binding domains with suitable titers from tumor or B cell clones. The polynucleotide encoding the fragment is cloned. The modified (e.g., chimeric, humanized) polynucleotide is inserted into a suitable vector. This is then introduced into a host cell to produce the antibody of the present disclosure. The antibodies and antigen-binding fragments thereof provided in claim 1 are cultured in host cells and subsequently expressed in the host cells. By isolating and purifying the cells or culture fluid (e.g., supernatant), a substantially pure and can be obtained in homogeneous form. For this purpose, conventional methods used for purifying polypeptides can be used.

[0132] In some embodiments, the isolated polynucleotide encodes a heavy chain variable region, Selected from the group consisting of SEQ ID NOs: 93, 95, 97, 99, 101, 103 and 105 Sequences that are at least 80% (e.g., at least 85%, 88%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) sequence identity. In some embodiments, the isolated polynucleotide comprises a homologous sequence thereof having The light chain variable region is encoded by SEQ ID NOs: 94, 96, 98, 100, 102, 104 and 1 06, and at least 80% (e.g., at least 85 %, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, 99%) or more of sequence identity thereto. In certain embodiments, -Percentage of the variation is due to genetic code degeneracy, and the encoded protein sequence is subject to change. It remains the same.

[0133] Isolated polynucleotides encoding anti-PD-L1 antibodies and antigen-binding fragments thereof are provided. , for further cloning (amplification of the DNA) or for expression, as known in the art. The antibody can be inserted into a vector using known recombinant techniques. Monoclonal antibodies can be produced by homologous recombination, as known in the art. A is isolated using conventional procedures (e.g., by isolating the genes encoding the heavy and light chains of an antibody). By using an oligonucleotide probe capable of binding to Many vectors are available. The vector components are Typically, the sequence includes, but is not limited to, one or more of the following: a signal sequence (e.g., a translation signal or leader sequence), an origin of replication, one or more selectable marker genes, Hanser elements, promoters (e.g., SV40, CMV, EF-1α) and transcription termination array.

[0134] In some embodiments, vector systems include mammalian, bacterial, yeast systems, and the like. , including but not limited to pALTER, pBAD, pcDNA, pCal, pL, p ET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2 , pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, p UNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, p Pro18, pTD, pRS420, pLexA, pACT2.2, pCDM8, pCD NA1.1 / amp, pcDNA3.1, pRc / RSV, pEF-1, pCMV-SC RIPT.RTM., pFB, pSG5, pXT1, pCDEF3, pSVSPORT, Plasmids such as pEF-Bos and other laboratory and commercially available expression vectors Suitable vectors include plasmid or viral vectors (e.g., replication defective). Examples of viruses that can be used include viral vectors such as retroviruses, adenoviruses and adeno-associated viruses. Vectors may be maintained in a single or multiple copies or may be integrated into the host cell genome. A vector containing a polynucleotide sequence encoding an antibody or antigen-binding fragment. The vectors can be introduced into a host cell for replication or gene expression. Suitable host cells for cloning and expressing DNA in the prokaryotic cells described above. Suitable prokaryotes for this purpose are bacteria, yeast, insect cells or higher eukaryotic cells. and eubacteria, such as gram-negative or gram-positive organisms, e.g., Escherichia erichia, e.g., E. coli, Enterobacter, Eleuthero, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium um), Serratia, e.g. Serratia marcescans ans and Shigella, as well as B. subtilis and B. licheniformis Bacilli such as licheniformis, Pseudomonas aeruginosa, etc. Pseudomonas and Streptomyces, Bacillus, Streptococcus, Streptomyces [Streptomyces], Staphylococcus, Enterococcus Enterococcus, Lactobacillus, Lactococcus ], Clostridium, Geobacillus, Ocean Bacillus (Oceanobacillus), Pseudomonas (Pseudomonas), Salmonella (Sal monella, Campylobacter, Helicobacter , Flavobacterium, Fusobacterium, Ilyobacter, Neisseria and Ureaplasma Suitable insect cells include Drosophila melanogaster and Enterobacteriaceae. Suitable yeasts include Eschneider S2 cells and Sf9. methanolica, P. pastoris, S. cerevisiae or Examples of preferred mammalian cells include CHO cells, HEK29 In certain embodiments, the glycosylation of the antibody is and Fc effector functions are not required, antibodies can be produced in bacteria. good.

[0135] In addition to the above examples, Schizosaccharomyces pombe, e.g. K. lactis lactis, K. fragilis (ATCC12,424), K. bulgaricus 〔bulgaricus〕(ATCC16,045), K. wickeramii〔wickeramii〕(ATCC 24,178), K. waltii (ATCC 56,500), K. drosophila drosophilarum (ATCC36,906), K. thermotolerans Kluyveromyces ans and Kluyveromyces marxianus Hosts: Yarrowia (EP402,226), Pichia pastoris ia pastoris (EP183,070), Candida, Trichoderma reesea [Trichoderma reesia] (EP244,234), Neurospora crassa , Schwanniomyces occidentalis, etc. Schwanniomyces and, for example, Neurospora Filamentous fungi such as Penicillium and Tolypocladium and Aspergillus, such as A. nidulans and A. niger. Several other genera, species and strains, such as Aspergillus hosts, are included herein. Generally available.

[0136] A host suitable for expression of the glycosylated antibodies or antigen fragments provided herein. Chief cells are derived from multicellular organisms. Examples of vertebrate cells include plant and insect cells. Numerous baculovirus strains and variants, as well as Spodoptera frugiperda ( ra frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) (Drosophila melanogaster (fruiffly)) and corresponding tolerant insects such as the silkworm (Bombyx mori) Host cells have been identified. Various virus strains for transfection, e.g. L-1 variant of Autographa californica NPV and the silkworm (Bombyx mori) NPV strain Bm-5 are publicly available. Such viruses are referred to herein as viruses in accordance with the invention, in particular Spodoptera optera frugiperda cells. Plant cell cultures of locomotive, potato, soybean, petunia, tomato and tobacco are also hosts. It can be used as.

[0137] However, vertebrate cells have attracted much interest, and the development of vertebrate cells in culture (tissue culture) has been Propagation of vertebrate cells has become a routine procedure. Examples of useful mammalian host cell lines include: SV40-transformed monkey kidney strain CV1 (COS-7, ATCC CRL 1 651), human embryonic kidney line (293 or subcloned for growth in suspension culture 293 cells, Graham et al., J. Gen Virol. 3 6:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 1 0), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al. l., Proc. Natl. Acad. Sci. USA 77:4216 ( 1980)), mouse Sertoli cells (TM4, Mather, Biol. Repro d. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CC L 70), African green monkey kidney cells (VERO-76, ATCC CRL-158 7), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK , ATCC CCL 34), Buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocellular carcinoma Hepatoma cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATC C CCL51), TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)), MRC 5 cells, F S4 cells, PC12, mouse embryonic fibroblast cell line (3T3), NSO myeloma cells (any There are mouse myeloma cell lines that do not endogenously produce functional immunoglobulin chains. Host cells contain a variety of pathways for post-translational processing and modification of proteins and gene products. Therefore, a suitable cell line is required to ensure the correct modification and expression of the expressed antibody. to ensure proper transcription and processing (primary transcript, glycosylation, and phosphorylation, etc.). In some preferred embodiments, the host cell is selected from HEK 293T cells. In some preferred embodiments, the host cells are CHO cells. .

[0138] The host cell can then express the above expression or cloning vector for the production of the anti-PD-L1 antibody. The vector is transformed with a promoter, a transformant is selected, or a desired sequence is inserted. Cultivation in conventional nutrient media modified as necessary to amplify the coding gene In certain embodiments, the vector is transformed, electroporated, phosphate-activated, or into the host cells by methods known in the art, such as calcium treatment, lipofection, etc. In certain embodiments, the vector can be transfected into a eukaryote. The method involves calcium phosphate coprecipitation, microinjection, electroporation, and lipolysis. The eukaryotic host cell is transfected with a vector encoding the antibody. In certain embodiments, the two polynucleotides may be co-transformed. Host cells containing the vector may transiently express the anti-PD-L1 antibody.

[0139] Hosts used to produce the antibodies or antigen-binding fragments provided herein Cells may be cultured in a variety of media. Ham's F10 (Sigma), Minimum Essential Medium ( MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle's Medium (DMEM, Sigma), Luria Broth (LB) and Terrific Commercially available media such as TB Broth are suitable for culturing the host cells. m et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), US Japanese Patent Nos. 4,767,704, 4,657,866, 4,927,762, Nos. 4,560,655 and 5,122,469, WO90 / 03430 and WO87 30,985, or any of the media described in U.S. Pat. No. Re. Any of these media may be used as a culture medium for the host cells, as desired. , hormones and / or other growth factors (insulin, transferrin or epithelial growth factors), salts (such as sodium chloride, calcium, magnesium and phosphate), Buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics Substances (such as the drug GENTAMYCIN®), trace elements (usually in the micromolar range) (defined as the inorganic compound present at the final concentration) and glucose or an equivalent energy Any other necessary supplements would be known to those skilled in the art. The culture conditions, such as temperature, pH, etc., may be selected for expression. These have been used previously with various host cells and will be apparent to those skilled in the art.

[0140] When using recombinant techniques, the antibodies and antigen-binding fragments thereof are expressed in the periplasmic space. It can be produced intracellularly by ELISA or directly secreted into the medium. If the antibody is produced intracellularly, it may be either particulate debris, host cells or lysed fragments. is removed, for example, by centrifugation or ultrafiltration. Carter et al. , Bio / Technology 10:163-167 (1992) A procedure has been described for isolating antibodies that are secreted into the periplasmic space of E. coli. Briefly, the cell paste was diluted with sodium acetate (pH 3.5), EDTA and phenyl The cells are thawed in the presence of PMSF for approximately 30 minutes. Cellular debris can be removed by centrifugation. Antibodies and antigen-binding fragments thereof are When secreted into the soil, the supernatant from such an expression system is generally prepared using commercially available tanks. Protein concentration filters, e.g., Amicon or Millipore Pellic First, concentrate the sample using an ultrafiltration unit as described above to inhibit proteolysis. A protease inhibitor such as PMSF may be included in any of the steps to prevent extraneous contamination. Antibiotics may be included to prevent the growth of infectious agents.

[0141] The antibodies prepared from the cells can be purified by, for example, hydroxylapatite chromatography, Aqueous chromatography, reversed phase chromatography, absorption chromatography, filtration, ultrafiltration, solvent precipitation, solvent extraction, distillation, SDS-polyacrylamide gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, immunoprecipitation Using immunoprecipitation, isoelectric focusing, recrystallization and affinity chromatography The antibody can be purified by affinity chromatography, with affinity chromatography being the preferred purification technique. The stability of Protein A as an affinity ligand is such that it is compatible with any immunoglobulins present in the antibody. Protein A was used to identify human Fc domains, depending on the species and isotype. Antibodies based on gamma 1, gamma 2 or gamma 4 heavy chains can be purified (Lindma rk et al., J. Immunol. Meth. 62:1-13 (19 83) Examples of Protein A columns include Hyper D, POROS, and Seph. arose FF (GE Healthcare Biosciences) Protein G is specific to all mouse isotypes and to human γ-3. It is recommended to use a 100% RT-PCR assay (Guss et al., EMBO J. 5:1567 1575 (1986). The matrix to which the affinity ligand is attached is most often agarose. However, other matrices are available. Controlled pore glass or poly Mechanically stable matrices such as (styrenedivinyl)benzene are used with agarose. This allows for faster flow rates and shorter processing times than can be achieved with conventional antibody-binding proteins. For the H3 domain, Bakerbond ABX.TM. resin ( JT Baker, Phillipsburg, NJ) are useful. Columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, anions, or cation exchange resin (polyaspartic acid column, etc.) (Target) Chromatography, isoelectric focusing, SDS-PAGE and Other techniques for protein purification, such as fractionation by ammonium sulfate precipitation, also have been used to Depending on the antibody being tested, it is available.

[0142] After any preliminary purification step(s), the antibody of interest and any contaminants are The mixture is eluted using an elution buffer with a pH between about 2.5 and 4.5, preferably a low salt Low pH hydrophobic interaction chromatography, performed at low concentrations (e.g., about 0-0.25 M salt) A fee may be added to the fee.

[0143] kit The present disclosure relates to an anti-PD-L1 antibody or antigen-binding fragment thereof or a combination thereof provided herein. The present invention provides a kit comprising a pharmaceutical composition comprising an anti-PD-L1 antibody or an antigen-binding fragment thereof. In some embodiments, the kit detects the presence or absence of PD-L1 in a biological sample. The biological sample may comprise a cell or tissue. .

[0144] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprised in the kit. The fragment is conjugated with a detectable label (e.g., a fluorescent, radioactive or enzymatic label). In certain other embodiments, the kit comprises an unlabeled anti-PD-L1 antibody or its antigen. Binding Fragments or Pharmaceutical Compositions Containing Unlabeled Anti-PD-L1 Antibodies or Antigen-Binding Fragments Thereof and further comprising a secondary labeled antibody that is capable of binding to the unlabeled anti-PD-L1 antibody. The kit includes a means for detecting the label (e.g., a filter set for detecting a fluorescent label, an enzyme The kit may further comprise a substrate for carrying out a particular method, such as an enzyme substrate for a specific label. The kit may include additional reagents and buffers used in the preparation of the present invention. The kit may further include packaging that separates each of the components in the kit. The kit includes an immunoassay for detecting PD-L1 antibodies.

[0145] In certain embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof contained in the kit in sandwich assays such as ELISA, or in immunographic assays The present invention relates to a substrate or device useful in the manufacture of a pharmaceutical composition comprising: , microtiter plates and test strips.

[0146] In certain embodiments, kits for detecting PD-L1 protein levels are provided. In some embodiments, the kit is for predicting, diagnosing, prognosing, or determining whether a PD-L1-associated condition exists. Used to prevent or treat

[0147] Pharmaceutical Compositions and Methods of Treatment The disclosure relates to an anti-PD-L1 antibody or antigen-binding fragment thereof and one or more pharma- ceutical Further provided is a pharmaceutical composition comprising an acceptable carrier.

[0148] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein For example, a medicament may be a medicament, such as ... Antimicrobials, isotonicity agents, buffers, antioxidants, anesthetics, dispensing agents gents], sequestering or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances The present invention may include any of the above-mentioned compounds, other components known in the art, or various combinations thereof. can.

[0149] Suitable ingredients include, for example, antioxidants, humectants, bulking agents, binders, disintegrants, buffers, etc. Fur, preservatives, lubricants, flavoring agents, thickeners, coloring agents, emulsifiers or sugars and cyclodex Suitable antioxidants include, for example, methionine, phosphate esters, and other stabilizers. Nin, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, Cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydrochloride Hydroxanisol, butylated hydroxytoluene and / or gallic acid As disclosed herein, the antibody or antigen binding In a composition comprising a conjugate as provided herein, methyl The inclusion of one or more antioxidants, such as onion, may aid in the oxidation of the antibody or antigen-binding fragment. This reduction in oxidation prevents or reduces loss of binding activity or binding affinity. , thereby improving antibody stability and maximizing shelf life. In the form of one or more of the antibodies or antigen-binding fragments disclosed herein, and one or more antioxidants, such as methionine, are provided. or antigen-binding fragment thereof with one or more antioxidants, such as methionine. and preventing oxidation of the antibodies or antigen-binding fragments as provided herein. Methods for extending duration and / or improving efficacy are further provided. Suitable humectants include ethylene glycol, glycerin or sorbitol. Suitable lubricants include, for example, cetyl ester wax, hydrogenated vegetable oil, malic acid stearate, Magnesium, methyl stearate, mineral oil, polyoxyethylene-polyoxypropylene copolymer Polymers, polyethylene glycol, polyvinyl alcohol, sodium lauryl sulfate Suitable emulsifiers include carboma wax, white wax, or a mixture of two or more thereof. -, Polyoxyethylene-20-stearyl ether, Cetostearyl alcohol, Cetearyl alcohol Alcohol, Cholesterol, Diglycol Stearate, Glyceryl Stearate, Hydroxypropyl cellulose, hydroxypropyl methylcellulose, lanolin, poly Oxyethylene lauryl ether, methylcellulose, polyoxyethylene stearate , polysorbate, propylene glycol monostearate, sorbitan ester or Stearic acid is one example.

[0150] To further illustrate, pharma- ceutically acceptable carriers include, for example, sodium chloride injection. , Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection or Dextrose and and aqueous vehicles such as lactated Ringer's injection, hydrogenated oils of vegetable origin, cottonseed oil, corn oil, gum A non-aqueous vehicle such as sesame oil or peanut oil, an antimicrobial agent in a bacteriostatic or fungistatic concentration, salt Isotonic substances such as sodium chloride or dextrose, phosphate or citrate buffers buffers such as sucrose, antioxidants such as sodium bisulfate, and local anesthetics such as procaine hydrochloride. , sodium carboxymethylcellulose, hydroxypropyl methylcellulose or Suspending and dispersing agents such as polyvinylpyrrolidone, polysorbate 80 (TWEEN-8 0), EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol Sequestering or chelating agents such as ethanol, ethyl alcohol, polyethylene glycol Coal, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid The antimicrobial agents utilized as carriers are phenol or cresol, mercury, benzene, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates Complex containing esters, thimerosal, benzalkonium chloride and benzethonium chloride The pharmaceutical composition may be added in a multi-dose container. Suitable excipients include, for example, water, saline, or the like. Examples of suitable carriers include saline, dextrose, glycerol, or ethanol. Nontoxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers. Strengthening agent or sodium acetate, sorbitan monolaurate, triethanolamine oleate Examples of suitable agents include cyclodextrins and the like.

[0151] The pharmaceutical composition may be in the form of a liquid solution, suspension, emulsion, lotion, foam, pill, capsule, etc. tablets, sustained release preparations, ointments, creams, pastes, gels, sprays, aerosols or Oral formulations may be in the form of a powder. Pharmaceutical grades of mannitol, lactose, starch, stearyl alcohol, etc. Magnesium phosphate, polyvinyl pyrrolidone, sodium saccharin, cellulose, carbonate Standard carriers such as magnesium may be included.

[0152] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. The pharmaceutical composition may be, for example, a liquid solution, a suspension, an emulsion or a liquid solution, a suspension or The may be prepared in any conventional form, such as a solid form suitable for making an emulsion. As an injectable preparation, a sterile and / or non-pyrogenic solution ready for injection, subcutaneously Sterile anhydrous formulations, such as lyophilized powders, including tablets, ready to be mixed with a solvent immediately prior to use Soluble preparations, sterile suspensions ready for injection, ready to be mixed with the vehicle immediately before use Examples of suitable formulations include sterile anhydrous insoluble preparations and sterile and / or non-pyrogenic emulsions. The solutions can be either aqueous or non-aqueous.

[0153] In certain embodiments, the unit dose parenteral preparation contains an ampoule, vial or needle. All preparations for parenteral administration are packaged in a syringe having a It must be sterile and non-pyrogenic, as known and practiced in the field.

[0154] In certain embodiments, the antibodies or antigen-binding fragments disclosed herein are administered in a suitable solution. A sterile, freeze-dried powder is prepared by dissolving the powder in a solvent. Excipients that improve the stability or other pharmacological components of the reconstituted solution prepared from Excipients that may be used include, but are not limited to, water, dextromethorphan, sugar, sorbitol, fructose, corn syrup, xylitol, glycerin, In one embodiment, the solvent may be: Citric acid, sodium or potassium phosphate or similar known to those skilled in the art, at approximately neutral pH. Under standard conditions known to those of skill in the art, the buffer may include other such buffers. Subsequent sterile filtration of the solution at 25° C. followed by lyophilization provides the desired formulation. In an embodiment, the resulting solution is apportioned into vials for lyophilization. Each vial contains , a single dose or multiple doses of an anti-PD-L1 antibody or antigen-binding fragment thereof, or a composition thereof A small amount (e.g., about 10% of what is needed for a dose or set of doses) may be added. ) overfilled vials facilitate accurate sample withdrawal and accurate dosing The lyophilized powder may be stored under appropriate conditions, such as at about 4° C. to room temperature.

[0155] Reconstitution of the lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, sterile and / or non-pyrogenic water or other suitable solvent is used for reconstitution. A suitable liquid carrier is added to the lyophilized powder. The exact amount is determined based on the selected formulation. The dosage will vary depending on the therapy used and can be empirically determined.

[0156] A subject is administered a PD-L1 antibody or antigen-binding fragment thereof as provided herein. A therapeutically effective amount is administered, thereby treating an associated condition or disorder associated with PD-L1. Therapies for treating PD-L1-associated conditions, including administering or preventing In another embodiment, a subject in need thereof is administered a PD- The present invention relates to a method for treating cancer, the method comprising administering a therapeutically effective amount of an L1 antibody to a patient having a ... A method for treating a condition in a deaf subject is provided.

