Antibody against PD-L1 and method of use thereof

Abstract

This invention provides an antibody against PD-L1 (programmed death ligand 1) and a method for using the same antibody. [Solution] The present invention relates to a human monoclonal antibody that binds to PD-L1. This antibody can be used to treat cancer and chronic viral infections.

Description

[Technical Field]

[0001] This application is an international application claiming priority to U.S. Provisional Patent Application No. 62 / 942,455, filed on 2 December 2019, each of which is incorporated herein by reference in its entirety.

[0002] All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. To more fully describe the latest art known to those skilled in the art to the date of the invention described herein and claimed, the disclosures of these publications are incorporated herein by reference.

[0003] This patent disclosure includes materials that are protected by copyright. The copyright holder reserves all copyrights to the patent document or patent disclosure, except as they appear in the patent files or records of the United States Patent and Trademark Office, and does not object to facsimile reproduction of either the patent document or the patent disclosure.

[0004] Government interests This invention was made possible with government support under Grant No. 1 R56 AI109223-01A1, awarded by the National Institutes of Health, USA. The government has certain rights to this invention.

[0005] Field of Invention This invention relates to an antibody against PD-L1 (also known as programmed cell death ligand 1 or B7H1) and a method of using the same. [Background technology]

[0006] Background of the Invention Programmed cell death-1 (PD-1) is a cell surface membrane protein of the immunoglobulin superfamily. This protein is expressed in pro-B cells and is thought to play a role in their differentiation. A member of the CD28 family, PD-1 is upregulated in activated T cells, B cells, and monocytes. PD-1 has two identified ligands from the B7 family: PD-L1 (also known as programmed cell death-1 ligand 1, differentiation cluster 274 (CD274), or B7 homolog 1 (B7-H1)) and PD-L2. PD-L1 is a 40 kDa type I transmembrane protein. Binding of PD-L1 to PD-1 or B7.1 transmits an inhibitory signal that reduces the proliferation of CD8+ T cells in lymph nodes. PD-L2 expression tends to be more restricted and is mainly found in activated antigen-presenting cells (APCs), while PD-L1 expression is widespread, including in hematopoietic cells (including activated T cells, B cells, monocytes, dendritic cells, and macrophages) and peripheral non-lymphoid tissues (including cardiac, skeletal, muscular, placental, lung, renal, and liver tissues). The widespread expression of PD-L1 indicates its important role in regulating peripheral immune tolerance via PD-1 / PD-L1. [Overview of the project]

[0007] One aspect of the present invention relates to an isolated monoclonal antibody that binds to human programmed death ligand 1 (PD-L1). In some embodiments, the antibody may be its antigen-binding fragment that binds to human programmed death ligand 1 (PD-L1). In other embodiments, the antibody or fragment may comprise a heavy chain, a light chain, or a combination thereof. In one embodiment, the heavy chain is G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 47), G-(X1)-T-(X2)-(X 13 X 14 )-(X3X4)(Sequence No. 205), G-(X1)-TF-(X 13 X 14 CDR1 containing )-Y-(X4)(Sequence ID 206), I-(X8X9X 10 X 11 )-G-(X 12)-A (Sequence No. 51) or II-(X 15 )-IFG-(X 16 )-A (Sequence No. 207)-containing CDR2 and / or an ARGRQMFGAGIDF (Sequence No. 6), ARVHAALYYGMDV (Sequence No. 14), TTGGLGLVYPYYNYIDV (Sequence No. 99), AKVHPVFSYALDV (Sequence No. 100), AEEGAFNSLAI (Sequence No. 101), ARDGSGYDSAGMDD (Sequence No. 102), ARGFGGPDY (Sequence No. 103), ARVHGALYYGMDV (Sequence No. 104), ASGSIVGAAYAFDI (Sequence No. 105), ARDRSEGGFDP (Sequence No. 106), or AEEGAFNSLAI (Sequence No. 107)-containing CDR3. In another embodiment, the light chain is SGSIDSNY (Sequence No. 18), S-(X 17 X 18 )I-(X 19 )-SNY (Sequence No. 208), or NIG-(X5)-K-(X 20 )(Sequence No. 48)-containing CDR1, EDN (Sequence No. 20), (X 21 )-DN (Sequence No. 209), (X 22The formulation includes CDR2 containing )-NN (SEQ ID NO: 210) or DD-X6 (SEQ ID NO: 49), and / or CDR3 containing QSYDSNNRHVI (SEQ ID NO: 22), QVWDS-(X7)-SDHWV (SEQ ID NO: 50), QVWDSSGDLWV (SEQ ID NO: 126), AAWDDSLNGLV (SEQ ID NO: 127), QSYDGITVI (SEQ ID NO: 128), QSYDSSNHWV (SEQ ID NO: 129), AVWDDSLSGVV (SEQ ID NO: 131), MIWHSSAYV (SEQ ID NO: 132), NSRDISDNQWQWI (SEQ ID NO: 134), or QSYDSSNHVV (SEQ ID NO: 135). In some embodiments, the antibody is a fully human antibody or has been humanized. In other embodiments, the antibody is monospecific, bispecific, or multispecific. In further embodiments, the antibody is a single-chain antibody. In some embodiments, the antibody has a binding affinity of at least 3.3 × 10⁻⁹ M. In embodiments, the antibody or fragment may further include a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In embodiments, X1, X2, X3, or X4 are nonpolar amino acid residues. In other embodiments, X1, X2, X 3、 Alternatively, X4 is glycine (G), tyrosine (Y), phenylalanine (F), leucine (L), or alanine (A). In some embodiments, X1, X2, or X4 are hydrophobic amino acid residues, for example, X1, X2, or X4 are glycine (G), leucine (L), or alanine (A). In some embodiments, X3 is a hydrophilic polar amino acid residue. In one embodiment, X3 is histidine (H). In some embodiments, X1 is phenylalanine (F), glycine (G), or tyrosine (Y). In further embodiments, X2 is phenylalanine (F) or leucine (L). In other embodiments, X3 is histidine (H) or tyrosine (Y). In yet another embodiment, X4 is serine (S), glycine (G), or alanine (A). In one embodiment, X8, X9, X 10 , or X 11 X8, X9, X10 , or X 11 isoleucine (I), proline (P), alanine (A), or phenylalanine (F). In other embodiments, X8, X 10 , or X 12 These are polar hydrophilic amino acid residues. In yet another embodiment, X8, X 10 , or X 12 X8 is histidine (H), serine (S), asparagine (N), or threonine (T). In one embodiment, X8 is alanine (A), isoleucine (I), or serine (S). In one embodiment, X9 is tyrosine (Y), serine (S), proline (P), or alanine (A). In one embodiment, X 10 is tyrosine (Y), aspartic acid (D), isoleucine (I), or histidine (H). In one embodiment, X 11 is glycine (G), leucine (L), asparagine (N), or phenylalanine (F). In one embodiment, X 12 X5 is isoleucine (I), arginine (R), threonine (T), or histidine (H). In some embodiments, X5 is a nonpolar hydrophobic amino acid residue. In one embodiment, X5 is glycine (G). In other embodiments, X5 is a polar hydrophilic amino acid residue. In one embodiment, X5 is serine (S), asparagine (N), or aspartic acid (D). In further embodiments, X6 is a nonpolar amino acid residue. In one embodiment, X6 is tyrosine (Y). In some embodiments, X6 is a polar hydrophilic amino acid residue. In one embodiment, X6 is threonine (T), serine (S), or arginine (R). In some embodiments, X7, X 15 , X 16 , X 17 , X 19 , X 20 , or X 21 These are nonpolar hydrophobic amino acid residues. In one embodiment, X7, X 17 , or X 20 This is glycine (G). In other embodiments, X7, X 13 , X 14, X 15 , X 16 , X 17 , X 18 , X 19 , or X 21 These are polar hydrophilic amino acid residues. In one embodiment, X7, X 14 , or X 21 is serine (S) or arginine (R). In one embodiment, X 13 is serine (S) or threonine (T). In one embodiment, X 15 is proline (P). In one embodiment, X 15 , X 17 , or X 20 is serine(S). In one embodiment, X 16 is threonine (T) or arginine (R). In one embodiment, X 16 isoleucine(I). In one embodiment, X 18 is serine (S) or asparagine (N). In one embodiment, X 19 is glycine (G) or alanine (A). In one embodiment, X 19 is aspartic acid (D). In one embodiment, X 21 is alanine (A). In one embodiment, X 21 This is glutamic acid (E).