[0157] A therapeutically effective amount of an antibody or antigen-binding fragment as provided herein (alone or or when used in combination with other agents, such as chemotherapeutic agents, are known in the art. Various factors, such as the type of disease to be treated, the type of antibody, body weight, age, past illnesses, etc. history, current medications, subject's health status, immune status and potential cross-reactions, allergies, Susceptibility and adverse side effects as well as route and type of administration, severity and occurrence of disease and In certain embodiments, the method described herein may vary depending on the discretion of the attending physician or veterinarian. The antibody or antigen-binding fragment as provided herein may be administered in an amount of from about 0.01 mg / kg to about 100 mg / kg. kg, once or multiple times per day (e.g., about 0.01 mg / kg, about 0.3 mg / kg g, approx. 0.5mg / kg, approx. 1mg / kg, approx. 3mg / kg, approx. 5mg / kg, approx. 10m g / kg, approx. 15mg / kg, approx. 20mg / kg, approx. 25mg / kg, approx. 30mg / kg , about 35mg / kg, about 40mg / kg, about 45mg / kg, about 50mg / kg, about 55 mg / kg, about 60mg / kg, about 65mg / kg, about 70mg / kg, about 75mg / k g, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or The compound may be administered at a therapeutically effective dose of about 100 mg / kg, one or more times per day. In certain embodiments, the antibody or antigen-binding fragment is administered at a dosage of about 50 mg / kg or less. In particular, the dosage is 20 mg / kg or less, 10 mg / kg or less, 3 mg / kg or less or less, 1 mg / kg or less, 0.3 mg / kg or less, or 0.1 mg / kg or less. In certain embodiments, the dosage of administration may vary over the course of treatment. In embodiments, the dosage of the initial administration may be higher than the dosage of subsequent administrations. In certain embodiments, the dosage of administration may vary over the course of treatment depending on the subject's response. do.

[0158] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). In certain embodiments, an antibody or antigen-binding fragment as provided herein is or administered to a subject over a course of treatment. The antibodies or antigen-binding fragments as provided may be administered one or more times depending on the type and severity of the disease. The drug may be administered to a subject in one or more separate doses, or by continuous infusion. See, for example, U.S. Patent Nos. 4,657,760, 5,206,344, and 5,222,461. This can be found in issue 5,212.

[0159] The antibodies and antigen-binding fragments disclosed herein can be administered via any of the methods known in the art. parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or cutaneous) intravenous injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) It may be administered by any route.

[0160] In certain embodiments, the antibodies and antigen-binding fragments disclosed herein are administered in a sustained release formulation. Sustained release parenteral preparations can be administered in the form of implants, oily injections, or granular systems (e.g. , microspheres, microparticles, microcapsules, nanocapsules, nanospheres and nanoparticles ) (Banga, AJ, Therapeutic Pep Tides and Proteins: Formulation, Process ing, and Delivery Systems, Technomic Pub lishing Company, Inc., Lancaster, Pa., ( 1995);Kreuter, J., Colloidal Drug Delivery ry Systems, J. Kreuter, ed., Marcel Dekk. er, Inc., New York, NY, pp. 219-342 (1 994);Tice & Tabibi, Treatise on Controlle d Drug Delivery, A. Kydonieus, ed., Marcel Dekker , Inc. New York, NY, pp. 315-339, (1992)). In certain embodiments, the PD-L1 antibodies and antigen-binding fragments disclosed herein , can be administered in a degradable or non-degradable polymer matrix (Langer, Ac See counts Chem. Res. 26:537-542, 1993. ).

[0161] The condition associated with PD-L1 may be an immune-related disease or disorder. The condition may be a solid tumor, a hematological disorder, an infectious disease, an autoimmune disease, or a fibrotic disease. In certain embodiments, the solid tumor may be, for example, non-small cell lung cancer (squamous / non-squamous). squamous cell), small cell lung cancer, renal cell carcinoma, colorectal cancer, colon cancer, ovarian cancer, breast cancer (basal breast cancer, ductal carcinoma of the breast and lobular carcinoma of the breast), pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head Cervical cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, melanoma, myeloma , mycoses fungoids, Merkel cell carcinoma, hepatocellular carcinoma (HCC), fibrosarcoma , myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovium, mesothelioma, euthyroidoma Lymphatic tumors, leiomyosarcoma, rhabdomyosarcoma, lymphatic tumors, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, thyroid Adenomedullary carcinoma, papillary thyroid carcinoma, pheochromocytic sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic Lung cancer, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular cancer, and seminoma Blood disorders include classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, Lymphoma, T-cell / histiocyte-rich B-cell lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia Myeloid leukemia, acute myeloid leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and Erythroleukemia, chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, Polycythemia vera, mast cell-derived tumors, EBV-positive and -negative PTLD and diffuse Large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell Lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, non-Hodgkin's lymphoma, multilineage Myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, myelodysplastic syndrome, Central nervous system (CNS) tumors, such as hairy cell leukemia, myelodysplasia, and primary CNS lymphoma Neoplasms of the spinal cord, tumors of the spinal cord, brain stem gliomas, astrocytomas, medulloblastomas, craniopharyngiomas [cr aniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningeal These include menangioma, melanoma, neuroblastoma, and retinoblastoma. In certain embodiments, the tumors and cancers are metastatic, particularly metastatic tumors that express PD-L1. In an embodiment, the tumor is melanoma or colon cancer.

[0162] In certain embodiments, the PD-L1 associated condition or disorder is systemic lupus erythematosus. (SLE), intestinal mucosal inflammation, debilitating disease with colitis, multiple sclerosis, viral infections , rheumatoid arthritis, osteoarthritis, Crohn's disease and inflammatory bowel disease, psoriasis These include autoimmune or inflammatory diseases such as sclerosis, systemic sclerosis, and autoimmune diabetes. In certain embodiments, the PD-L1 associated conditions and disorders include infectious diseases, such as fungal infections. Disease, parasitic / protozoan infection or chronic viral infection, e.g., Coccidioideum Coccidioiodmycosis immitis, histoplasmosis, onychomycosis, asthma Aspergilosis, Blastomycosis, Candida albicans paracoccidioidomycosis, microsporidia Acanthamoeba keratitis, amebiasis, ascariasis, babesiosis, balantidiosis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia chliomyia], cryptosporidiosis, diphyllobothriasis, dracunculiasis, echinococcosis, Elephantiasis, enterobiasis, fascioliasis, fascioliasis, filariasis, giardiasis, gnathostomiasis, membranous Tapeworm infection, isosporosis, Katayama fever, leishmaniasis, Lyme disease, malaria, tumefaciens , myiasis, onchocerciasis, lice infestation, scabies, schistosomiasis, sleeping sickness, fecal matter disease, Tapeworm disease, toxocariasis, toxoplasmosis, trichinosis, trichuriasis, trypanosomiasis, helminths Infection, Hepatitis B (HBV), Hepatitis C (HCV), Herpes virus, Epstein-Barr virus -Viruses, HIV, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type I pesvirus I, human papillomavirus, adenovirus, human immunodeficiency virus I, Human immunodeficiency virus II, Kaposi-West sarcoma-associated herpesvirus infection, simian Thin ring virus (Torque tenovirus), human T Human T-lymphotrophic virus I, human T-lymphotrophic virus II, varicella zoster, JC virus or viral infection with BK virus. In certain embodiments, PD-L1-associated Conditions include glomerulonephritis, neural scarring, skin scarring, and pulmonary fibrosis. ry fibrosis, lung fibrosis, radiation-induced fibrosis, liver fibrosis, bone marrow fibrosis and other fibrotic diseases.

[0163] How to use The disclosure further provides methods of using the anti-PD-L1 antibodies or antigen-binding fragments thereof. .

[0164] In some embodiments, the disclosure provides a method of treating a PD-L1-associated condition in a subject. 23. A method comprising administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof. In certain embodiments, the subject is provided with a PD-L1 antagonist. In certain embodiments, the present disclosure provides a method for treating PD A method for preventing, detecting, or diagnosing an -L1-associated condition, comprising the steps of: The provided PD-L1 antibodies, or antigen-binding fragments thereof, can be administered to a subject suspected of having a PD-L1-associated condition. Biological samples obtained from subjects who have, have, or are at risk of having and contacting the biological sample with a PD-L1 antibody that binds to PD-L1 in the biological sample. or an antigen-binding fragment thereof.

[0165] For treatment of a PD-L1 associated condition, the subject tests positive for PD-L1 expression. or are tested to have elevated levels of PD-L1 expression. Thus, the presence or level of PD-L1 can be determined in a test biological sample obtained from an individual. For example, a test biological sample may be subjected to binding to expressed PD-L1 protein, The subject can be exposed to an anti-PD-L1 antibody or antigen-binding fragment thereof that detects PD-L1. Alternatively, PD-L1 can also be detected by qPCR, reverse transcriptase PCR, microarray, SAGE, Detection can be at the nucleic acid expression level using methods such as FISH. In the present invention, the test sample may be a cancer cell or tissue (e.g., a biopsy tissue obtained from an organ), a tumor infiltrate, or a combination thereof. Derived from immune cells or bodily fluids (e.g., blood or serum). The presence or upregulated levels of PD-L1 in biological samples predicts the likelihood of response. The term "upregulated" as used herein refers to a level of upregulated expression detected using the same antibody. As used herein, the PD-L1 protein level is compared to a reference sample, such as PD in a test sample as detected using the provided antibody or antigen-binding fragment. -10%, 15%, 20%, 25%, 30%, 35% at the protein level of L1; 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or more A reference sample refers to a control sample obtained from a healthy or non-diseased individual. A control sample or a healthy or non-diseased sample obtained from the same individual from which the test sample was obtained. The sample may be a sample obtained from the same individual at a later time during the treatment of the condition, or at an earlier time during the treatment of the condition. For example, the reference sample may be adjacent to or adjacent to the test sample (e.g., a tumor). The samples may be non-diseased samples located in the vicinity of the sample.

[0166] The antibodies or antigen-binding fragments disclosed herein may be used alone or in combination with one or more of the above. The compound may be administered in combination with any of a number of additional therapeutic procedures or agents. The disclosed antibodies or antigen-binding fragments can be used in radiation therapy, chemotherapy, targeted therapy, gene therapy, and the like. chemotherapy, immunotherapy, hormonal therapy, anti-angiogenic therapy, palliative care, surgery for the treatment of cancer (e.g. , tumor removal), one or more secondary therapies for complications resulting from chemotherapy Antiemetics or other treatments or cancer or any medical condition mediated by PD-L1 A second therapeutic agent, e.g., another antibody, a therapeutic polynucleotide, for use in treating a chronic disorder. thrombolytic drugs, chemotherapy agents (single or multiple), angiogenesis inhibitors, cytokines, and other cell In combination with a cytotoxic agent(s) or a growth inhibitory agent(s), In certain of these embodiments, the compound may be administered in combination with one or more additional therapeutic agents. The antibody or antigen-binding fragment disclosed herein that is administered may comprise one or more of the following: In certain of these embodiments, the antibody or antigen-binding fragment may be administered simultaneously with a therapeutic agent comprising The fragment and additional therapeutic agent(s) may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment administered "in combination with" another therapeutic agent may be administered It does not have to be administered simultaneously with the agent or in the same composition. It may be administered before or after another agent. The administered antibody or antigen-binding fragment may be a different antibody or antigen-binding fragment and a second agent. However, the term may be used interchangeably herein, even if the subject is administered by any route other than the above. Whenever possible, the term "combination" refers to a drug that is administered "in combination with" that drug. Additional therapeutic agents administered in combination with the disclosed antibodies or antigen-binding fragments include According to the schedule listed on the drug product information sheet for the therapeutic agent, or ians' Desk Reference 2003 (Physicians' D esk Reference, 57th Ed; Medical Economics s Company; ISBN: 1563634457; 57th editorial n (November 2002)) or according to protocols well known in the art. In certain embodiments, the PD-L1 antibodies provided herein are administered in combination with The angiogenesis inhibitors administered in combination are bevacizumab (VEGF antibody) and IMC-1C1. 1 or DC101 (VEGFR-2 antibody), mF4-31C1 (VEGFR-3 antibody) ) and vitaxin (integrin α v β 3 Monoclonal antibodies for antivascular therapy It is a clonal antibody.

[0167] In certain embodiments, the therapeutic agent may induce or boost an immune response against cancer. For example, tumor vaccines can be used to induce an immune response against a particular tumor or cancer. Additionally, cytokine therapy can be used to enhance tumor antigen presentation to the immune system. Examples of interferon therapies include, but are not limited to, interferon-α, -β and -γ Interferons such as macrophage-CSF, granulocyte-macrophage CSF and Colony-stimulating factors such as granulocyte-CSF, insulin growth factor (IGF-1), vascular endothelium Cell growth factor (VEGF), transforming growth factor (TGF), fibroblast proliferation Factor (FGF), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, I such as L-6, IL-7, IL-8, IL-9, IL-10, IL-11 and IL-12. Interleukins such as TNF-α and TNF-β, tumor necrosis factors or their Any combination may be used. Agents that inactivate immunosuppressive targets, such as IL-1-antibody. agonists (IL-1A), VEGFR2 antagonists (e.g., vatalanib, sunitinib, nib, sorafenib, pazopanib), TGF-β inhibitors, FGFR antagonists, hemoglobin Platelet-derived growth factor receptor (PDGFR) antagonists (e.g., imatinib, sunitinib , sorafenib, pazopanib), epidermal growth factor receptor (EGFR, ErbB) antagonists stimulants (e.g., gefitinib, lapatinib, canertinib), IL-10 inhibitors and F As ligand inhibitors or any combination thereof may also be used. Activating immune responsiveness to tumor or cancer cells, e.g., enhancing T cell activation (e.g., activation of T cell costimulatory molecules such as CTLA-4, ICOS, and OX-40) agonists) and those that enhance dendritic cell function and antigen presentation.

[0168] Screening and evaluation of the in vivo efficacy of anti-PD-L1 antibodies The in vivo efficacy (e.g., binding activity or Inoculation of non-human animals to screen and / or evaluate the binding affinity (or binding affinity) of the In certain embodiments, the method further comprises generating non-human tumor cells that express a human PD-L1 protein. The non-human tumor cells are rodent (e.g., mouse, rat, or hamster) cells. In certain embodiments, the non-human tumor cells are a melanoma cell line (B16) or a mouse The colon cancer cell line (MC38) is a non-human tumor cell that expresses endogenous non-human PD-L1 gene sequences. The present invention relates to a method for the preparation of a human PD-L1 protein comprising administering to said patient a therapeutically effective amount of said human PD-L1 protein. Targeted gene inactivation can be achieved by gene disruption, mutation, addition, or deletion of protein-coding sequences. Gene silencing (e.g., RNAi, gene antisense) or at the endogenous locus Gene deletions (e.g., coding sequences or coding sequences including flanking regions) a partial or complete deletion of the non-human target gene or functionally inhibits expression of a polypeptide that is not bound by its ligand. Generates an inactive / truncated polypeptide. The region of interest can be in the range of about 1 bp to about 500 bp at both the 5' and 3' ends. Alternatively, the regions flanking each end may be longer than 500 bp, but as described herein "Gene disruption" as used herein does not include the inactivation of other genes according to It refers to the addition of one or more nucleotides or amino acids to a naturally occurring sequence. Gene disruption involves the addition or insertion of a marker / reporter gene into a protein coding sequence. In certain embodiments, the inactivation is irreversible. Inactivation results in cells that have no detectable activity for the target gene or gene product. Targeted gene inactivation can be achieved by any suitable means in the art, for example, homologous recombination, This is carried out using RNA interference (RNAi) or the CRISPR / Cas9 system. In a specific embodiment, the human PD-L1 gene segment encoding the human PD-L1 protein is The ment is operably inserted into the endogenous non-human PD-L1 locus (gene replacement). In certain embodiments, the inserted polynucleotide encoding human PD-L1 is In yet a further embodiment, the human The polynucleotide encoding PD-L1 may be present as an episome, e.g., a separate segment of DNA. The episomal DNA is stably maintained in the cell in the form of an episomal segment, and replication of the episomal DNA is Independent of or synchronized with the host cell cycle.

[0169] Non-human tumor cells with complete inactivation of the targeted gene were subjected to FACS analysis of cell surface expression. Human PD-L1 can be identified by target gene transcription or by detecting target gene transcription. The polynucleotide encoding the protein can be expressed in any suitable expression vector described in this disclosure. lentivirus vectors) and other methods such as homologous recombination and transgenic methods. The gene may be introduced into the tumor cells by any suitable method known in the art. Tumor cells with complete inactivation of and / or expression of the introduced gene of interest are By FACS analysis of cell surface expression or by any suitable method in the art. The target gene can be identified by detecting target gene transcription via

[0170] Screening the in vivo efficacy of antibodies or antigen-binding fragments against human PD-L1 The method comprises the steps of: detecting or assessing a polynucleotide sequence encoding a human PD-L1 protein; Inoculating tumor cells containing the peptide into a non-human animal and producing antibodies in the non-human animal. and determining the tumor burden of the tumor cells. In the present specification, "tumor burden" refers to the amount of tumor cells in an individual, which can be measured by tumor volume, number, or weight. Tumor cells can be solid tumor cells or non-solid tumor cells (e.g., hematological cells). The tumor cells can be human tumor cells or non-human tumor cells. In this study, tumor cells were transferred to syngeneic non-human animals to generate syngeneic tumor models. In certain embodiments, the tumor cells are passaged several times and then inoculated into a non-human animal. In certain embodiments, the non-human animal has a human immune system. In one embodiment, the in vivo efficacy of a PD-L1 antibody or antigen-binding fragment thereof is compared to a control. In comparison, the inhibition of tumor volume growth in non-human animals treated with PD-L1 antibodies It is determined.

[0171] The following examples are provided to better illustrate the invention as claimed. All of the following specific examples are intended to be illustrative and not to be construed as limiting the scope of the present invention. Certain compositions, materials, and methods, in whole or in part, fall within the scope of the present invention. The particular compositions, materials and methods are not intended to limit the invention but merely to It is intended to illustrate specific embodiments falling within the scope of the present invention. equivalent compositions, materials and methods may be used without departing from the spirit of the present invention. Methods may be developed that are consistent with the methods described herein while still falling within the scope of the present invention. It will be understood that numerous variations can be made in the procedure. It is the intention of the inventors that such modifications are included within the scope of the present invention. EXAMPLES

[0172] Example 1. Preparation and characterization of PD-L1 protein Human PD-L1 / CD274 protein: Recombinant human PD-L1 / CD274 protein The substrate (accession no. NP_054862.1) (hPD-L1-his) was transfected with human 293 cells ( Briefly, the C-terminal 6xHis tag was expressed in HEK293. The coding region of the human PD-L1 gene derived from Phe19-Arg238 was transduced The supernatant was purified using a His-tag affinity column. The purified protein was characterized using SDS page gel. , purchased from ACRO Biosystem (PD1-H5229).

[0173] Human PD-L1 / CD274 with C-Fc tag: Recombinant human with C-Fc tag PD-L1 / CD274 (accession number NP_054862.1) (hPD-L1-Fc) , and expressed in human 293 cells (HEK293). Human PD-L1 derived from Phe19-Arg238 fused to the Fc fragment of IgG1 The coding region of the gene was used for transfection. Fc-tag affinity column The supernatant was purified using SDS page gel. The protein was purchased from ACRO Biosystem (PD1-H 5258).

[0174] Mouse PD-L1 / CD274 protein: recombinant mouse PD-L1 / CD274 protein Protein (mPD-L1-his) Phe19-Arg238 (accession number NP_06869 3) was fused to a 6xhis tag at the C-terminus and produced in human 293 cells (HEK293). The transfection supernatant from HEK293 cells was incubated with His-tag affinity The purified protein was analyzed using an SDS page gel. The protein was purchased from ACRO Biosystem (PD1-M 5220).

[0175] C-Fc tagged mouse PD-L1 / CD274: C-Fc tagged recombinant mouse PD-L1 / CD274 Mouse PD-L1 / CD274 protein (mPD-L1-Fc) Phe19-Arg23 8 (accession number NP_068693) was fused to the Fc fragment of human IgG1 at the C-terminus to obtain human The transfected antibody was produced in HEK293 cells. The transfection supernatant was purified using an Fc-tag affinity column. The quality was characterized using SDS page gels. The protein was purified from ACRO Bios Purchased from the system (PD1-M5251).

[0176] Cynomolgus PD-L1 / CD274 with His tag: Recombinant cynomolgus PD-L1 / CD274 L1 / CD274 protein (cPD-L1-His) Phe19-Arg238 (contract The nucleotide sequence of the nucleotide sequence (number F6VEW6) was fused to a polyhistidine tag at the C-terminus and transformed into human 293 cells (HEK The transfection supernatant from HEK293 cells was The purified protein was purified using a His-tag affinity column. The protein was purchased from ACRO Biosystems. I entered (PD1-C52H4).

[0177] Cynomolgus PD-L1 / CD274 with C-Fc tag: Recombinant cynomolgus monkey PD-L1 / CD274 protein (cyno PD-L1-Fc) Phe19-Arg238 (accession number F6VEW6) is a C-terminal Fc fragment of human IgG1. The fragment was fused to the GFP gene and produced in human 293 cells (HEK293). The transfection supernatant obtained from the fusion was purified using an Fc-tag affinity column. The purified protein was characterized using SDS page gel. Purchased from RO Biosystem (PD1-C5253).

[0178] The above PD-L1 proteins were used in the following experiments.

[0179] [Example 2] Antibody production 1. Antigen Conjugation and Immunization For immunization, recombinant hPD-L1-Fc (or mPD-L1-Fc) protein The proteins were conjugated to various MabSpace immune enhancing peptides. A 2- to 8-fold molar excess of the peptide was added to sulfo-SMCC (sulfosuccinimidyl 4-[N- Maleimidomethyl]cyclohexane-1-carboxylate, Peirce No. 2232 2) Mix with activated hPD-L1-Fc protein and incubate at room temperature for 1 hour. The reaction was stopped and the conjugated proteins were analyzed using SDS-PAGE gels. Analyzed and quality controlled.

[0180] The conjugated hPD-L1-Fc and mPD-L1-Fc proteins and Freund's complete adjuvant (Pierce) in a 1:1 ratio, respectively. The proteins were then emulsified and then immunized subcutaneously and intraperitoneally into C57B / L6 mice. To preserve the native conformation of the ribozyme, further immunization was performed using CpG and Alum. Immunizations were performed at least every 2 weeks, and anti-P was detected by ELISA assay. Antisera were collected from mice after the first immunization for D-L1 titer analysis. Serum titers were To determine the binding of the high binding clear polymer, 20 μl of mouse serum was prepared from each immunized mouse. Restyrene 96-well plates (Nunc) were coated with high pH coating buffer (0.1 Mouse or human PD in 6% Na2CO3, 0.3% NaHCO3, pH 9.8 -Coated with 100 μl / well of a 1 μg / ml solution of L1-his The plates were incubated overnight at 4°C and then washed with washing buffer PBS+0. Plates were washed once with 1% Tween 20 (Sigma) using an automated plate washer. Add 200 μl of blocking buffer (PBS + 1% BSA + 1% goat serum) to each well. +0.05% Tween 20) was added and incubated at room temperature for 2 hours. Aspirate off the blocking buffer and add 100 μl of dilution buffer (PBS + 1% B Serially diluted sera in SA+1% goat serum+0.01% Tween 20 were used for ELI The plate was then transferred to each well of the SA plate and incubated at room temperature for 60 minutes. The plates were washed three times using the method described above. Then, 100 μl / well of dilution buffer was added The solution of HRP-conjugated goat anti-mouse Fc antibody (Abcam) was diluted in The ELISA plate was then incubated at room temperature for 60 minutes. After incubation, the plate was washed 3 times with 250 μl / well wash buffer. Finally, 100 μl / well of TMB was added to each well, and 0.64 M H 2 SO 4 of The reaction was terminated using a Thermo Multiscan filter at 450 nM. n Read with FC.

[0181] 2.Fusion Four days before fusion, each mouse received unconjugated hPD-L1-F in PBS. On the day of fusion, the spleen was then intraperitoneally boosted with mPD-L1-Fc protein. The organs were aseptically harvested and processed into single cell suspensions. Red blood cells were lysed and spleen cells were purified using DME. Viable, log-phase growing myeloma cells (SP2 / 0) were cultured in myeloma cells and washed with PBS (Gibco). The cells were then washed twice and then fused with PEG. After fusion, the cells were washed with DMEM and then diluted with 10% FBS + HFCS + OPI + 1X HA. The cells were suspended in cell growth medium supplemented with T. 200 μl of this cell suspension per well was added to 9 Plate in 6-well cell culture plates and incubate at 37 °C in a humidified 10% CO 2 Incubate The cultures were incubated for 7 days and then transferred to growth medium. The medium was aspirated from the wells and replaced with fresh growth medium. The incubation was started 2 to 3 days after the medium change.

[0182] [Example 2] Antibody screening 1. Screening for PD-L1 binders by ELISA assay High-binding clear polystyrene 96-well plates (Nunc) were filled with 100 μl / well of 0.5μg / ml hPD-L1-his or mP in high pH coating buffer D-L1-his and incubated overnight at 4°C. The rate was measured using washing buffer (PBS + 0.1% Tween 20 (Sigma)). Each well was washed once with an automatic plate washer using 200 μl of blocking buffer. - (PBS + 1% BSA + 1% normal goat serum + 0.05% Tween-20) added The mixture was incubated at room temperature for 2 hours. 100 μl of the hybridoma supernatant was added to the ELISA kit. The plate was then transferred to each well and incubated at room temperature for 60 minutes. The plates were washed three times using 100 μl / well of diluted blocking solution. A solution of HRP-conjugated goat anti-mouse Fc antibody (Abcam) was added to the plate. The ELISA plate was then incubated at room temperature for 60 minutes. The plate was then washed three times with 250 μl / well of wash buffer. Finally, 100 μl / well of TMB was added to each well, and 0.64 M H 2 SO 4 of The plate was scanned at 450 nM using a Thermo Multiscan Cells from ELISA-positive hybridoma wells were then read by FC. The cells were expanded in cell culture for further characterization studies.

[0183] 2. Hybridoma supernatants that inhibit PD-1 binding to PD-L1 in ELISA Evaluation of blocking activity of High-binding clear polystyrene 96-well plates (Nunc) were filled with 100 μl / well , 2 μg / ml hPD-L1-Fc (Acrobiotic) in high pH coating buffer osystems, catalog no. PD1-H5258) and incubated at 4°C. The plates were then washed with washing buffer (PBS + 0.1% T The plate was washed once using an automatic plate washer with 200 ml of 100% ethanol (Sigma). μl of blocking buffer (PBS + 1% BSA + 1% normal goat serum + 0.05% Tween-20) was added to each well and incubated at room temperature for 2 hours. 1 μl of hybridoma supernatant was transferred to each well of the ELISA plate and incubated at room temperature for 60 min. The plate was then washed three times using the method described above. 1 μg / ml hPD-1-his (ACRO Biosystems, Cat. No. P D1-H5221) was added to each well together with the dilution buffer. Then, 100 μ 1 / well of anti-his-HRP diluted in blocking solution (1:4000 dilution, CW A solution of the antibody (BIO, Cat. No. CW0285) was added to each well of the plate. Afterwards, the ELISA plate was incubated at room temperature for 60 minutes, and then the plate was The plate was washed three times with 100 μl / well of washing buffer. TMB was added to each well, 0.64MH 2 SO 4 The reaction was terminated using pre The assay was read on a Thermo Multiscan FC at 450 nM. ELISA Cells from positive hybridoma wells were then cultured for further characterization studies. and expanded in culture (see Fig. 1A-C ).

[0184] 3. FACS analysis of hybrids that block the binding of PD-1 to PD-L1 on tumor cells Assessment of blocking activity of lysoma supernatants Log-phase IFN-r stimulated (500 U / ml, 2 days) HCC827 cells were collected and cultured. The cells were resuspended in rocking buffer (5% BSA + PBS). 2 × 10^5 cells were The tube was then washed once with PBS (1500 rpm, 5 min, room temperature). 100 μl / tube of hybridoma supernatant and blocking buffer were added to the corresponding The cells were then added to a tube containing 1 ml of 1% PBS and incubated at 4°C for 1 hour. Wash twice with PBS, followed by 100 μl / tube of 3 μg / ml biotinylated hPD-1-Fc-N297A (ACRObiosystems) was added and incubated at 4°C for 1 hour. After washing three times with PBS, 100 μl / tube of streptomycin was added. Ptoavidin-PE (EBiosciences), 1:20 in blocking buffer 0 was added to each tube. The cells were incubated at 4°C for 1 hour, then PBS was added. Subsequently, cells were resuspended in 150 μl of PBS for each sample and washed twice with The cells were then transferred to FACS tubes and analyzed by flow cytometry (ACEA Bio The binding of the antibody to the cells was detected using a cellular immunofluorescence assay (NCA) using a cellular immunofluorescence assay (NCA).

[0185] 4. Screening for pH-dependent PD-L1 binding by ELISA High-binding clear polystyrene 96-well plates (Nunc) were filled with 100 μl / well of Coating was performed with 0.5 μg / ml hPD-L1-his in high pH coating buffer. The plates were then washed with washing buffer P and incubated overnight at 4°C. 1x in an automated plate washer using BS+0.1% Tween 20 (Sigma) Washed with 200 μl of blocking buffer (PBS + 1% BSA + 1% normal goat Serum + 0.05% Tween-20) was added to each well and incubated at room temperature for 2 hours. 100 μl of hybridoma supernatant was transferred to each well of the ELISA plate, and The mixture was incubated at room temperature for 60 minutes at either pH 7.4 or pH 6.0. The method described above was used to generate the plasmid p53 using either a pH 7.4 or pH 6.0 buffer. The plates were washed three times. Then, 100 μl / well of blocking solution, pH 7.4, was added. HRP-conjugated goat anti-mouse Fc antibody (Abcam) diluted in ELISA kit ) was then added to each well of the plate. The ELISA plate was then incubated at room temperature for 24 hours. Allow to incubate for 60 minutes and then plate was washed with 200 μl / well pH 7.4 or The plates were washed three times with pH 6.0 wash buffer. Finally, 100 μl / well of TM B was added to each well, and 0.64MH 2 SO 4 The reaction was terminated using the plate was read on a Thermo Multiscan FC at 450 nM. ELISA positive The cells obtained from the hybridoma wells were then cultured in cell culture for further characterization studies. Clone 23A11 (Figure 2A) and clone 23F11 (Figure 2B) The results are shown.

[0186] [Example 3] Subcloning of positive hybridoma clones and small-scale antibody production 1. Subcloning of positive hybridoma clones ELISA positive hybridoma wells with desired binding profile and blocking activity Cells from each were selected and plated using limiting dilution in 96-well plates. These cells were grown for 7 days. Once a suitable cell mass was reached, each well was The supernatants were collected from the cultures and rescreened for antigen-binding ability (see the screening in Example 2). (see ping).

[0187] From each 96-well plate, clones with the highest antigen-binding activity were identified and subjected to limiting dilution. Further, 96-well plates were cultured with 200 μl of hybridoma growth medium per well. After 7 days, cells from the 96-well plate were analyzed for antigen binding. Subcloning was performed more than twice. More than 90 of the wells showed a positive binding signal. When the clones showed the highest antigen binding activity, two clones were identified and cultured. The cells were transferred to a 24-well plate and grown for an additional 2 days. Once the cells were cultured, they were transferred to 6-well plates. After 5 days of incubation, A portion was frozen. The rest of the cells were transferred to flasks and expanded. When the flasks became confluent, Once the cells reached the desired concentration, half of the cells were frozen for further backup (3 cultures per clone). The other half was further expanded in flasks containing medium for antibody production. Isotypes were determined using standard methodologies.

[0188] 2. Small-scale Antibody Production Hybridoma cells were inoculated into roller bottles and 200-300 ml of hybridoma cells were The hybridoma cells were cultured for 14 days with culture medium (Invitrogen). PD-L1 monoclonal antibodies (mAbs) were purified from the samples as follows. The purification process was carried out at room temperature. A single purification scheme was used to purify the various mAbs. , affinity chromatography was used.

[0189] The host cell culture fluid (CCF) was centrifuged to remove cell debris. The CCF supernatant was then The mixture was filtered, diluted, and then purified using Protein G High Performance (Bio- Load and equilibrate the Protein G chromatography medium in the form of a column (Rad) did.

[0190] After loading, the Protein G column was run based on the absorbance of the flow-through at 280 nm. The PD-L1 was then isolated using glycine, pH 2.5. The mAb was eluted from the column with 50 μL of 1 M Tris base per mL of elution volume. The mixture was immediately neutralized by adding a storage solution. The absorbance of the eluate at 280 nm was monitored. The fractions were filtered and protein-containing fractions were collected to create the Protein A pool.

[0191] After purification, the eluate was transferred to a 10,000 MWCO membrane (Pierce Slide-A-Lys). PD-L1 mAb was prepared in PBS by dialysis using a dialysis tube or dialysis tube. After formulation, the PD-L1 mAb was filtered.

[0192] [Example 4] Analysis of purified PD-L1-binding antibodies High-binding clear polystyrene 96-well plates (Nunc) were filled with 100 μl / well of Using 0.5μg / ml hPD-L1-his in high pH coating buffer The plates were then washed with washing buffer. (PBS + 0.1% Tween 20 (Sigma)) in an automated plate washer. Wash once with 200 μl of blocking buffer (PBS + 1% BS A+1% normal goat serum+0.05% Tween-20) was added to each well and incubated at room temperature. The mixture was incubated for 2 hours. Then, the dilution buffer (PBS + 1% BSA + 1% normal yam) was added. Purified antibodies serially diluted with 100% guinea pig serum + 0.01% Tween-20 were added and incubated at room temperature. The plates were then washed three times using the method described above. 100 μl / well of HRP-conjugated goat anti-mouse in dilution buffer A solution of Fc antibody (Abcam) was added to each well of the plate. Then, ELISA The plates were allowed to incubate at room temperature for 60 minutes and then the plates were refilled with 250 μl / well. Finally, 100 μl / well of TMB was added to each well. Add 0.64MH to the well. 2 SO 4 The plates were terminated using 450 nM Read on a Thermo Multiscan FC (see Figure 3).

[0193] [Example 5] Evaluation of blocking activity of purified antibodies that inhibit the binding of PD-1 to PD-L1 1. Evaluation by ELISA High-binding clear polystyrene 96-well plates (Nunc) were filled with 100 μl / well of Coating solution consisting of 0.5 μg / ml hPD-L1-Fc was used overnight at 4°C. Then, the cells were washed with washing buffer PBS + 0.1% Tween 20 (Sig Each well was washed once with an automatic plate washer using 200 μl of blocking solution. King's buffer (PBS + 1% BSA + 1% normal goat serum + 0.05% Tween -20) was added and incubated at room temperature for 2 hours. + 1% BSA + 1% normal goat serum + 0.01% Tween-20) and serially diluted. Add the antibody (10 μg / ml to 0.0006 μg / ml) and incubate at room temperature for 1 hour. The plates were then washed three times using the method described above. Add tinned hPD-1-Fc-N297A (Acrobiosystem) and incubate at room temperature. The plate was washed three times and then 100 μl / well of , HRP-conjugated neutravidin antibody ( Pierce) solution was added to each well of the plate. Then, the ELISA plate The plate was then washed with 250 μl / well of PBS and incubated for 60 min at room temperature. The plate was washed three times with buffer. Finally, 100 μl / well of TMB was added to each well. Add 0.64MH 2 SO 4 The reaction was terminated using 450 nM T The results were read using Hermo Multiscan FC (see Figure 4A). T is the benchmark antibody (MPDL-3280) disclosed in U.S. Pat. No. 8,217,849. A).

[0194] 2. Evaluation of tumor cells by FACS Log-phase IFN-r stimulated (500 U / ml, 2 days) HCC827 cells were collected and cultured. The cells were resuspended in rocking buffer (5% BSA + PBS). 2 × 10^5 cells were The tube was then washed once with PBS (1500 rpm, 5 min, room temperature). 100 μl / tube of antibody diluted in blocking buffer was added at 10 μg / ml. into the corresponding tubes to contain a final concentration of 1 ml and incubate for 1 hr at 4 °C. The cells were then washed twice with 1 ml PBS, followed by 100 μl / ml Tube containing 3 μg / ml biotinylated hPD-1-Fc-N297A (Acrobios The plate was incubated at 4°C for 1 hour and washed three times with PBS. Then, 100 μl / tube of streptavidin diluted 1:200 in blocking buffer was added. Avidin-PE (EBiosciences) was added to each tube. The cells were incubated at 4°C for 1 The cells were incubated for 2 h and then washed twice with PBS. The cells were then transferred to FACS tubes and resuspended in 150 μl of PBS. Using flow cytometry (ACEA Bioscience Novocyte) Binding of the antibody to the cells was detected by immunoblotting (see FIG. 4B).

[0195] Example 6: Dose-dependent response of purified PD-L1 antibody 1. Binding of purified PD-L1 antibodies to HCC827 cells as measured by FACS Dependency Response Log-phase IFN-γ stimulated (500 U / ml, 2 days) HCC827 cells were collected and cultured. The cells were resuspended in rocking buffer (5% BSA + PBS). 2 × 10^5 cells were The tube was then washed once with PBS (1500 rpm, 5 min, room temperature). 100 μl / tube of serially diluted hybridoma The purified PD-L1 antibody from the supernatant was added to the corresponding tube and incubated at 4℃ for 1 hour. The cells were then washed twice with 1 ml PBS, followed by 100 μl Secondary antibody (1:400 anti-mIgG (H+L) in blocking buffer / tube -PE, Cell signaling) was added. The cells were incubated at 4°C for 1 hour. The cells were then washed twice with PBS, and then transferred to 150 mL of The cells were then transferred to FACS tubes and the antibodies were allowed to bind to the cells. Binding was detected using flow cytometry (BD Accuri C6) (Figure 5 (See A.)

[0196] 2. hPD-1 binding to hPD-L1 on HCC827 as measured by FACS Dose-dependent activity of purified PD-L1 antibodies to block Log-phase IFN-γ stimulated (500 U / ml, 2 days) HCC827 cells were collected and cultured. The cells were resuspended in rocking buffer (5% BSA + PBS). 2 × 10^5 cells were The tube was then washed once with PBS (1500 rpm, 5 min, room temperature). 100 μl / tube of serially diluted hybridization buffer was used. PD-L1 antibodies purified from doma supernatant were added to the corresponding tubes and incubated at 4℃ for 1 hour. The cells were then washed twice with 1 ml PBS, followed by 10 Add 0 μl / tube of 3 μg / ml biotinylated hPD-1-N297A and incubate at 4°C. The plate was incubated for 1 hour. After washing three times with PBS, 100 μl / tube of Diluted 1:200 in blocking buffer (rabbit anti-hIgG-PE, Santa Cruz) was added to each tube. The cells were incubated at 4°C for 1 hour and then The cells were then washed twice with PBS and resuspended in 150 μl of PBS for each sample. The cells were then transferred into FACS tubes and analyzed by flow cytometry (BD A ccuri C6) was used to detect the binding of the antibody to the cells (see Figure 5B). .

[0197] [Example 7] Cloning and sequencing of hybridoma antibodies The sequences of the mouse anti-human PD-L1 antibody light and heavy chain variable regions were determined by 5'RACE (cDNA by the polymerase chain reaction (PCR) amplification technique, also known as rapid amplification of A-terminal 4B6 / 23A11 / 23F11 / 22C9 / 26F5 / 21F11 / 18G4 antibodies Total RNA was extracted from somatically produced hybridoma cells using Trizol (Invitrogen). A was isolated and synthesized using the Superscript first-strand synthesis system (Invitrogen) and cDNA was extracted using the oligo(DT)12-18 primer (Invitrogen). The variable region of the mouse IgG gene was synthesized as MuIgG VH3 for the heavy chain variable region. The '-2' and MuIg-5' leader primers were used for the light chain variable region, MuIgK VL3'-1 and MuIg-5" leader primers (NOVAGEN) were used. The resulting bands for each antibody were cloned using TOPO TA cloning. The DNA from over 10 clones was sequenced. The sequences were determined using an ABI DNA sequencing instrument (Perkin Elmer). Vector NTI Advance 10 software (Invitrogen) was used to determine the consensus sequence.

[0198] Generation of chimeric antibodies: After sequence analysis and confirmation, the above-mentioned method was used for antibody production and purification. The variable regions of each gene were cloned into a recombinant expression vector, e.g., The sequence of the light chain variable region (VL) was cloned into pCP-hIgG1, and the sequence of the heavy chain variable region (V H) was cloned into pCP-hIgG1.

[0199] [Example 8] Recombinant chimeric antibody expression and purification The recombinant chimeric antibody protein produced above was expressed and purified by the following method. Conducted: 1x10 6 cells / ml with 10% Pluronic F-68 HEK293E cells cultured in Freestyle 293 expression medium were incubated with 0.5 μg / m Equal amounts of heavy and light chain vector DNA and 1.0 μl final concentration 100 μg / ml PEI (polyethyleneimine-linear, Polyscience) was used for the transfection. The DNA to PEI ratio was 1:2. The time period for PEI complex formation should be 15 minutes at room temperature. The transfected cells were incubated at 4 °C in 5% CO 2 The flask was incubated at 37°C and a shaking speed of 125 rpm. 22-26 hours after transfection, 1% peptone medium was added. On the sixth day, the conditioned medium was collected and the supernatant was centrifuged at 3000 rpm for 30 minutes. The clarified conditioned medium was then loaded onto a Protein A column (G. E. Healthcare) and loaded with PBS and 0.1% triton-X. 100 and finally the bound IgG was removed by centrifugation with 0.1 M glycine, pH 3.5. The eluted antibody protein was dialyzed against PBS and eluted with a solution containing The purified protein was purified using Hitrapremove to remove endotoxins and stored at -80°C. The resulting antibody was further processed by passing it through a DEAE Sepharose FF column. , size exclusion chromatography (Superdex 200 5 / 150 GL, G. The purity level was determined by analysis using a ELISA kit (E. Healthcare).

[0200] Example 9. Purified PD-L1 antibodies and cynomolgus monkey and rodent PD-L1 proteins Cross-reactivity and binding selectivity of hPD-L1 to hPD-L2 1. Cross-species binding to cynomolgus monkey PD-L1 protein High-binding clear polystyrene 96-well plates (Nunc) were filled with 100 μl / well , 5 μg / ml human or cynomolgus monkey (cyno) in high pH coating buffer The cells were coated with PD-L1-his and incubated overnight at 4°C. The plate was washed with washing buffer (PBS + 0.1% Tween 20 (Sigma)). The plate was washed once using an automatic plate washer with 200 μl of blocking buffer (P BS + 1% BSA + 1% normal goat serum + 0.05% Tween-20) was added to each well. and incubated at room temperature for 2 hours. Then, dilution buffer (PBS + 1% Antibodies serially diluted in BSA + 1% normal goat serum + 0.01% Tween-20 and incubated at room temperature for 1 hour. The plates were then plated using the method described above. Then, 100 μl / well of mouse hybridization buffer in dilution buffer was added to the wells and washed three times. For the doma antibody, HRP-conjugated goat anti-mouse Fc antibody (Abcam ) or Goat anti-human IgG Fc-HRP pre-adsorbed for 4B6 chimeric antibody (Ab A solution of cam) was added to each well of the plate. The ELISA plate was then incubated at room temperature. and then the plate was washed with 250 μl / well wash buffer. Finally, 100 μl / well of TMB was added to each well. 0.64MH 2 SO 4 The reaction was terminated using 450 nM The The results were read using rmo Multiscan FC (see Figures 6A-6E).

[0201] Chimeric antibody 4B6-C has a binding affinity similar to that of its parent antibody 4B6. The binding affinities for human and monkey antigens were compared with those of 4B6-C, as shown in FIG. 6A. Similarly, the binding affinity of 4B6-C in mice was 4B6 It is similar to that of

[0202] 2. Cross-species binding to mouse PD-L1 protein High-binding clear polystyrene 96-well plates (Nunc) were filled with 100 μl / well of Coating was performed with 1μg / ml mouse PD-L1-his in high pH coating buffer. The plates were then washed with washing buffer ( PBS + 0.1% Tween 20 (Sigma)) in an automated plate washer. Wash once with 200 μl of blocking buffer (PBS + 1% BSA + 1% normal Goat serum + 0.05% Tween-20) was added to each well and incubated at room temperature for 2 hours. Then, the cells were incubated in a dilution buffer (PBS + 1% BSA + 1% normal goat serum + 0 Purified hybridoma mouse antibodies were added at 20 μg / ml in 0.01% Tween-20. The plates were then washed with 250 μl / well of The plate was washed three times with washing buffer. Then, 100 μl / well of A solution of HRP-conjugated goat anti-mouse Fc antibody (Abcam) was added to the plate. The ELISA plate was then incubated at room temperature for 60 minutes. The plate was then washed three times with 250 μl / well of wash buffer. Finally, 100 μl / well of TMB was added to each well, and 0.64MH 2 SO 4 The reaction was terminated using a Thermo Multisc an FC was read (see Figure 6F).

[0203] 3. Cross-linking with hPD-L2 protein Purified antibodies (4B6, 23A11, 26F5, 23F11 and 22C9) human PD- Binding to L2 protein was assayed by ELISA in a similar manner as described above for cross-species binding. A. Briefly, high-binding transparent polystyrene 96-well plates (N unc) and high pH coating buffer (0.16% Na2CO3+0 100 μl / well of 0.5 μg / ml in 0.3% NaHCO3, pH 9.8 of human PD-L1-Fc (Acrobiosystems, Cat. No. PD1-H52 58) or 100 μl / well of 1 μg / ml hPD-L2-Fc (Acrobio Systems, Cat. No. PD2-H5251) and incubated at 4°C. The plates were then washed with washing buffer (PBS + 0.1% Tween 100 and 100 mM NaCl). The plate was washed once using een 20 (Sigma) in an automatic plate washer. l blocking buffer (PBS + 1% BSA + 1% normal goat serum + 0.05% Tween-20) was added to each well and incubated at room temperature for 2 hours. of dilution buffer (PBS + 1% BSA + 1% normal goat serum + 0.01% Twe 2μg / ml of purified PD-L1 antibody with 100μg / ml ELISA kit was added and incubated at room temperature for 1 hour. The plate was then washed three times with washing buffer. 100 μl / well of HRP-conjugated goat anti-mouse I in dilution buffer A solution of antibody against IgG (1:20000, Abcam) was added to each well of the plate. After this, the ELISA plate was allowed to incubate at room temperature for 60 minutes, and then the plate was Then, the plate was washed three times with 250 μl / well of washing buffer. Finally, the plate was washed three times with 100 μl / well of TMB was added to each well for 15 minutes, and 50 μl / well of 0.16 M / L sulfuric acid was used. The reaction was terminated by 500 nM incubation at 450 nM using a Thermo Multiscan F C (see Figure 6G).

[0204] As shown in Figure 6G, all of the tested antibodies were able to bind to hPD-L1, but It cannot bind to PD-L2.

[0205] Example 10. Characterization of the binding of purified anti-PD-L1 antibodies to PD-L1 on tumor cells: FACS binding assay (HCC827 cells) Log-phase IFN-γ stimulated (500 U / ml, 2 days) HCC827 cells were collected and cultured. The cells were resuspended in rocking buffer (5% BSA + PBS). 2 × 10^5 cells were The tube was then washed once with PBS (1500 rpm, 5 min, room temperature). 100 μl / tube of serially diluted biotinylated antibody in blocking buffer was added. PD-L1 antibody was added to the corresponding tubes and incubated at 4℃ for 1 hour. Next, the cells were washed twice with 1 ml PBS, followed by 100 μl / tube of Secondary antibody (or 1:200 rabbit anti-human IgG-PE (S Anta Cruz) was added. The cells were incubated at 4°C for 1 hour and then The cells were then washed twice with PBS and resuspended in 150 μl of PBS for each sample. The cells were then transferred to FACS tubes and analyzed by flow cytometry (ACEA The antibody binding to the cells was detected using a ELISA kit (Bioscience Novocyte). (See FIG. 7). The antibodies marked with a "C" represent chimeric antibodies, and the antibody "4B6-H "3L3" refers to the combination of heavy chain H3 and light chain L3 as produced in Example 15. This refers to humanized 4B6 having the following structure:

[0206] [Example 11] Purified anti-PD-1 antibody inhibits the binding of PD-1 to PD-L1 on tumor cells Assessment of blocking activity of L1 antibody Log-phase IFN-γ stimulated (500 U / ml, 2 days) HCC827 cells were collected and cultured. The cells were resuspended in rocking buffer (5% BSA + PBS). 2 × 10^5 cells were The tube was then washed once with PBS (1500 rpm, 5 min, room temperature). 100μl / tube of serially diluted PD-L1 in blocking buffer was Antibodies (see Figure 8) are added to the corresponding tubes and incubated for 1 hour at 4°C. The cells were then washed twice with 1 ml PBS, followed by 100 μl / tube Add 3 μg / ml of biotinylated hPD-1-Fc-N297A to the antibody and incubate at 4°C for 1 hour. After washing three times with PBS, 100 μl / tube of blotting paper was added. Streptavidin-PE 1:200 in King's buffer (EBioscience s) was added to each tube. The cells were incubated at 4°C for 1 hour, and then PBS was added. Subsequently, cells were resuspended in 150 μl of PBS for each sample and washed twice with The cells were then transferred to FACS tubes and analyzed by flow cytometry (ACEA Bio The binding of the antibody to the cells was detected using a cellular immunofluorescence assay (NCA) using a cellular immunofluorescence assay (NCA).

[0207] As shown in Figure 8, the FACS results show that anti-PD-L1 antibodies inhibited the expression of hPD-1 in tumor cells. The results showed that PD-L1 binding to HCC827 cells was inhibited in a concentration-dependent manner. showed.

[0208] Example 12: Epitope binning of selected anti-PD-L1 antibodies by competitive ELISA High-binding clear polystyrene 96-well plates (Nunc) were filled with 100 μl / well of Coating solution consisting of 0.5 μg / ml hPD-L1-Fc was used at 4°C overnight. The plate was then washed with washing buffer (PBS + 0.1% Tween n 20 (Sigma)) in an automated plate washer, followed by 200 μl of blocking buffer (PBS + 1% BSA + 1% normal goat serum + 0.05% Tween-20) was added to each well. The plate was incubated at room temperature for 2 hours. Then, the cells were diluted with dilution buffer (PBS + 1% BSA + 1% normal goat serum + 0.01% Serially diluted competing antibodies 4B6, 23A11, 23F11, 22C9 and 21F11 were added and the plate was incubated at room temperature for 1 hour. The plate was then washed three times with wash buffer. Biotinylated anti-PD-L1 antibodies (e.g., 18G4, 2 3A11, 4B6) was added and incubated at room temperature for 1 hour. The plate was washed three times. Then, 100 μl / well of HRP-conjugated plate diluted with dilution buffer was added. A solution of neutralized neutravidin antibody (Pierce) was added to each well of the plate. The ELISA plate was then incubated at room temperature for 60 minutes, and then 250 μl The plate was washed three times with 100 μl / well of washing buffer. Finally, the plate was washed with 100 μl / well of TMB was added to each well, and 0.64MH 2 SO 4 The reaction was terminated using the plate was read on a Thermo Multiscan FC at 450 nM (Figures 9A to 9C (see

[0209] [Example 13] Characterization of pH-dependent binding of recombinant chimeric antibodies High-binding clear polystyrene 96-well plates were filled with 100 μl / well of high pH coated 0.5 μg / ml of hPD-L1-his (Acrobiosys) in incubation buffer The plates were then coated with TEMS and incubated at 4°C overnight. The coated plates were washed with PBS + 0.1% Tween 20 (Sigma) The plate was washed once with an automatic plate washer. Then, 200 μl of blocking buffer was added to the plate. was added to each well and the plate was then incubated at room temperature for 2 hours. After aspirating the buffer, add serially diluted chimeric antibodies in dilution buffer at pH 7.4. The plates were then incubated at room temperature for 40 minutes. The wells are washed once with a wash buffer having a pH of 7.4 or 5.5, then Add 100 μl of antibody dilution buffer, pH 7.4 or pH 5.5, and incubate the ink. Incubation was continued for 2 hours at room temperature. The plate was then washed with 250 μl of washing buffer. Wash three times with 10 sec shaking between washes, then 100 μl / well of pH 7.4 Dilute goat HRP conjugated 1:20000 in dilution buffer A solution of anti-human Fc antibody (Abcam) was added to each well of the plate and incubated at room temperature for 1 hour. The plate was then washed three times with 250 μl of wash buffer. Finally, 100 μl / well of TMB was added to each well. Add 0.64MH 2 SO 4 The reaction was terminated using 450 nM T The sequences were read using hermo Multiscan FC (see Figures 10A-F).

[0210] As shown in Figures 10A-10F, chimeric antibodies 23F11, 23A11 and 22C 9 (Figures 10A-10C) showed pH-dependent binding to hPD-L1 (e.g., pH 5. 5), whereas chimeric antibodies 21F11, 4B6, and 26 F5 (Figures 10D-10F) did not show pH-dependent binding to hPD-L1 (pH5 .5 / similar binding affinity at pH 7.4).

[0211] [Example 14] In vivo evaluation of anti-PD-L1 antibodies The in vivo activity of the anti-PD-L1 antibodies obtained above was evaluated using modified syngeneic mouse xenografts. The study was evaluated using a xenograft tumor model. Mice were injected with the murine colon cancer cell line MC38 and black The mouse PD-L1 gene was replaced with human PD-L1 in the chromosome 16 cell line, B16. The mice were subcutaneously inoculated with mouse tumor cells. Anti-human PD-L1 antibody was administered at 3 mg / kg, 1 0 mg / kg and / or 30 mg / kg, intraperitoneally (IP), three times weekly, or The doses were 1, 3 or 10 mg / kg and were injected intravenously once a week. The compound BM-GT (i.e., in U.S. Pat. No. 8,217,149 (Genentech) Disclosed YW243.55.S70 or MPDL3280A) and BM-ME ( i.e., 2.14H9OP of U.S. Patent No. 8,779,108) was used as a positive control. The tumors in the vehicle group were 1000 mm 3 Once the tumor volume reached 100 μg / ml, the effect of the antibody on tumor growth was Tumor volumes and body weights were measured twice weekly as described below to assess tumor growth. The effect of antibodies on tumor growth was calculated as tumor growth inhibition (TGI) relative to the vehicle group.

[0212] 1. Generation of mouse tumor cell lines expressing human PD-L1 Using the highly efficient CRISPR / Cas9 system that we recently developed, The endogenous CD2 In brief, mouse CD274 / PD-L1 was knocked out. Design sgRNAs targeting the first coding exon of genes and generate hit markers. Cells were transfected with a novel CRISPR / Cas9+sgRNA construct and We selected knockout cells that completely knock out endogenous CD274 / PD-L1. The cells were analyzed by FAC for cell surface expression of CD274 / PD-L1 at steady state. S analysis or stimulated with interferon gamma, followed by targeting The gene was confirmed by TA cloning and sequencing of the genomic region involved in the expression of human CD274. / To generate PD-L1 replacement cell lines, human CD274 / PD-L1 cDNA The coding sequence of was then cloned into a lentiviral vector derived from FG12. Mouse CD274 / PD-L1 knockout cells express human CD274 / PD-L1 We infected established cell lines with lentivirus targeting human CD274 / PD-L1 High-level and stable expression of B16 and MC38 was confirmed by FACS analysis. The genetically engineered cells were B16-hPD-L1 KI, MC38-hPD-L1 KI, and MC38-hPD-L1 KI, respectively. It was named L1 KI.

[0213] 2. Antitumor activity of PD-L1 antibodies in the B16 / human PD-L1 knock-in tumor model Activity B16 cells were engineered to replace the mouse PD-L1 gene with human PD-L1. The clone was engineered to be B16-hPD-L1 KI and named B16-hPD-L1 KI. L1 KI cells were subcultured within 5 passages and then inoculated into mice. 1 cell / 0.2 mL was injected SC into each of 10 female SPF grade C57BL / 6 mice. The first injection of antibodies (e.g., BM-GT, 4B6-C, and 23A11) was performed. The dose (3mpk or 10mpk) was injected one day after the tumor cells were injected. Size was measured twice weekly in two dimensions using calipers (INSIZE). Tumor growth inhibition The TGI percentage was calculated based on the values ​​measured on day 29. The results were analyzed using Prism Graph. Analyses were performed using phPad and presented as mean ± SEM. Comparisons were made and differences were considered significant when P was <0.05 (see Table 1). (see reference).

[0214] [Table 1]

[0215] 3. PD-L1 antibody in MC38 / human PD-L1 knock-in (KI) tumor model Antitumor activity of the body M, engineered to replace the mouse PD-L1 gene with human PD-L1 The C-38 cells are designated MC38-hPD-L1 KI. MC38 / hPDL1 -KI tumor cells were cultured in air, 5% CO 2 Heat-inactivated 10% at 37°C under an atmosphere containing Activated fetal bovine serum (ExCell Biology), 100 U / ml penicillin, and RPMI 1640 medium supplemented with 100 μg / ml streptomycin (Hyclone) Tumors were maintained in vitro as monolayer cultures in 100% ethanol at 37 °C for 24 h (Thermo Fisher). Cells were routinely passaged twice weekly by trypsin-EDTA treatment (Hyclone). Cells growing in logarithmic growth phase were harvested and counted for tumor inoculation. Each SPF grade C57BL / 6 mouse was injected with 2×10^6 cells SC in the right flank. Approximately 10 days after inoculation, the tumor volume was approximately 100 mm^3. Forty mice were selected and randomized into five groups (see scheme in Table 2). Mice were treated with PBS, purified antibody hybridomas formulated in PBS at 1 mg / ml. 10% of the mouse antibodies 4B6, 23A11, 23F11, and 26F5 were injected IP. Treatment was continued for 4 weeks, 3 times a week from the first injection. The animals were then placed in a CO 2 The animals were sacrificed at the end of the study using inhalation. Tumor size was measured using a vernier caliper (I NSIZE) twice a week in two dimensions and the volume is calculated using the formula: V = 0.5a × b 2 (formula In the figure, a and b are the long and short diameters of the tumor, respectively) in mm 3 Results were analyzed using Prism GraphPad and expressed as mean ± SEM Comparisons between two groups were performed by T-test, with P values ​​of *<0.05 and **<0. Differences were considered significant when they were .01 (see Table 3).

[0216] [Table 2]

[0217] [Table 3]

[0218] As shown in Table 3 and Figures 11A-11C, at the end of the study, the mean tumor size in the control group was 1.0-1.2 mmHg. The tumor volume was 6.7 times larger than the volume at the starting point. 4 / 8, 6 / 8, 7 / 8 and 7 / 8 of mice treated with 25F5 and 23F11 , had no detectable tumor and could therefore be considered a complete response. Two-eight of the mice in this study had >70% tumor reduction (see FIG. 11B). Our data show that these antibodies are highly potent inhibitors of established tumor growth and in mice It has been shown to have the ability to eliminate tumors in the majority of cases.

[0219] 4.PH-dependent PD-L1 in the MC38 / human PD-L1 knock-in tumor model Antitumor activity of antibodies To evaluate whether pH-dependent antibodies have any advantage in tumor inhibition in vivo To evaluate the pH-dependent and non-pH-dependent binding of the antibody 23F11, the present inventors The activity of one of the antibodies, 4B6-C, was compared (see Figures 12A and 12B). The antibody has strong neutralizing activity and can inhibit tumors at high doses. To clarify the potential advantages, we administered the antibody at a low dose: 1 mg by IP injection. Briefly, female SPF grade C57BL / 6 mice were each administered 2× 10^6 cells (i.e., MC38-hPD-L1 KI) were injected SC in the right flank. Approximately 10 days after inoculation, 4 mice with tumor volumes of approximately 200 mm^3 were inoculated. 0 mice were selected and randomized into 5 groups. The mice were then inoculated with 1 mg / ml PBS. The purified antibodies were formulated and injected IP at 1 mg / kg body weight in PBS, 4B6 - Chimera (4B6-C), 23F11, benchmark antibody BM-GT (U.S. Patent No. 821 Also named YW243.55.S70 or MPDL3280A in issue 7149 ) and BM-ME (also known as 2.14H9OPT in U.S. Patent No. 8,779,108). Treatment was administered three times a week and continued for three weeks after the first injection. Animals were then incubated with CO 2 Mice were sacrificed at the end of the study by inhalation. Tumor size was measured using a Vernier caliper (INS The volume was measured twice a week in two dimensions using the IZE and calculated using the formula: V = 0.5a × b 2 (In the formula, a and b are the long and short diameters of the tumor, respectively) in mm 3 In table Results were analyzed using Prism GraphPad and presented as mean ± SEM. Comparisons between two groups were performed using the T-test, and differences were observed when P was <0.05. This is considered to be intentional.

[0220] The data in Figures 12A and 12B show that antibodies with pH-dependent binding properties bind These antibodies were shown to inhibit tumor growth much more potently than antibodies without the dependent binding properties. Treatment with antibody 23F11 at a dose of 1 mg / kg reduced tumors in 3 of 8 mice. The treatment resulted in complete disappearance of the tumor, with one out of eight mice showing a 70% decrease in tumor volume compared to the PBS-treated group. Treatment with the non-pH-dependent binding antibody 4B6-C had a significant reduction in tumor volume. The results were not significant, with no mice having tumors larger than their starting volume. Thus, antibodies with pH-dependent binding may be able to exert a pH-independent role in inhibiting tumor growth. The antibody may work at lower doses than the dependent binding antibody.

[0221] Antibody 18G4 in the MC38 / human-PD-L1 KI tumor model (Figure 12C and 12D) and 22C9 (see Figures 12E and 12F) tumor growth The inhibition of MC38 / hPD-L1 was also examined when delivered intravenously. Tumor cells were inoculated into C57 / BL6 mice as before and allowed to grow for 6 days. At this point, it is about 120 mm 3 Select mice with an average tumor volume of 10 ... Each test antibody was administered at 1 or 3 mg / kg in equal volumes of hybridoma cells. Purified antibodies 18G4 and 22C9 were purified from cultures at day 0 and subsequently up to 25 days. Individual mice were dosed IV on day 1. The animals were then dosed IV with CO 2 The animals were sacrificed at the end of the study using inhalation. Tumor size was measured twice weekly in two dimensions using calipers (INSIZE) and volumetric V = 0.5a × b 2 (where a and b are the long and short diameters of the tumor, respectively) (diameter) in mm 3 (See Table 4 and Figures 12C to 12F) Tumor volume was < 100% of the tumor volume at the start of treatment without decreasing below measurable size. A 50% reduction in tumor mass was considered partial (PR) and a tumor mass that was no longer palpable was considered partial (PR). The patient recorded tumor regression as complete (CR) (see Table 5). Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparisons between two groups were performed using the T-test, and differences were considered significant when P was <0.05. The data show that at doses of 1 or 3 mg / kg, 18G4 (Tables 4 and 5 and Figures 12C and 12D) and 22C9 (Tables 4 and 5 and Figures 12E and 12F) were highly active in inhibiting tumor growth under the same conditions. It was shown that.

[0222] [Table 4]

[0223] [Table 5]

[0224] 5. Chimeric pH-dependent P2T3 expression in the MC38 / human-PD-L1 knock-in tumor model Antitumor activity of D-L1 antibody Antibodies with pH-dependent antigen-binding properties do not have pH-dependent binding in vivo Furthermore, it has been shown that the IL-16 / ... inhibitor inhibits tumor growth better than the IL-16 / IL-16 inhibitor, eliminating the potential effect of the Fc domain. To verify this, the present inventors performed a cellular marker analysis in HEK293 cells (i.e., BM-GT). Produced with the same IgG isotype as the benchmark antibody (i.e., human IgG1) The chimeric antibodies (i.e., 23A11-C and 23F11-C) were retested.

[0225] Briefly, MC38 / hPD-L1 KI cells were inoculated and tumors were grown to a volume of 100 mm 3 When the antibody was administered, treatment with the antibody was started by IP injection at 1 mg / kg, and the antibody was administered 3 times a week. The results were analyzed using Prism GraphPad and the mean The results are expressed as ±SEM. Comparisons between two groups (test vs. vehicle) were performed by T-test with P = 0.05. Differences were considered significant when <0.05.

[0226] As shown in Figures 13A and 13B, the 1 mpk chimeric pH-dependent antibody (i.e. , 23A11-C, and 23F11-C) Tumor volume in the mouse tumor model was significantly increased with the administration of 1 mpk of the benchmark antibody (i.e., BM-GT ) was smaller than that of

[0227] [Example 15] Generation and characterization of humanized antibodies 1. Generation, expression and purification of humanized antibodies The sequences of the variable domains of the mouse antibodies 4B6, 23A11 and 23F11 were used to Germline sequences with the highest homology to the mouse framework were identified. Using TA modeling, humanized variants were created using CDR grafting and back mutations. was designed.

[0228] 4B6 For CDR grafting, human germline framework for the light chain, The sequences VK / 1D-13 and for the heavy chain VH / 1-2 were used.

[0229] Direct grafting of the three CDRs into the germline sequence (SEQ ID NO: 87) and HC variant 3 (SEQ ID NO: 65) K12V, T28V, V68A, R72V, T 74K, S77R and T28V, R72V, S7 Backmutation of 7R gave rise to heavy chain (HC) variants 3 and 4 (see FIG. 14A). and).

[0230] Germline sequence of 4B6 HC: VH / 1-2 (4B6-germline, SEQ ID NO:87): QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQA PGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAY MELSRLRSDDTAVYYCAR

[0231] VH / 1-2 variant 3 (4B6-Hzd-HC-V3, SEQ ID NO: 65): QVQLVQSGAEVVKPGASVKISKASGYVFTDYYMNWVKQA PGQGLEWIGDINPNNGGTSYNQKFQGRATVTVDKSTRTAY MELSRLRSDDTAVYYCVKWGDGPFAYWGQGTLVTVSS

[0232] VH / 1-2 variant 4 (4B6-HC-V4, SEQ ID NO:67): QVQLVQSGAEVKKPGASVKVSCKASGYVFTDYYMNWVRQA PGQGLEWIGDINPNNGGTSYNQKFQGRVTVTVDTSIRTAY MELSRLRSDDTAVYYCVKWGDGPFAYWGQGTLVTVSS

[0233] Direct grafting of the three CDRs into the germline sequence (SEQ ID NO: 88) and LC variant 3 (SEQ ID NO: 66) L4M, P8Q, L78M, Y87F and L The back mutations L4M, Y87F in C variant 4 (SEQ ID NO: 68) resulted in a light chain (LC) variant Diants 3 and 4 were obtained (see Figure 14B).

[0234] Germline sequence of 4B6 LC: VK / 1D-13 (4B6 LC germline, SEQ ID NO:88) DIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKP GKAPKLLIYDASSLESGVPSRFSGSGSGTFTLTISSLQP EDFATYYCQQFNNYP

[0235] VK / 1D-13 variant 3 (4B6-Hzd-LC-V3, SEQ ID NO:66) DIQMTQSQSSLSASVGDRVTITCQASQNVGAAVAWYQQKP GKAPKLLIYSASNRYTGVPSRFSGSGSGTDFTLTISSMQP EDIATYFCQQYSNYPTFGSGTKLGIK

[0236] VK / 1D-13 variant 4 (4B6-LC-V4, SEQ ID NO:68) DIQMTQSPSSLSASVGDRVTITCQASQNVGAAVAWYQQKP GKAPKLLIYSASNLYTGVPSRFSGSGSGTDFLTISSLQP EDIATYFCQQYSNYPTFGSGTKLGIK

[0237] 23A11 For CDR grafting, human germline framework for the heavy chain, The sequences VH / 1-2 and for the light chain VK / 7-3 were used.

[0238] Direct grafting of the three CDRs into the germline sequence (SEQ ID NO: 89) and HC variant 3 (SEQ ID NO: 71) V20L, M48I, V68A, M70L, R 72V, T75K, R87T and HC variant 5 (SEQ ID NO: 189, SEQ ID NO: 69 The first amino acid L is converted to Q in the M48I, M70L, and R72V reverse mutations. Thus, heavy chain (HC) variants 3 and 5 were obtained (see FIG. 15A).

[0239] Germline sequence of 23A11 HC: VH / 1-2 (23A11-HC-germline, SEQ ID NO: 89): QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQA PGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAY MELSRLRSDDTAVYYCAR

[0240] VH / 1-2 variant 3 (23A11-HC-V3, SEQ ID NO:61): QVQLVQSGAEVKKPGASVKLSCKASGYIFTTYWIHWVKQR PGQGLEWIGMIQPNSGGTKYNDQFKNRATLTVDKSISTAY MELSRLTSDDTAVYYCARGAGTVDYFDYWGQGTTLSISS

[0241] VH / 1-2 variant 5 (23A11-HC-V5, SEQ ID NO: 189): QVQLVQSGAEVKKPGASVKVSCKASGYIFTTYWIHWVRQA PGQGLEWIGMIQPNSGGTKYNDQFKNRVTLTVDTSISTAY MELSRLRSDDTAVYYCARGAGTVDYFDYWGQGTTLSISS

[0242] Direct grafting of the three CDRs into the germline sequence (SEQ ID NO: 90) and HC variant 3 (SEQ ID NO: 72) P15V, P47S, Q54R, V62I, G 72R, N89T, L93H and P15V, P4 of HC variant 5 (SEQ ID NO: 70) The back mutations 7A, Q54R, V62I, G72R, N89T, and L93Q result in light chain variants. Ants 3 and 5 were obtained (Figure 15B).

[0243] Germline sequence of 23A11 LC VK / 7-3 (23A11-LC-germline, SEQ ID NO: 90): DIVLTQSPASLAVSPGQRATICRASESVSELGINLIHWY QQKPGQPPKLLIYQASNKDTGVPARFSGSGSGTDFTLTIN PVEANDTANYYCLQSKNFP

[0244] VK / 7-3 variant 3 (23A11-LC-V3, SEQ ID NO:72): DIVLTQSPASLAVSVGQRATITCRASESVDIYGNSFMHWY QQKPGQSPKLLIYRASNLESGIPARFSGSGSRTDFLTIN PVEANDTATYYCHQSNDDPYTFGGGTKLETK

[0245] VK / 7-3 variant 5 (23A11-LC-V5, SEQ ID NO:70): DIVLTQSPASLAVSVGQRATITCRASESVDIYGNSFMHWY QQKPGQAPKLLIYRASNLESGIPARFSGSGSRTDFLTIN PVEANDTATYYCQQSNDDPYTFGGGTKLETK

[0246] 23F11 For CDR grafting, human germline framework for the heavy chain, The sequences VH / 1-2 and for the light chain VK / 7-3 were used.

[0247] Direct grafting of the three CDRs into the germline sequence (SEQ ID NO: 91) and HC variant 4 (SEQ ID NO: 75) V20L, M48I, R67K, V68A, M 70L, R72V, T74K, R87T and M4 of HC variant 6 (SEQ ID NO: 77) The reverse mutations 8I, R67K, V68A, M70L, and R72V cause heavy chain (HC) barrier Compounds 4 and 6 were obtained (see FIG. 16A).

[0248] Germline sequence of 23F11 HC: VH / 1-2 (23F11 germline HC, SEQ ID NO: 91): VQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAP GQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYM ELSRLRSDDTAVYYCAR

[0249] VH / 1-2 variant 4 (23F11-HC-V4, SEQ ID NO:75): QVQLVQSGAEVKKPGASVKLSCKASGYIFTTYWMHWVKQR PGQGLEWIGMIQPNSGGTKYNEKFKKKATLTVDKSISTAY MELSRLTSDDTAVYYCARGAGTVDYFDYWGQGSTLTVSS

[0250] VH / 1-2 variant 6 (23F11-HC-V6, SEQ ID NO:77): QVQLVQSGAEVKKPGASVKVSCKASGYIFTTYWMHWVRQA PGQGLEWIGMIQPNSGGTKYNEKFKKKATLTVDTSISTAY MELSRLRSDDTAVYYCARGAGTVDYFDYWGQGSTLTVSS

[0251] Direct grafting of the three CDRs into the germline sequence (SEQ ID NO: 92) and LC variant 4 (SEQ ID NO: 76) P15V, Q54R, V62I, N89T, L 93Q and P15V, Q54R, V62I, N8 of LC variant 6 (SEQ ID NO: 78) Back mutation of 9T, L93H gave rise to light chain variants 4 and 6 (see FIG. 16B). thing).

[0252] Germline sequence of 23F11 LC VK / 7-3 (23F11 germline LC, SEQ ID NO: 92): DIVLTQSPASLAVSPGQRATICRASESVSELGINLIHWY QQKPGQPPKLLIYQASNKDTGVPARFSGSGSGTDFTLTIN PVEANDTANYYCLQSKNFP

[0253] VK / 7-3 variant 4 (23F11-LC-V4, SEQ ID NO:76): DIVLTQSPASLAVSVGQRATITCRASESVDIYGNSFMHWY QQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFLTIN PVEANDTATYYCQQSTEDPYTFGGGTKLEIK

[0254] VK / 7-3 variant 6 (23F11-LC-V6, SEQ ID NO:78): DIVLTQSPASLAVSVGQRATITCRASESVDIYGNSFMHWY QQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFLTIN PVEANDTATYYCHQSTEDPYTFGGGTKLEIK

[0255] The heavy and light chain cDNAs described above were synthesized and the effector genes of the Fc region were expressed as known in the art. Human I IgG1 with the N297A mutation in the Fc region to reduce or minimize inhibitor function The heavy chain residue numbering described herein is the same as that of the cover According to the EU index of protein synthesis (Kabat et al., "Proteins and f Immunological Interest”, US Dept. of H (See Health & Human Services (1983)). The heavy and light chain variable regions of the antibody gene were synthesized and cloned into an expression vector. , PureYield™ Plasmid Maxiprep system from Promega Large scale DNA preparation was performed using ExpiFectamini (Invitrogen). Transfection was performed using ne™ 293 reagent according to the manufacturer's protocol. The supernatant was harvested when cell viability was approximately 50%. Protein A The beads and the cleaned supernatant were incubated with shaking at 4° C. for 2 hours, after which The Protein A beads inside the column were washed with PBS and then washed for 100 ml. The antibody was eluted using M glycine buffer (pH 3.0) and then diluted with PBS buffer. -(137mM NaCl, 2.7mM KCl 10mM Na 2 HPO 4 , 2 mM KH 2 PO 4The solution was dialyzed overnight at 4°C against Pierce Hi gh Capacity Endotoxin Removal Resin(Invi Endotoxins were removed using SDS-PAG. The purified antibodies were characterized by E and SEC-HPLC.

[0256] 2. Binding of humanized antibodies to hPD-L1 in ELISA High-binding clear polystyrene 96-well plates (Nunc) were filled with 100 μl / well of 0.5μg / ml human PD-L1-his(Acrin) in high pH coating buffer Obiosystems, Catalog No. PD1-H5229) The plate was then incubated overnight at 4° C. The plate was then washed with washing buffer (PBS+0.1 % Tween 20 (Sigma)) and washed once using an automatic plate washer. 200 μl of blocking buffer (PBS + 1% BSA + 1% normal goat serum + 0. 05% Tween-20) was added to each well and incubated at room temperature for 2 hours. Dilution buffer (PBS + 1% BSA + 1% normal goat serum + 0.01% Tween- 20), serially diluted humanized antibodies (produced in 293 cells) were added to the chamber. The plate was then washed three times with washing buffer. 100 μl / well of HRP-conjugated goat anti- antibody in dilution buffer. A solution of human Fc antibody (Abcam) was added to each well of the plate. The SA plates were allowed to incubate at room temperature for 60 minutes and then the plates were reconstituted with 200 μl The plate was washed three times with 100 μl / well of pH 7.4 wash buffer. Add 0.64 M TMB to each well. 2 SO 4 Terminate the reaction using Plates were read on a Thermo Multiscan FC at 450 nM ( See Figures 17A and 17B).

[0257] 3. Blockade of hPD-1 binding to hPD-L1 in ELISA High-binding clear polystyrene 96-well plates (Nunc) were filled with 100 μl / well of Coat the plates overnight at 4°C using a coating solution consisting of 1 μg / ml hPD-L1-Fc. The plate was then washed with washing buffer (PBS + 0.1% Tween The plate was washed once with 200 μl of bromine. Mixture of PBS + 1% BSA + 1% normal goat serum + 0.05% Tween n-20) was added to each well and incubated at room temperature for 2 hours. The medium used was PBS + 1% BSA + 1% normal goat serum + 0.01% Tween-20. Serially diluted antibodies (i.e., humanized 4B6, 23F produced in 293 cells) were 11, 23A11, and the benchmark antibody BM- GT and BM-ME) were added and incubated at room temperature for 1 hour. The plate was then The plate was washed three times using the method described above. 1 μg / ml biotinylated hPD-1-N297 A (Acrobiosystem) was added and incubated at room temperature for 1 hour. After washing the plate three times, 100 μl / well of 1:5000 diluted PBS in dilution buffer was added. A solution of HRP-conjugated neutravidin antibody (Pierce) was added to the plate. The ELISA plate was then incubated at room temperature for 60 minutes. The plate was then washed three times with 250 μl / well of wash buffer. Finally, 100 μl / well of TMB was added to each well, and 0.64MH 2 SO 4 The reaction was terminated using a Thermo Multisc an FC (see Figures 18A to 18D).

[0258] The data in Figure 16 show that some of the humanized antibodies inhibited the binding of human PD-1 to human PD-L1. It was shown to be significantly more potent than the others in terms of harm.

[0259] 4. hPD-L1 antibody with hPD-L1 on HCC827 measured by FACS Combination of Log-phase IFN-γ stimulated (500 U / ml, 2 days) HCC827 cells were collected and cultured. The cells were resuspended in rocking buffer (5% BSA + PBS). 2 × 10^5 cells were The tube was then washed once with PBS (1500 rpm, 5 min, room temperature). 100μl / tube of serially diluted humanized PD-L1 in blocking buffer Antibodies (produced in 293 cells) were added to the corresponding tubes and incubated at 4°C for 1 hour. The cells were then washed twice with 1 ml PBS, followed by 100 μl / tube of secondary antibody (1:200 rabbit anti-human Ig G-PE) was added. The cells were incubated at 4°C for 1 hour and then washed with PBS. After washing twice, the cells were resuspended in 150 μl of PBS for each sample. Then, the cells were transferred to FACS tubes and analyzed by flow cytometry (ACEA Biosciences). The antibody binding to the cells was detected using a fusion protein (ELISA) (see FIG. 19). thing).

[0260] 5. hPD-1 binding to hPD-L1 on HCC827 cells as measured by FACS Blocking the connection Log-phase IFN-γ stimulated (500 U / ml, 2 days) HCC827 cells were collected and cultured. The cells were resuspended in rocking buffer (5% BSA + PBS). 2 × 10^5 cells were The tube was then washed once with PBS (1500 rpm, 5 min, room temperature). For small-scale production in the following experiments, 100 μl / tube of blocking buffer was used. Humanized PD-L1 antibodies serially diluted in buffer (antibodies expressed in 293 cells were The one produced in CHO cells was named "old" and the one produced in CHO cells was named "new". , 4B6-H3L4, 4B6-H4L3, 23A11-H3L5 293 and 23F1 1 H4L4 293 is produced in 293 cells, 23A11-H3L5 CHO and 23F11 H4L4 CHO were produced in CHO cells) with the corresponding The tube was then incubated for 1 hour at 4° C. The cells were then diluted with 1 ml of PBS. followed by 2 washes with 100 μl / tube of 3 μg / ml biotinylated hP D-1-N297A was added and incubated at 4°C for 1 hour. The cells were washed three times with PBS. After washing, 100 μl / tube of streptavidin- PE 1:200 was added to each tube. The cells were then incubated for 1 hour at 4°C. The cells were then washed twice with PBS, and then 150 μl of each sample was added. The cells were then transferred to FACS tubes and analyzed by flow cytometry. The antibody binding to cells was confirmed using ACEA Bioscience Novocyte. The combination was detected (see Figures 20A and 20B).

[0261] [Example 16] Purified humanized IgG1 that stimulates T cell activation in the mixed lymphocyte reaction (MLR) Assessment of antibody potency Anti-PD-L1 antibodies in reducing inhibition of PD-1 / PD-L1-mediated T cell activation To assess the efficacy, we performed a mixed lymphocyte reaction (MLR) assay. In the first experiment, DCs were cultured from adherent peripheral blood mononuclear cells (PBMCs) isolated from fresh human blood. PBMCs were cultured in serum-free medium at a density of 2×10^6 cells / ml in six wells. After 2 hours, the suspension cells were discarded and the adherent cells were plated in 10 The cells were treated with 30 ng / ml GM-CSF and 30 ng / ml IL-4. The medium was replaced every day, and 20 ng / ml of TNFα was added on the sixth day to mature DC cells. On day 7, anti-CD11c-PE (Biolegend) and anti-HLA-DR-APC ( The phenotype of DCs was detected by FACS using a CD4 + Fresh blood from a second donor was used to enrich for T cells (Stem Cell Inc.). CD4 from the fluid + T cells were isolated. DCs were treated with mitomycin C (10 μg / ml for 2 h). After treatment with + T cells and PD-L1 antibody were administered to DC 1: CD4 + Add 200 μl / well at a T ratio of 10 and incubate at 37°C in 5% CO 2 Incubus The cells were incubated in a 100% ethanol solution for 5 days. The IL-2 and IFN-γ in the supernatant were then L-2 duo-set (R&D) and IFN-γ detection kit (Peprot The proliferation of T cells was detected using a ELISA kit (see Figures 21A and 21B). CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega) was used to measure cell viability. It was determined.

[0262] In the second experiment, a 1:1 volume of Ficoll solution was added, followed by centrifugation for 30 minutes. After the injection, peripheral blood was collected and PBMCs were isolated. Monocytes and T cells were isolated using CD4-negative and CD4-positive magnetic beads. The monocytes obtained above were incubated with 500 U / ml IL-4 (R&D) and 250 U / ml GM -CSF (R&D) for 7 days, followed by 100 ng / ml TNFα (R &D) was added and the culture was continued for another 3 days to allow differentiation. Count the DC cells and culture 10,000 DC cells in a volume of 100 μl per well. , mixed with 100,000 T cells in a volume of 50 μl, and then the resulting volume of 50 μl The antibodies or control IgG were added, and human PD-L1 (1 μg / ml final concentration) was also added. The supernatants were collected and analyzed by ELISA (R&D ) was used to quantify the concentrations of IL-2 and IFNγ (see Figures 21C and 21D). thing).

[0263] Example 17: Evaluation of the binding affinity of selected humanized anti-PD-L1 antibodies by Biacore CM5 sensor chips were loaded with freshly prepared 1:1 50 mM N-hydroxysuccinimide Imide (NHS): 200 mM 1-ethyl-3-(3-dimethylaminopropyl)- Activation was performed in each flow cell by a 7 min injection of EDCA (10 μl / min). Then, the mixture was diluted to a concentration of 10 μg / ml in 10 mM sodium acetate buffer, pH 5.0. Anti-human Fc antibody (GE Healthcare) was injected onto the activated chip at 10 μl / min. The running buffer was 10 mM HEPES, 150 mM HBS-EP. M NaCl, 3.4mM EDTA, 0.005% surfactant P20, pH 7.4). Remaining active coupling sites were depleted by a 7 min injection of 1 M ethanolamine at 10 μl / min. The immobilization level of each flow cell was approximately 9000 RU. Anti-human Fc IgG (GE Healthcare) was used to measure the antibody concentration. 0 RU. FC1 was used as the reference cell. After antibody capture, antigen was added at various concentrations. The association time of the antibody-bound antigen was 180 seconds. The surface regeneration conditions were Gly p H1.5 at 10 μl / min for 120 seconds. The signals of the captured antibodies were analyzed using the Biacore X100 evaluation software. Calculations were performed using Biacore 2.0.

[0264] [Table 6]

[0265] The interaction between human PD-1 and hPD-L1 is weak, with a K of approximately 7500 + / - 2200 nM. D (N=6, Cheng X et al., Struc ture and interactions of the human progr. ammed cell death 1 receptor, J Biol Chem 2013;288(17):11771-85). Therefore, the present invention shown in Table 6 Our antibody binds to hPD-L1 with approximately 100-fold higher affinity than prior art PD-1. It has sexuality.

[0266] Example 18: Evaluation of pH-dependent binding of selected PD-L1 neutralizing humanized antibodies High-binding clear polystyrene 96-well plates were filled with 100 μl / well of high pH coated The cells were coated with PD-L1-his at 0.5μg / ml in washing buffer. , and incubated overnight at 4° C. The coated plates were then washed with PBS+0. The plates were washed once with 1% Tween 20 (Sigma) using an automatic plate washer. Next, 200 μl of blocking buffer was added to each well and the plate was then The mixture was incubated at room temperature for 2 hours. After aspirating the blocking buffer, the plate was filled with 100 mL of 1000 mM NaCl. Serially diluted antibodies (i.e., antibodies produced by CHO or 293 cells) in dilution buffer The plate was incubated at room temperature for 40 minutes. The plates were then washed with 250 μl / well of pH 7.4 wash buffer. The plate was washed once with buffer and then 100 μl / well of pH 7.4 or pH 5 0.5 Wash Buffer was added and incubation was continued for 2 hours at room temperature with shaking. The plate was then washed three times with pH 7.4 wash buffer and 100 μl 1 / well of HRP-conjugated goat anti-human IgG in pH 7.4 dilution buffer. A solution of anti-Fc antibody (Abcam) was added to each well of the plate. The plate was incubated at room temperature for 60 minutes, followed by 200 μl / well of pH 7.4 wash buffer. Finally, 100 μl / well of TMB was added to each well. And 0.64MH 2 SO 4 The reaction was terminated using 450 nM Th The data were read using ermo Multiscan FC (see Figures 22A to 22E). .

[0267] Characterization of pH-dependent hPD-L1 binding across a wide range of pH The pH-dependent binding affinity was measured at pH 4, pH 4.5, pH 5.0, pH 5.5, and pH 6.0. The ELISA assay was performed according to the above method. A high-binding transparent polystyrene 96-well plate was filled with 100 μl / well of hPD-L1-his at 0.5μg / ml in high pH coating buffer. The plates were then coated with 100% ethanol and incubated overnight at 4°C. , automated plate washing with PBS + 0.1% Tween 20 (Amresco) The plate was washed three times with shaking for 10 seconds in a shaking machine. Then, 200 μl of blocking buffer was added. (1% BSA (Solarbio) + 1% normal goat serum (Solarbio) + 0.0 5% Tween 20 (Amresco) was added to each well and the plate was then The plates were incubated at room temperature for 2 hours and washed three times as above. Blocking buffer After aspirating the lysate, 100 μl of dilution buffer (pH 7.4, 1% BSA (Sola rbio) + 1% normal goat serum (Solarbio) + 0.01% Tween 20 (Amresco)) in serial dilutions of antibodies (i.e., 2 3A11 H3L5, 23F11 H4L4 and 22C9-C) were added to the plate. Incubated at room temperature for 40 minutes. Plates were then washed with 250 μl / well of pH 7 .4, pH6.0, pH5.5, pH5.0, pH4.5 or pH4.0 cleaning buffer Wash once with a wash filter, then add 100 μl / well of pH 7.4 or pH 5.5 wash solution. Wash buffer was added and incubation was continued for 2 hours at room temperature with shaking. Afterwards, the plate was washed three times with pH 7.4 wash buffer and 100 μl / well of pH 7.4 HRP-conjugated goat anti-hIgG HRP antibody (1 A solution of 1:20,000 (Abcam) was added to each well of the plate. The plates were incubated at room temperature for 60 minutes, followed by addition of 250 μl / well of pH 7.4 The plates were washed three times with washing buffer. Finally, 100 μl / well of TMB was added to each well. for 15 min, and the reaction was terminated using 50 μl / well of 0.16 M / L sulfuric acid. Plates were read on a Thermo Multiscan FC at 450 nM (Figure 1). 23A-23F). Measurements were performed in triplicate.

[0268] For each antibody, the binding signal at low pH was lower relative to that at pH 7.4. The lower the pH, the lower the binding signal for a given concentration of antibody. At pH 5, high concentrations, e.g. For example, the binding signals of the three antibodies tested at 0.1, 0.3 or 1 μg / ml varied with pH At pH 5.5, the signal was less than 50% of that at 7.4, and the signal at pH 5.5 was significantly higher for the three antibodies tested. Gunal is less than 75% of that at pH 7.4.

[0269] Example 18: Epitope binning of selected anti-PD-L1 by alanine scanning 1. Generation of mutant human PD-L1 recombinant proteins cD of extracellular human PD-L1 (amino acids 19-238) and the Fc fragment of human IgG1 The NA coding was synthesized in vitro (SEQ ID NO: 107 is the amino acid sequence and SEQ ID NO: 108 is the corresponding DNA sequence). Variants of human PD-L1 with single amino acid changes at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, By such overlap PCR, the primers shown in Table 7 (Dr. Oligo The resulting fragments were amplified using a PCR kit (BLP-192, Biolytic). , using restriction enzymes Hind III and BamH I at the 5' and 3' ends. The PCR products were then digested with Syno Assembly Mix Reagent (Synbio). , and transformed into pcDNA3.1(+ The plasmid was cloned into the purified QIAGEN plasmid It was a Mega kit (QIAGEN).

[0270] Amino acid sequence (SEQ ID NO:107): METDTLLLWVLLLWVPGSTGFTVTVPKDLYVVEYGSNMTI ECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQH SSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISY GGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAE GYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLR INTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER ENLYFQGAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK

[0271] DNA sequence (SEQ ID NO: 108): AAGCTTgccgccaccATGGAAACCGACACTCTGCTGCTGT GGGTGCTGCTGCTGTGGGTGCCAGGGTCAACCGGGTTCAC CGTGACAGTGCCCAAGGACCTGTACGTGGTGGAGTACGGC AGCAACATGACCATCGAGTGCAAGTTCCCCGTGGAGAAGC AGCTGGATCTGGCCGCCCTGATCGTGTATTGGGAGATGGA GGACAAGAACATCATCCAGTTCGTGCACGGCGAAGAGGAC CTGAAGGTGCAGCACAGCAGCTACAGGCAGAGGGCCAGAC TGCTGAAGGACCAGCTGTCTCTGGGAAACGCAGCTCTGCA GATCACCGACGTGAAGCTGCAGGACGCAGGAGTCTACCGC TGCATGATCAGCTACGGCGGAGCCGACTACAAGAGGATCA CCGTGAAGGTCAACGCCCCCTACAACAAGATCAACCAGAG AATCCTGGTGGTGGACCCCGTGACCAGCGAGCACGAGCTG ACTTGTCAGGCAGAGGGCTACCCCAAGGCCGAAGTGATTT GGACCAGCAGCGACCATCAGGTGCTGAGCGGAAAGACCAC CACCACCAACAGCAAGCGGGAGGAGAAGCTGTTCAACGTG ACCAGCACCCTGCGGATCAACACCACCACCAACGAGATCT TCTACTGCACCTTCCGGAGACTGGACCCAGAGGAGAACCA CACAGCCGAGCTGGTCATCCCAGAACTGCCTCTGGCTCAC CCTCCTAACGAGAGAGAGAATCTGTATTTTCAGGGAGCAC CAGAACTGCTGGGAGGACCATCCGTGTTCCTGTTTCCACC CAAACCTAAGGACACCCTGATGATTAGCAGAACACCAGAA GTCACTTGCGTGGTCGTGGACGTGTCCCACGAGGACCCCG AAGTCAAATTCAACTGGTACGTGGATGGCGTCGAGGTGCA TAATGCTAAGACCAAACCAAGAGAGGAACAGTACAACAGC ACCTATAGGGTCGTGTCCGTCCTGACAGTGCTGCACCAGG ACTGGCTGAACGGAAAGGAGTATAAATGCAAGGTGTCTAA CAAGGCCCTGCCAGCTCCCATCGAGAAGACTATTAGTAAA GCTAAGGGCCAGCCCCGCGAACCTCAGGTGTACACCCTGC CTCCATCCCGAGACGAGCTGACCAAGAACCAGGTCTCTCT GACTTGTCTGGTGAAGGGATTCTATCCATCAGATATCGCA GTGGAGTGGGAAAGCAATGGCCAGCCCGAGAACAATTACA AGACTACCCCCCCTGTGCTGGACTCCGATGGCTCTTTCTT TCTGTATTCTAAACTGACCGTGGATAAGAGTCGGTGGCAG CAGGGGAATGTCTTTTCATGCAGCGTGATGCACGAGGCAC TGCACAATCATTACACTCAGAAGTCCCTGTCACTGTCACC TGGAAAGtagGGATCC

[0272] The wild-type PD-L1 plasmid (SEQ ID NO: 107 and 108) prepared above was used as a template. The two segments of the integrated sequence were then amplified using megaprimers (Table 7). The fragments were then sequenced to identify variants. After screening individual positive colonies by PCR, the products were transformed into pcDNA3.1(+ ) vector and found that the PD-L1 mutants were successfully created. The PCR procedures and conditions were as follows:

[0273] Step 1: To generate the two mega-fragments of the variant μl ddH2O 35 5x S15 PCR Buffer 10 10mM dNTP 1 F primer 1 R primer 1 PCR product 1 S15 polymerase 1 --------------------------------- 50 First denaturation: 98℃ 1 min Denaturation: 98℃ 15 seconds Annealing: 58℃ 30 seconds Extension: 72°C 30 sec per Kb Final extension: 72°C 3 min, 30 cycles Step 2: Join the two pieces together Set up the following reaction on ice (homologous recombination): Syno assembly mix 10μl Sequence product 1 2 μl Sequence product 2 2 μl Deionized H2O 6 μl -------------------------------- Total volume 20μl

[0274] [Table 7-1] [Table 7-2]

[0275] The other 20 variants are being developed by another company, Genewiz (Suzhou, China) a) was constructed by Synbio to synthesize PD-L1 variants. A similar method is carried out using Tech. Also, the megaprimer used for the mutant site is The results are listed in Table 8.

[0276] [Table 8-1] [Table 8-2]

[0277] These plasmids of mutant and wild-type PD-L1 were then transfected into 293T (ATCC (R) CRL3216) cell line. First, 5×10 6 pcs 293T cells were seeded in a 60 mm dish, and the primary ratio was Then, add 10 μg DNA to 400 μl of Add 10 μl of 25 μl of 1x HBS to the above mixture and incubate for approximately 5 min. 100 kDa linear PEI transfection reagent (1 mg / ml dissolved in 1x HBS) Stock solution) to ensure a DNA / PEI ratio of 1:2.5. The mixture was added dropwise to a 293T dish. After about 6 to 8 hours, the medium was changed and After 72 hours, the cells were filtered using a 0.22 μm filter. The culture supernatant was collected and then stored at -80°C for use.

[0278] 2. PD-L1 Antibody Binding by ELISA Supernatants were used to assay PD-L1 antibody binding by ELISA as described below. Detected.

[0279] 2.1 Mouse Ab: High-binding clear polystyrene 96-well plates (Nunc) were filled with 100 μl / well of Coating consisted of 0.5 μg / ml anti-human Fc antibody (Abcam) in high pH buffer. The plates were then coated with the washing solution at room temperature for 1 hour. Automated plate washing was performed using a PBS + 0.1% Tween 20 (Sigma) The plate was washed once with a purifier, followed by 200 μl of blocking buffer (PBS + 1% BS A + 1% normal goat serum + 0.05% Tween-20) was added to each well. The plates were incubated overnight at 4°C. The plates were then washed once with washing buffer. Wash and incubate 160 μl of various mutant human PD-L1-Fc proteins or DMEM DMEM supernatant containing 500ng / ml wild-type human PD-L1-Fc protein , was added to each well and incubated at room temperature for 1 hour. The plate was then washed with the above method. The plate was washed three times using 0.5μg / ml mouse anti-PD-L1 antibody and incubated at room temperature for 12 h. Incubated for 1 hour. Plates were washed with 200 μl / well of pH 7.4 wash buffer. After washing three times using a centrifuge, 100 μl / well of HR diluted in dilution buffer was added. A solution of P-conjugated mouse IgG antibody (1:20000, Pierce) The ELISA plate was then incubated at room temperature for 60 minutes. Incubate and then wash three times with 200 μl / well of pH 7.4 wash buffer. Finally, 100 μl / well of TMB was added to each well, and 0.64 M H 2 SO 4 The reaction was terminated using Thermo Mul The results were read on a tiscan FC (see FIG. 24).

[0280] 2.2 For Abs with human Fc (chimeric or humanized antibodies): High-binding clear polystyrene 96-well plates (Nunc) were filled with 100 μl / well of Antibodies that can be paired with other PD-L1 antibodies at 0.5 μg / ml in high pH buffer A coating solution consisting of human Fc (for mouse antibodies) or anti-hPD-L1 antibody The plate was then washed with washing buffer (PBS+ Wash once with 0.1% Tween 20 (Sigma) in an automated plate washer. Then, 200 μl of blocking buffer (PBS + 1% BSA + 1% normal Allantoin serum + 0.05% Tween-20) was added to each well. The plate was incubated at 4°C overnight. The plate was then washed once with washing buffer and incubated for 160 μl. l of various mutant human PD-L1-Fc proteins or 500 ng / ml in DMEM 1 of DMEM supernatant containing wild-type human PD-L1-Fc protein was added to each well. The plates were then washed three times using the method described above. The cells were purified with 0.5 μg / ml of the biotinylated humanized anti-PD-L1 antibody (23 F11-H4L4-bio and 23A11-H3L5-bio) or PD-1 protein The plate was then filled with 200 μl / well of protein and incubated at room temperature for 1 hour. After washing three times with H7.4 wash buffer, 100 μl / well of Anti-mouse Fc antibody conjugated with HRP (Abcam) diluted in dilution buffer ) or HRP-conjugated neutravidin (1:5000, Pierce The solution of e) was added to each well of the plate. Then, the ELISA plate was incubated at room temperature. Incubate for 60 min, then add 200 μl / well of pH 7.4 wash buffer. Finally, 100 μl / well of TMB was added to each well. 0.64MH 2 SO 4 The reaction was terminated using 450 nM The Read on rmo Multiscan FC (see Figure 24).

[0281] Table 9 lists the individual antibodies tested against human PD-L1 in the ELISA assay. The key residues on hPD-L1 required for each are summarized. The PD-L1 protein binding signal was significantly reduced. Amino acid residue mutations are noted.

[0282] [Table 9]

[0283] The data in Table 9 demonstrate that antibodies with pH-dependent binding properties are We showed that the enzyme utilizes many of the following amino acids:

[0284] R125 and S80 are compatible with both types of PD-L1 antibodies (e.g., pH-dependent and non-pH-dependent). The PD-1 binding domains provided herein are important for PD-1 binding to the PD-1 receptor (pH dependent). The importance of S80 for binding of the -L1 antibody has not been previously reported.

[0285] E58 and R113 are 22C9, 23A11, 23F11 and their humanized variants. For the purposes of this study, non-pH-dependent binding abs (including benchmark antibodies such as BM-ME) E58 is more important for antibodies 22C9, 23A11, and 23F11. This is a novel site discovered by the present inventors' analysis, and is involved in PD-1 binding to PD-L1. (not shown by the prior art because it is not important).

[0286] M115 and K124 are unique to 22C9, but these two residues are involved in the PD-L 1, are known to be important for PD-1 binding to PD-1.

[0287] N63 and Y81 are 23F11 and its humanized versions 23F11 and 23A1. 1 (but not 23A11), whereas they are specific to PD-L1 and PD- 1 binding and is not important for benchmark antibodies that bind hPD-L1 It is not important and is therefore a novel site.

[0288] I64 is unique to 23F11 and its humanized versions, and has been reported in the prior art It is not known to be important for PD-1 binding to PD-L1.

[0289] K62 is unique to the humanized version of 23A11 and is not associated with PD-L 1 is not known to be important for PD-1 binding to .

[0290] Y123 is the most critical for 4B6 and the benchmark antibody, but is also a pH-dependent antibody. This is less critical for other antibodies, including 22C9, another pH-dependent binding antibody. (This is also important.)

[0291] Y56 is unique to 21F11 and has been implicated in PD-1 binding to PD-L1 in the prior art. It is not known to be important for

[0292] In anti-PD-L1 antibodies, mutations at either R113 or R125 to A are This has been reported to result in loss of its binding ability to PD-L1 ( U.S. Patent No. 87791 (See No. 08), Lin DY et al., Proc Natl Acad Sci US A. 2008 Feb. 26;105(8):3011-6 As reported in the literature, they are known to be important for PD-1 binding to PD-L1. There are.

[0293] Example 19: Large-scale production of anti-PD-L1 antibodies for in vivo studies Antibodies were produced using transient expression in CHO-K1 cells. Protein A affinity The produced antibody was purified and desalted using a hydrophilic column, yielding 5 mg / mL of antibody in PBS. The resulting antibody was formulated in S Purity was characterized using DS-PAGE gels and SEC-HPLC.

[0294] Transient expression and purification of recombinant antibodies using CHO-K1 cells The heavy and light chain variable regions of the selected antibody gene are synthesized and cloned into an expression vector. The resulting expression construct was used to adapt the strain to grow in serum-free medium. CHO-K1 cells were transfected with 10L of Applikon Bioreactor. After 10 days of growth in the aquaria, the culture medium was harvested and the cells and The cellular debris was removed, and the clarified supernatant was concentrated by ultrafiltration to give prepared Protein A. (human IgG) or G-Sepharose (mouse IgG) columns. Wash with equilibration buffer to baseline under UV monitoring. Afterwards, the column was then eluted with 0.1 M citric acid, pH 3.5, and the eluted antibody was Immediately neutralize with 1.0 M Tris-HCl buffer, pH 8.0, and then resuspend in PBS, H7.2 (Invitrogen) was incubated overnight at 2–8°C with two buffer changes. The purified antibody was filtered through a 0.22 μm sterile syringe filter and diluted with -8 The solution was stored in aliquots at or below 0°C.

[0295] [Example 20] In vivo evaluation of anti-PD-L1 humanized antibodies The in vivo activity of humanized anti-PD-L1 antibodies was assessed as described in Example 14. did.

[0296] 1. Humanized PD-L1 antibody in the MC38 / human-PD-L1 knock-in tumor model Antitumor activity of the body Humanized PD-L1 antibody 4B in the MC38 / human-PD-L1 knock-in tumor model The antitumor activity of 6-H3L3-N297A was tested as described in Example 14. The results are shown in Table 10 and Figure 25 (PBS, BM-GT and 4B6-H3L3- Results of treatment with N297A were shown).

[0297] [Table 10]

[0298] 2. Inhibition of MC38 / hPD-L1 KI tumor growth using IP injection of humanized antibodies The study consisted of 1 mg / kg, 3 mg / kg, or 10 mg / kg i.p. To evaluate the efficacy of a humanized anti-PD-L1 antibody in inhibiting tumor growth when delivered Briefly, 2×10^6 MC38 / hPD-L1 tumor cells were Cells were inoculated as previously in C57 / BL6 mice and allowed to grow for 6 days. At that time, mice with an average tumor volume of 80-100 mm^3 were selected and assigned to each group. Each test antibody was administered to individual mice at 1 mg / kg, 23F1 at 3 mg / kg or 10 mg / kg (for BM-GT) or an equivalent volume Mice were dosed with 1-H4L4 (1 mg / kg) intraperitoneally, three times a week, starting on day 6. , CO 2Mice were sacrificed at the end of the study (day 25) using inhalation. Tumor size was measured using a caliper. (INSIZE) was used to measure volume twice a week in two dimensions and the volume was calculated using the formula: V = 0.5a × b 2 where a and b are the long and short diameters of the tumor, respectively. m 3 Tumor volume was measured at 0.05 mg / kg / day, without decreasing below measurable size, and was expressed as A partial reduction (PR) was defined as a reduction in tumor volume of <50% or a reduction in tumor burden that was no longer palpable. If the tumor regression was complete (CR), it was recorded (see Table 11). , analyzed using Prism GraphPad and expressed as mean ± SEM. Comparisons between two groups were performed using the T-test, and differences were considered significant when P was <0.05. FIG. 26E shows the results of the analysis of various antibodies (BM-GT, BM-ME, 23A11-H3L5, and Tumor mass measured in individual mice treated with 23F11-H4L4 and 23F11-H4L4 The RTV is the relative tumor volume compared to the tumor volume measured on day 1 in the same mouse. represents tumor volume, and TV is the tumor volume measured on the last day of the study (i.e., day 25). Represents.

[0299] [Table 11]

[0300] 3. MC38 / HPD-L1 KI Tumor Growth Using Intravenous Injection of Humanized PD-L1 Antibody Long Inhibition The study is looking into the use of humanized antibodies delivered intravenously as a potential method for administering them to patients. The study was designed to evaluate the efficacy of humanized antibodies in inhibiting tumor growth when compared to placebo. Briefly, MC38 / hPD-L1 tumor cells were cultured in C57 / BL6 mice. They were inoculated as before and allowed to grow for 10 days, at which point they reached 180–220 mm 3 Mice with a mean tumor volume of 1000 mg / kg were selected and randomized into groups with 12 mice in each group. Each test antibody was administered IV to individual mice at equal volumes of 1 mg / kg on days 0 and 15. The animals were dosed on day 1 of CO 2 Mice were sacrificed at the end of the study by inhalation. ,Measured twice a week in two dimensions using calipers (INSIZE) and volume was calculated using the formula: V = 0.5 a×b 2 where a and b are the long and short diameters of the tumor, respectively. Use mm 3 The tumor volume was expressed as 0.01 mg / kg / day. A partial reduction (PR) was defined as a reduction in tumor volume to <50% of the initial tumor volume, or a reduction in tumor burden that was palpable. If this is no longer possible, tumor regression is recorded as complete (CR) (see Table 12). Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparisons between two groups were performed using the T-test, and differences were considered significant when P was <0.05. It is thought that there is.

[0301] The data in Figure 29 shows that 23A11-H3L5 and 23F11-H4L4 were administered at 1 mg / kg dose was significantly more effective than other doses in inhibiting tumor growth under the same conditions. Figure 27A shows that 1 mg / kg of 23F11-H4L4 or 23A11-H3L5 caused 91% and 82% tumor growth inhibition, respectively; Meanwhile, 1 mg / kg of the benchmark antibody inhibited tumor growth by 41.6% versus vehicle control. Figure 29A shows that 1 mg / kg of 23F11-H4L4 or 2 mg / kg of 23F11-H4L4 caused adverse effects. 3A11-H3L5 inhibited MC38 / hPD-L1 knock-in tumor growth at 10 mg / kg The tumor-inhibiting activity of these two drugs was similar to that of BM-GT of the same type under the same conditions. This indicates that there was no statistically significant difference. The antibodies exemplified by 23A11-H3L5 and 23A11-H3L5 have a pH-independent binding to antigens. Approximately 10 times higher than the Thymark antibody BM-GT (also named MPDL-3280A). It has good in vivo activity (see Figures 29A and 29B).

[0302] [Table 12]

[0303] The change in tumor size in each mouse at the study endpoint is shown in a waterfall graph. The changes in tumor size were expressed as changes from the starting point (see Figures 28A to 28D). Mice that showed >0% increase in tumor size were designated 100%, whereas mice that showed <100% increase in tumor size were designated 100%. The reduction from the starting point was expressed as a percentage exact change.

[0304] The above results indicate that 23F11-H4L4 and 23A11-H3L5 inhibited tumor growth. was significantly more potent than the benchmark antibody BM-GT at 1 mg / kg Furthermore, the effect of 2 mg / kg on tumor growth inhibition was also shown (Figures 27 and 28). The potency of 3A11-H3L5 and 23F11-H4L4 was compared with that of BM-G at 10 mg / kg. (See Figures 29A and 29B.) Furthermore, at 10 mg / kg 23A11-H3L5 was significantly increased in mice dosed at 1 mg / kg compared to those dosed at 1 mg / kg. or 23H11-H4L4, while there was no further increase in tumor growth inhibitory activity. There is a significant increase in activity for BM-GT. Thus, it is possible to identify a mAb with pH-dependent antigen binding. The antibody may be of a novel class of antibody capable of inhibiting tumor growth at extremely low doses.

[0305] 4. Tumor Rechallenge Studies Mice that achieved complete tumor clearance with IV treatment with PD-L1 antibody showed sustained In order to understand whether it is possible to mount an immune response and control the emergence of new tumors, They administered either 1 mg / kg of 23A11-H3L5 or 23F11-H4L4. In a subset of mice that achieved a complete response when treated from days 10 to 35 with The mice were then rechallenged with fresh tumor cells. Thirty-five days after inoculation, 2×10^6 fresh tumor cells were injected. Freshly injected tumor cells Upon injection, the tumors were able to grow for the first 5-10 days, but then began to decrease in size. Therefore, by day 50 (15 days after rechallenge), no mice had any measurable tumors. This is because these mice have developed memory against the same tumor cells, and the antibodies react to them. The tumor inhibitory response demonstrates durability, even though it was not presented by et al. See Figure 30).

[0306] [Example 21] Evaluation of tumor penetration of selection-dependent neutralizing antibodies Antibodies need to penetrate into the tumor to exert their effect. pH-dependent antibodies Whether it has an advantage in penetration into the tumor and during its residence time in the tumor To assess whether there are differences in Used.

[0307] Briefly, the test antibodies BM-GT and 23F11-H4L4 were detected by near-infrared (NIR) imaging. Inferred] Fluorescent labeling dye (CF750, Catalog: 99952, Lot: 12M0413 The antibody was labeled using 0.1 mg of PBS. 0.8 ml of each antibody solution was then added to prewarmed 10x antibody buffer in a 1:9 ratio. Pipette into the reaction buffer vial so that the antibody solution is in 1x reaction buffer. The solution was mixed by pipetting up and down several times. Afterwards, all the solution was transferred from each vial to the vial containing the CF dye and the vials were held for a few seconds. Vortex and incubate the vial in the dark for 30 minutes. 0.2 ml of the antibody-dye solution was injected intravenously into each mouse.

[0308] MC38 / hPD-L1 syngeneic tumor model 2×10^6 MC38 / hPD-L1 cells were subcutaneously injected into 8-week-old C57BL / 6 mice. Twelve mice were randomized into four groups. Tumors grew to approximately 220 mm^3. At adulthood, the animals were injected intravenously with 0.2 ml of the antibody-dye solution.

[0309] 1 hour, 4 hours, 24 hours, 48 ​​hours, 96 hours, 7 days, 11 days, 15 days, 18 days after administration After anesthesia at days 1 and 21, the tumor sites of each mouse were visualized using IVIS Lumina. Briefly, each mouse was imaged using the IVIS prior to IVIS imaging at each time point. The animals were anesthetized with 50 mg / kg sodium pentobarbital. At that point, the animal was placed in the observation box (tumor side up), the door was closed, and then Each mouse was imaged using Living Image software.

[0310] Examples of radiated signals are shown for BM-GT and humanized 23F11 (i.e., 23F11 -H4L4) (see FIG. 31A). The mean emission signal from each mouse for each time point was The nulls were quantified and analyzed using GraphPad prism, and are shown in Figure 31B. The results showed that antibody 23F11-H4L4 gave a significantly higher emission signal than the benchmark antibody. Thus, significantly more of the antibody is retained in the tumor than the benchmark antibody. In response to more antibody in the tumor over time, 23F11-H4L4 Tumors were significantly reduced in two of three mice treated with BM-GT, whereas tumors were significantly reduced in two of three mice treated with BM-GT. None of the mice treated with rituximab had significant tumor reduction (see FIG. 31C). Therefore, 23F11-H4L4, an antibody with pH-dependent properties, offers unique advantages. and are more effective in inhibiting tumor growth. -2, 2-3, 4-1, 4-2 and 4-3 are the individual mice in groups 2 and 4, respectively. The cursor will indicate 1, 2 or 3.

[0311] Example 22: Combination therapy of selected humanized anti-PD-L1 antibodies with other tumor modulating agents Checkpoint inhibitors: anti-PD-L1 and anti-VEGFR2 antibodies (anti-angiogenesis) To assess whether the combination could better control tumor growth, C38 tumors were treated with DC101, an antibody with neutralizing activity against mouse VEGFR2, at 20 mg / kg. g, or PD-L1 antibody-23A11-chimera at a suboptimal dose of 0.3 mg / kg, or Briefly, 2×10^6 MC38 / hPD-L1 tumor cells were treated with either Tumor cells were inoculated as previously described in C57 / BL6 mice and allowed to grow for 3 days. At that point, the mice were randomized into groups with 8 mice in each group. Each test antibody was administered individually. Mice were treated with 0.3 mg / kg 23A11-chimera or 20 mg / kg DC101. or both, intraperitoneally, starting on day 3, three times a week for three weeks. 2 Mice were sacrificed at the end of the study by inhalation. Tumor size was measured using a vernier caliper (INSIZE). The volume was calculated by the formula: V = 0.5a × b 2 (In the formula, a and b are are the long and short diameters of the tumor, respectively) in mm 3 The results are expressed as Analyses were performed using Prism GraphPad and presented as mean ± SEM. Comparisons between the two groups were performed using the test, and differences were considered significant when P was <0.05. The data show that the combination treatment is significantly more effective in inhibiting tumor cell growth. (See Figs. 32A to 32C).

[0312] To further demonstrate that combination therapy is more effective than monotherapy, the inventors performed The tumor volume was approximately 180 mm 3 Treatment was started when the patient was 18 years of age or older. However, they were not highly sensitive to the humanized PD-L1 or VEGFR2 antibody DC101. In mice of different strains, the mice were dosed in pairs at either 1 mg / kg or 3 mg / kg. The treatments were administered intravenously once a week for 2-3 weeks. Data showed that the combination of both treatments significantly reduced tumor growth. The results showed that the IgG1-specific ... See 2E.)

[0313] Thus, the inventors have demonstrated that humanized PD-L1 antibodies have anti-angiogenic properties, such as VEGFR2 antibodies. Combination with a proliferative inhibitor for the treatment of established tumor growth or progression of newly formed tumors. This has demonstrated that it can be an effective approach.

[0314] [Example 23] Thermal stability of PD-L1 antibody Capillary cell differential scanning calorimetry (DSC) was used to measure the change in temperature of samples and The thermal stability of the protein is evaluated by detecting the thermal differentiation between the target and the reference. To measure the thermal transition midpoint (Tm), which is an indicator of the relative stability of a protein in liquid, The antibodies 23F11-H4L4 or 23A11-H3L5 (in CHO cells) The His-His-HCl buffer (20 mM His-His-HCl, 5% sucrose) was added to The solution was diluted to 1 mg / ml using 100% ethanol (pH 6.0) and added to a DSC 96-well tray. Micro with a temperature range of 10~110°C and a scan rate of 200°C / hour Cal VP-DSC Capillary Cell Microcalorimeter (GE) was used to sample The results were analyzed using MicroCal VP capillary DSC automated analysis software. The capillary cell DSC test results are shown in Table 13 below and in FIG. and 33B.

[0315] As shown in Table 13, the 23F11-H4L4 antibody showed a significantly increased Tm value. (i.e., Tm2 corresponds to the Tm of the Fab fragment, while Tm1 corresponds to the Tm of the CH2 fragment. (equivalent to).

[0316] [Table 13]

[0317] [Example 24] Epitope mapping of E58, an anti-PD-L1 antibody The importance of the E58 residue for binding of the anti-PD-L1 antibodies described herein The antibodies were assayed using an ELISA-based binding assay as described in Example 18. Either the wild-type anti-PD-L1 protein or the mutant harboring E58A were used to analyze The antibodies were produced and the relative binding of the antibodies to two proteins was carried out on the same plate. The results are shown below. The results are shown in Table 14.

[0318] [Table 14]

[0319] The data show that residue E58 is critical for 23F11 and 23F11-H4L4. When this residue is mutated to alanine in human PD-L1, the binding is reduced by 9 Similarly, the binding of 23A11-H3L5 and 22C9 was reduced by more than 0%. The binding signals of T and PD-L1 protein were reduced to 8.3% and 17.2%, respectively. Binding of BM-GT or BM-ME was significantly reduced when E58 was mutated to Ala compared with wild-type PD The binding of the antibody to 23A11, 26 The binding of F5 and 4B6 was also 51.7%, 60.6%, and 70.0% of that of the wild-type PD-L1 protein. Most notably, 18G4 binding to PD-L1 was reduced by 69.6% compared with E5 When 8 was mutated to Ala, the activity was not reduced at all. Thus, E58 has a variety of anti- Differential requirements for binding to the PD-L1 protein in the body, 23F11, 23F1 The critical residues were 1-H4L4 and 22C9.

[0320] In addition, 18G4 was the most abundant amino acid among 39 different amino acids analyzed by alanine scanning. Other anti-PD-L1 antibodies disclosed herein that require only S80 binding in These antibodies have very different epitope binding profiles for the body. It was noted to be extremely potent in inhibiting tumor growth in vivo.

[0321] [Example 25] Internalization of humanized PD-L1 antibody To assess whether 23F11-H4L4 (derived from CHO cells) can be internalized The present inventors carried out the following experiment. The dye was first labeled with a reactive dye (Sigma, G9845), a pH-sensitive dye. The dye is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. The antibody binds to the intracellular endosomes (pH 6.0-6.5) and lysosomes (pH 4.5-5.5 When the MC38- hPD-L1 cells (MabSpace) were cultured in 10% fetal bovine serum (Hyclone). The log-phase growing cells were isolated by centrifugation in RPMI-1640 containing 100 mM NaCl. The cells were collected and plated in a 96-well plate at 2 x 10^4 cells / well (50 μl / well). The cells were seeded at a density of 100x and allowed to attach overnight. Then, 2 μg / ml of pHAb amine reactant was added. Goat anti-human Fc (Jackson ImmunoResearch) labeled with a chromogenic dye h, Catalog No. 109-005-098) (25 μl / well) was added to each well. Then, serially diluted 23F11-H4L4 or hIgG1 (25 μl / well) was added. Antibodies were added to each well and incubated with the cells at 37°C for 6 or 24 hours. At the end of the incubation, the culture medium was removed and 100 μl of PBS was added. The antibody signal in each well of the plate was monitored using a Varioska n Flash (Thermo Scientific) was used to measure the Ex 532 nm. The data in the graph was determined by reading the spectrum at 560 nm. After 6 or 24 hours, there was a significant increase in the fluorescent signal in a concentration-dependent manner in the cells. These results indicate that 23F11-H4L4 inhibits the proliferation and proliferation of MC38 / hPD-L1 cells. It was demonstrated that it can be efficiently internalized upon binding to hPD-L1 on the surface ( See Figure 35).

[0322] Example 26: Binding of humanized PD-L1 antibodies to activated T cells in vitro Peripheral blood monolayers were isolated from leukapheresis packs using Ficoll-Paque density gradient centrifugation. Spheres were isolated and resuspended in PBS containing 2% FBS. Cells were diluted 0.5×1 in culture medium. The cells were diluted to a density of 0^7 cells / ml and then incubated with an anti-human CD3 antibody (OKT3, ebios The plate was coated with 100% glycerol (Civilian, Cat. No. 16-0037). (1 μg / ml, 1 ml / well). Anti-human CD28 (BD, Cat. No. 555725 ) was added to each well, and the cells were then incubated in RPMI 1640 with 10% FBS. The cells were cultured in 6-well plates at 37°C for 3 days. Then, the cells were harvested and placed in 96-well plates. The bottom plate was placed in FACS buffer (PBS + 5% BSA). After centrifugation, the supernatant Discard the diluted 23F11-H4L4 antibody or hIg with the N297A mutation. G1 was added as a negative control. The plates were incubated at 4°C for 1 hour and then The cells were washed three times with PBS. Then, the cells were diluted with 2.5 μl of PBS. Then, the plate was washed three times with 2.5C human IgG-APC (Biolegend, Catalog No. 409306) to each well and incubate the plate at 4°C for 1 hour. The cells were then washed three times and then loaded onto a flow cytometer (Beckman Cytoflex ) was used for the analysis.

[0323] The data in Figures 36A and 36B show that 23F11-H4L4 inhibits the activation of T cells by Binds to expressed PD-L1 in a dose-dependent manner, but binds only slightly to non-activated T cells It was shown that the cations of the nucleosomes exhibited only weak binding.

[0324] [Example 27] hPD-1 knock-in mouse model using MC38 / hPD-L1 cells In vivo efficacy study of PD-L1 antibody in Previous experiments (e.g., Examples 14 and 20) have demonstrated that PD-1 The protein is of murine origin and immunosuppression is mediated by the binding of hPD-L1 to murine PD-1. If mouse PD-1 is changed to hPD-1, it can be transmitted to human patients. The situation exists in the MC38 / hPD-L1 syngeneic model, but antibody treatment may have some effect. To further evaluate whether there were differences, we compared MC38 / hPD-L1 tumor The cells were transplanted into PD-1 knock-in mice. Specifically, MC38 / hPD-L1 tumors Cells were incubated at 4 °C in air and 5% CO2 The cells were incubated at 37°C in a 10% heat-inactivated fetal bovine serum (E xCell Biology), 100U / ml penicillin and 100μg / ml streptomycin. RPMI 1640 medium (Thermo F) supplemented with leptomycin (Hyclone) was used. Tumor cells were maintained in vitro as monolayer cultures on a 100% PBS (Isher). They were routinely subcultured twice a week by EDTA treatment (Hyclone). Cells growing in the culture medium were harvested and subjected to FACS analysis to confirm hPD-L1 expression and tumor inoculation. Five to six-week-old PD-1 knock-in female mice (C57BL / 6-P dcd / tm1 (hPDCD1), Beijing Biocytogen Co., Ltd) were cultured at 100x for PD-L1 expression (2 × 10 6 ) Patients were inoculated subcutaneously in the right flank with MC38 / hPD-L1 tumor cells that had been confirmed to be resistant to chemotherapy. The tumor size is approximately 100 mm 3 The treatment was started approximately 7 days after inoculation, reaching a total dose of 100 mg / kg / day. Groups consisted of 10 tumor-bearing mice (see FIG. 37A). That is, 23F11-H4L4) was aspirated with 200 μl of a stock solution of 3 mg / ml concentration. Then, add 1.8 ml of formulation buffer (20 mM histidine, sucrose and diluted to pH 6.0 ± 0.2). Prepare a working solution of 0.3 mg / ml and inject it into mice by intravenous injection. Tumor size was measured twice weekly in two dimensions using calipers (INSIZE). , the volume is calculated using the formula: V = 0.5a × b 2 (where a and b are the long diameter and (the short diameter) in mm 3 Tumor volume was less than measurable size. If the tumor volume decreased to <50% of the volume at the start of treatment without any decline, partial recovery (PR) was determined. If the tumor volume was no longer palpable, tumor regression was recorded as complete (CR). The tumor sizes in each treatment group are shown in Table 15 and Figures 37B and 37C. These data show that treatment with antibody 23F11-H4L4 led to significant tumor growth inhibition. demonstrated tumor eradication in 5 of 10 treated mice. or complete response, whereas saline treatment resulted in all growth and mice None of the patients had tumor regression.

[0325] [Table 15]

[0326] To further compare in vivo tumor growth inhibition efficacy, we Similarly, 5- to 6-week-old PD-1 knock-in female mice (C57BL / 6-Pdcd / tm1 (hPDCD1), Beijing Biocytogen Co., Ltd.) were incubated with 0.1 ml of PBS for PD-L1 expression (2× 10 6 MC38 / hPD-L1 tumor cells, which have been confirmed to be effective against PD-L1, were inoculated subcutaneously into the right flank. Treatment was performed on tumors measuring approximately 100 mm 3 Approximately 7 days after vaccination, Each group consisted of eight tumor-bearing mice. The test article was administered at a concentration of 3 mg / ml. Aspirate 0.128 ml, 0.384 ml, and 1.28 ml of the stock solution and , 3.456 and 2.58 ml of sterile PBS (Hyclone, Cat. No. SH3 0256.01) to 0.1mg / ml, 0.3mg / ml or 1mg / ml and inject 200 µl into each mouse intraperitoneally twice a week. The doses were then administered to achieve doses of 1, 3, and 10 mg / kg. SPACE, produced in CHO cells) and control Ig with the N297A mutant G (CrownBio, CHO cell-derived AB160160) was added to 1.280 mL of 3 ml Aspirate 2.5g / ml of CHO cell-derived BM-GT and place it in 2.560mL of PBS. or aspirate 0.662 ml of 5.8 mg / ml stock and 3.178 ml of sterile Prepare 200 μl of the final diluted solution by placing it in PBS and administer it to each mouse, twice a week. The tumors were administered by intraperitoneal injection at 10 mg / kg IP twice weekly to achieve dosing at 10 mg / kg IP twice weekly. Tumor size was measured twice weekly in two dimensions using calipers (INSIZE) and the volume was calculated using the formula: :V=0.5a×b 2 where a and b are the long and short diameters of the tumor, respectively. (mm) 3 The tumor volume was reduced to below measurable size. If no improvement was observed and the tumor volume was reduced to <50% of the volume at the start of treatment, the tumor was considered partial (PR). If the tumor volume is no longer palpable, tumor regression is recorded as complete (CR) (Table 16 and See Figures 38A-38C.

[0327] [Table 16]

[0328] The data from the above experiments showed that MC38 mice expressing human PD-L1 were transplanted with PD-L1-positive mice. 1) 23F11-H4L4 induced strong dose-dependent tumor growth in knock-in mice The inhibitory activity was 12.5%, 62.5% and 10 mg / kg, respectively. and 100% overall response (PR+CR) rate (see Figure 38A); 2) 3 mg / k 23F11-H4L4 at 10 mg / kg was at least as potent as BM-GT at 10 mg / kg. 4) 3) administered by IP or IV (see Figures 38B and 38C) 23F11-H4L4 at 100 mg / kg showed similar activity, achieving complete tumor regression (50%). (See Figures 37A-37B and 38A-38C), 3) at a dose of 10 mg / kg 23F11-H4L4 achieved complete tumor regression as well as a reduction in overall tumor size. The number of mice that developed BM-GT was also significantly higher than that of BM-GT at 10 mg / kg (6 / 8 vs. 3 / 8). was significantly more potent (see FIG. 38C), thus demonstrating pH-dependent antigen binding and The associated recycling properties and therefore longer drug residence time in the tumor are particularly The maximally effective dose of GT has been shown to translate into better tumor control and tumor regression. Ta.

[0329] [Example 28] Receptor occupancy and mouse pharmacokinetics / pharmacodynamics Target protein human in MC38 / hPD-L1 tumors upon a single intravenous injection of the antibody To measure the duration of antibody binding to PD-L1, we used FACS: Available target protein binding in single cells prepared from tumors at different times after antibody injection Specifically, MC38 / hPD-L1 tumor cells were incubated in air at 5% CO 2The cells were incubated at 37°C in a 10% heat-inactivated fetal bovine serum (ExCell Biol ogy), 100 U / ml penicillin and 100 μg / ml streptomycin (Hy Monolayer culture was performed in RPMI 1640 medium (Thermo Fisher) supplemented with Tumor cells were maintained in vitro as a culture medium. Lone) were routinely subcultured twice a week. Cells growing in logarithmic phase were The cells were harvested and counted for FACS analysis to confirm hPD-L1 expression and for tumor inoculation. Each ~6-week-old female C57B / L6 mouse was cultured in 0.1 ml of PBS for tumor development. PD-L1 expression (2 × 10 6 ) was confirmed using MC38 / hPD-L1 tumor cells. The tumors were inoculated subcutaneously in the right flank. 3 Reached The mice were dosed with 23F11-H4L4 at 3 mpk starting approximately 12 days after vaccination. (See Table 17.) Receptor occupancy was assessed on days 2, 7, and 14 after dosing. Tumors were harvested on day 1. Tumors were harvested, minced into small pieces, and then placed in ACCUMAX™ Cell detachment solution (STEMCELL Catalog No. 07921) was added at a ratio of 10 ml / 0.5 g. The tumor mass suspension was then gently shaken at room temperature for 1-2 hours to allow the ink to dry. During incubation, cells were further dissociated by pipetting up and down repeatedly. At the end of incubation, the cell suspension was passed through a 40 μm nylon mesh. The cells were washed three times with cold PBS and centrifuged at 1500 rpm for 5 min. The collected cells were resuspended in 1-2 ml of PBS and the total cell number was counted. 1 μl of the cell suspension (approximately 1 million cells) was added to each 96-well plate. The cells were washed twice with FACS buffer (1% BSA in PBS). Cells were incubated with 2 μg / ml of 23F11-H4L4 antibody or PBS or isotype control The mixture was incubated at 2-8°C or on ice for at least 30 min. The cells were washed three times with FACS buffer and protected from light. Then, the mixture was centrifuged at 1500 rpm for 5 minutes. Anti-human IgG FC-FITC secondary antibody Add 100 mM NaCl and incubate at 2-8°C in the dark for 30 minutes. Then, the cells are FACS buffered. Finally, the cells were resuspended in 0.5 ml of FACS buffer. The mixture was then analyzed using a flow cytometer. The isotopic analysis was performed using software (Cytoflex) and GraphPad Prism. Percentage and mean of PD-L1 positive cells compared to type-matched control antibody stained cells The molecular weight fluorescence intensity (MFI) was calculated. 11-H4L4 or BM-GT (to detect available PD-L1 binding sites) To detect in vivo binding, the isotype control hIgG1-N197A (Cro Preincubate in vitro with a saturating dose of 1000 mM NaCl (wnBio, AB160160). The percentage change in relative mean fluorescence intensity (RMFI) between treated and untreated cells was used to estimate receptor occupancy. (RO) was estimated. Values ​​were positive relative to the appropriate isotype control hIgG1-N297A. The data (see Table 18) are for a single dose of 3 mg / kg 23F11-H4L4. showed that a single injection could lead to sustained tumor PD-L1 occupancy for at least 7 days. On the other hand, BM-GT at 3 mg / kg showed tumor PD-L1 occupancy for more than 2 days, but not after the first Despite the higher occupancy, the incubation time was less than 7 days, which is consistent with the pH-dependent The PD-L1-dependent binding properties of the antibody allow it to persist in tumors for a long time and bind to PD-L1 on tumor cells. -L1.

[0330] [Table 17]

[0331] [Table 18]

[0332] [Example 29] Tumor penetration results With MC38 / hPD-L1 tumors grown in normal syngeneic C57B / L6 mice Three mice were treated with 10 mg / kg of 23 mg / kg of 10 ... The tumor was treated with a single intravenous injection of F11-H4L4. Tumor penetration and tumor-infiltrating lymphocytes ( Tumors were harvested 7 days after antibody administration for TIL analysis. l Cutting Temperature(OCT)(Tissue Tek Tumor masses were prepared using a 50-mL PBS (pH 7.0) according to the manufacturer's instructions. 6-μm-thick tissue sections were prepared. The tissue slides were then fixed with methanol, followed by washing with PBS, and then The slides were then permeabilized with 0.2% TritonX-100 for 5 min. Wash with PBS and block with blocking buffer (1x PBS, 3.0% BSA). Block the slides at room temperature for 30-60 minutes, then rinse the slides with PBS-T. The cells were washed three times for 5 minutes each. Then, Alexa Fluor 488 diluted in PBS was added. Goat anti-human IgG (H+L) secondary antibody (Thermo, Cat. No. A11013, 23 F11-H4L4), Alexa Fluor® 594 anti-mouse CD31 antibody (Biolegend, Cat. No. 102520, detects vasculature ) and Alexa Fluor® 594 anti-mouse CD8a antibody (Bio legend, Catalog No. 100758, to detect CD8 T cells) was applied to the sections. The secondary antibody was drained and washed with PBS-T. After washing the slides three times (5 min each), the slides were counterstained with DAPI and then attached to a glass filter. The specimens were covered with a bar slip and stored in the dark at 4°C. Using this method, the locus and phenotype of 23F11-H4L4 and TILs were compared with control frozen tissue sections. The distribution was analyzed.

[0333] 2 in frozen MC38 / hPD-L1 CDx tumor sections, 7 days after dosing at 10mpk Representative images of 3F11-H4L4 infiltration and TILs are shown in FIG.

[0334] This disclosure will now be described with reference to certain embodiments, some of which are preferred embodiments. Although specifically shown and described, various changes in form and details will occur to those skilled in the art. No. 5,393,311, which may be made therein without departing from the spirit and scope of the present disclosure as set forth herein. It must be understood that

Claims

1. 1. An isolated PD-L1 antibody or antigen-binding fragment thereof, comprising a heavy chain HCDR1′, HCDR2′, and HCDR3′, and a light chain LCDR1′, LCDR2′, and LCDR3′ sequence, the HCDR1′ sequence comprises DYYMN (SEQ ID NO:23); The HCDR2' sequence is DINPNNGGTSYNX' １ K.F.X. ２ G (SEQ ID NO:24), the HCDR3′ sequence comprises VKWGDGPFAY (SEQ ID NO:25); The LCDR1' sequence is X' ３ ASQNVGAAVA (SEQ ID NO:26), The LCDR2' sequence is SASNX' ４ X' ５ T (SEQ ID NO:27), the LCDR3' sequence comprises QQYSNYPT (SEQ ID NO:28); X' １ is H, and X' ２ is K, and X' ３ is K, and X' ４ is R, and X' ５ is Y; X' 1 is Q, X' 2 is Q, X' 3 is Q, X' 4 is R and X' 5 is Y; or X' 1 is Q, X' 2 is Q, X' 3 is Q, X' 4 is L and X' 5 is Y; An isolated PD-L1 antibody or antigen-binding fragment thereof.

2. and a light chain framework sequence of LFR1', LFR2', LFR3' and LFR4', wherein the sequence of the heavy chain variable region is according to the formula: HFR1'-HCDR1'-HFR2'-HCDR2'-HFR3'-HCDR3'-HFR4' and the sequence of the light chain variable region is according to the formula: LFR1'-LCDR1'-LFR2'-LCDR2'-LFR3'-LCDR3'-LFR4'; The HFR1′ sequence is １ VQLX'a ２ QSGX'a ３ EX'a ４ X'a ５ KPGASVKX'a ６ SCKASGYVFT (SEQ ID NO:47); The HFR2' sequence is ７ QX'a ８ X'a ９ GX'a １０ X'a １１ Contains LEWIG (SEQ ID NO:48); The HFR3′ sequence is １２ X'a １３ TVTVDX'a １４ SX'a １５ X'a １６ TAYMELX'a １７ X'a １８ LX'a １９ SX'a ２０ DX'a ２１ Contains AVYYC (SEQ ID NO:49); The HFR4′ sequence is WGQGTLVTVSX′a ２２ (SEQ ID NO:50); The LFR1' sequence is DIX'a ２３ MTQSX'a ２４ X'a ２５ X'a ２６ X'a ２７ SX'a ２８ SVGDRVX'a ２９ ITC (SEQ ID NO:51); The LFR2' sequence is WYQQKPGX'a ３０ X'a ３１ Contains PKLLIY (SEQ ID NO:52); The LFR3′ sequence is GVPX′a ３２ RFX'a ３３ GSGSGTDFTLTISX'a ３４ X'a ３５ QX'a ３６ EDX'a ３７ AX'a ３８ YFC (SEQ ID NO:53); and The LFR4' sequence comprises FGSGTKLGIK (SEQ ID NO:54), Here, X′a １ is E, and X'a ２ is Q, and X′a ３ is P, and X′a ４ is L, and X'a ５ is V, and X′a ６ is I, and X'a ７ is K, and X'a ８ is S, and X'a ９ is H, and X′a １０ is K, and X'a １１ is S, and X'a １２ is K, and X'a １３ is A, and X'a １４ is K, and X'a １５ is S, and X'a １６ is R, and X′a １７ is L, and X'a １８ is S, and X'a １９ is T, and X'a ２０ is E, and X'a ２１ is S, and X'a ２２ is A, and X'a ２３ is V, and X′a ２４ is Q, and X′a ２５ is K, and X'a ２６ is F, and X′a ２７ is M, and X'a ２８ is T, and X'a ２９ is S, and X'a ３０ is Q, and X′a ３１ is S, and X'a ３２ is D, and X'a ３３ is T, and X'a ３４ is N, and X′a ３５ is M, and X'a ３６ is S, and X'a ３７ is L, and X'a ３８ is D; X'a １ is Q, and X′a ２ is V, and X′a ３ is A, and X'a ４ is V, and X′a ５ is V, and X′a ６ is I, and X'a ７ is K, and X'a ８ is A, and X'a ９ is P, and X′a １０ is Q, and X′a １１ is G, and X'a １２ is R, and X′a １３ is A, and X'a １４ is K, and X'a １５ is T, and X'a １６ is R, and X′a １７ is S, and X'a １８ is R, and X′a １９ is R, and X′a ２０ is D, and X'a ２１ is T, and X'a ２２ is S, and X'a ２３ is Q, and X′a ２４ is Q, and X′a ２５ is S, and X'a ２６ is S, and X'a ２７ is L, and X'a ２８ is A, and X'a ２９ is T, and X'a ３０ is K, and X'a ３１ is A, and X'a ３２ is S, and X'a ３３ is S, and X'a ３４ is S, and X'a ３５ is M, and X'a ３６ is P, and X′a ３７ is I, and X'a ３８ is T; X'a １ is Q, and X′a ２ is V, and X′a ３ is A, and X'a ４ is V, and X′a ５ is K, and X'a ６ is V, and X′a ７ is R, and X′a ８ is A, and X'a ９ is P, and X′a １０ is Q, and X′a １１ is G, and X'a １２ is R, and X′a １３ is V, and X′a １４ is T, and X'a １５ is I, and X'a １６ is R, and X′a １７ is S, and X'a １８ is R, and X′a １９ is R, and X′a ２０ is D, and X'a ２１ is T, and X'a ２２ is S, and X'a ２３ is Q, and X′a ２４ is Q, and X′a ２５ is S, and X'a ２６ is S, and X'a ２７ is L, and X′a ２８ is A, and X'a ２９ is T, and X'a ３０ is K, and X'a ３１ is A, and X'a ３２ is S, and X'a ３３ is S, and X'a ３４ is S, and X'a ３５ is M, and X'a ３６ is P, and X′a ３７ is I, and X'a ３８ is T; X'a １ is Q, and X′a ２ is V, and X′a ３ is A, and X'a ４ is V, and X′a ５ is V, and X′a ６ is I, and X'a ７ is K, and X'a ８ is A, and X'a ９ is P, and X′a １０ is Q, and X′a １１ is G, and X'a １２ is R, and X′a １３ is A, and X'a １４ is K, and X'a １５ is T, and X'a １６ is R, and X′a １７ is S, and X'a １８ is R, and X′a １９ is R, and X′a ２０ is D, and X'a ２１ is T, and X'a ２２ is S, and X'a ２３ is Q, and X′a ２４ is P, and X′a ２５ is S, and X'a ２６ is S, and X'a ２７ is L, and X′a ２８ is A, and X'a ２９ is T, and X'a ３０ is K, and X'a ３１ is A, and X'a ３２ is S, and X'a ３３ is S, and X'a ３４ is S, and X'a ３５ is L, and X′a ３６ is P, and X′a ３７ is I, and X'a ３８ is T; or X'a １ is Q, and X′a ２ is V, and X′a ３ is A, and X'a ４ is V, and X′a ５ is K, and X'a ６ is V, and X′a ７ is R, and X′a ８ is A, and X'a ９ is P, and X′a １０ is Q, and X′a １１ is G, and X'a １２ is R, and X′a １３ is V, and X′a １４ is T, and X'a １５ is I, and X'a １６ is R, and X′a １７ is S, and X'a １８ is R, and X′a １９ is R, and X′a ２０ is D, and X'a ２１ is T, and X'a ２２ is S, and X'a ２３ is Q, and X′a ２４ is P, and X′a ２５ is S, and X'a ２６ is S, and X'a ２７ is L, and X′a ２８ is A, and X'a ２９ is T, and X'a ３０ is K, and X'a ３１ is A, and X'a ３２ is S, and X'a ３３ is S, and X'a ３４ is S, and X'a ３５ is L, and X′a ３６ is P, and X′a ３７ is I, and X'a ３８ is T, The antibody or antigen-binding fragment thereof described in claim 1.

3. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region set forth in SEQ ID NO:65 and a light chain variable region set forth in SEQ ID NO:66; a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:68; a heavy chain variable region set forth in SEQ ID NO:65 and a light chain variable region set forth in SEQ ID NO:68; a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:66; or a heavy chain variable region set forth in SEQ ID NO:85 and a light chain variable region set forth in SEQ ID NO:

86.

4. The antibody or antigen-binding fragment thereof of claim 1 , wherein the antibody is a bispecific antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a recombinant antibody, a labeled antibody, or a bivalent antibody.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a bispecific antibody that further binds to an antigen other than PD-L1.

6. The antibody or antigen-binding fragment thereof is a diabody, scFv, scFv dimer, BsFv, dsFv, (dsFv) ２ , dsFv-dsFv', Fv fragment, Fab, Fab', F(ab') ２ 2. The antibody or antigen-binding fragment thereof of claim 1, which is an antigen-binding fragment selected from the group consisting of:

7. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in claim 1.

8. A kit comprising the antibody or antigen-binding fragment thereof of claim 1.

9. A polynucleotide encoding the antibody or antigen-binding fragment thereof described in claim 1.

10. A vector comprising the polynucleotide of claim 9.

11. An isolated host cell comprising the vector of claim 10.

12. 12. A method for producing a PD-L1 antibody, comprising culturing a host cell of claim 11 under conditions in which the polynucleotide is expressed, the host cell comprising a vector comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof of claim 1.

13. 8. The pharmaceutical composition of claim 7 for use in a method for treating a PD-L1 associated condition in a subject, the method comprising administering to the subject the antibody or antigen-binding fragment thereof of claim 1.

14. 14. The pharmaceutical composition of claim 13, further comprising a second therapeutic agent, and the method further comprises administering the second therapeutic agent to the subject.

15. 15. The pharmaceutical composition of claim 14, wherein the second therapeutic agent is an agent used in radiation therapy, chemotherapy, targeted therapy, gene therapy, immunotherapy, hormone therapy, anti-angiogenesis, palliative care, surgery or a combination thereof.

16. The pharmaceutical composition of claim 15, wherein the second therapeutic agent is a VEGFR2 antibody.

17. The pharmaceutical composition of claim 13, wherein the PD-L1 associated condition is a cancer or a tumor.

18. The pharmaceutical composition of claim 17, wherein the cancer or tumor is a metastatic tumor that expresses PD-L1.

19. The pharmaceutical composition of claim 17, wherein the cancer or tumor is a solid tumor.

20. 20. The pharmaceutical composition of claim 19, wherein the solid tumor is non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, colon cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, myeloma, mycosis fungoides, Merkel cell carcinoma, hepatocellular carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic lung carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, testicular tumor, or seminoma.

21. 18. The pharmaceutical composition of claim 17, wherein the cancer or tumor is non-small cell lung cancer, small cell lung cancer, breast cancer, bladder cancer, hepatocellular carcinoma, colorectal cancer, gastric carcinoma, esophageal cancer, melanoma or colon cancer.

22. The pharmaceutical composition of claim 13, wherein the PD-L1 associated condition is a hematological disorder, an infectious disease, an autoimmune disease, or a fibrotic disease.

23. Hematological disorders include classical Hodgkin's lymphoma, primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic and erythroleukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, polycythemia vera, obesity 23. The pharmaceutical composition of claim 22, wherein the tumor is a cell-derived tumor, EBV positive and negative PTLD and diffuse large B-cell lymphoma, plasmablastic lymphoma, extranodal NK / T-cell lymphoma, and HHV8-associated primary effusion lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, myelodysplastic syndrome, or hairy cell leukemia.

24. 14. The pharmaceutical composition of claim 13, wherein the subject has tested positive for expression of PD-L1 protein or has been tested to have an elevated level of expression of PD-L1 protein compared to healthy individuals or individuals not having a PD-L1 associated condition.

Images