[0008] One aspect of the present invention relates to an isolated antibody or fragment thereof that binds to the human programmed death ligand 1 (PD-L1) protein and comprises (a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 2, VH CDR2 containing the amino acid sequence of SEQ ID NO: 4, VH CDR3 containing the amino acid sequence of SEQ ID NO: 6, VL CDR1 containing the amino acid sequence of SEQ ID NO: 18, VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 22, or (b) VH CDR1 containing the amino acid sequence of SEQ ID NO: 10, VH CDR2 containing the amino acid sequence of SEQ ID NO: 12, VH CDR3 containing the amino acid sequence of SEQ ID NO: 14, VL CDR1 containing the amino acid sequence of SEQ ID NO: 48, VL CDR2 containing the amino acid sequence of SEQ ID NO: 49, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 50. In some embodiments, X5 of SEQ ID NO: 48 is a nonpolar hydrophobic amino acid residue. In one embodiment, X5 is glycine (G). In other embodiments, X5 is a polar hydrophilic amino acid residue. In one embodiment, X5 is serine (S), asparagine (N), or aspartic acid (D). In a further embodiment, X6 of SEQ ID NO: 49 is a nonpolar amino acid residue. In one embodiment, X6 is tyrosine (Y). In some embodiments, X6 is a polar hydrophilic amino acid residue. In one embodiment, X6 is serine (S), threonine (T), or arginine (R). In some embodiments, X7 of SEQ ID NO: 50 is a nonpolar hydrophobic amino acid residue. In one embodiment, X7 is glycine (G). In other embodiments, X7 is a polar hydrophilic amino acid residue. In one embodiment, X7 is serine (S) or arginine (R). In some embodiments, VL CDR1 contains the amino acid sequence of SEQ ID NO: 26, 33, 40, or 44. In embodiments, VL CDR2 contains the amino acid sequence of SEQ ID NO: 28, 35, or 45. In the embodiment, VL CDR3 contains the amino acid sequence of SEQ ID NO: 30 or 37.In some embodiments, the antibody of (b) described herein comprises VL CDR1 containing the amino acid sequence of SEQ ID NO: 26, VL CDR2 containing the amino acid sequence of SEQ ID NO: 28, and VL CDR3 containing SEQ ID NO: 30; or comprises VL CDR1 containing the amino acid sequence of SEQ ID NO: 33, VL CDR2 containing the amino acid sequence of SEQ ID NO: 35, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 37; or comprises VL CDR1 containing the amino acid sequence of SEQ ID NO: 40, VL CDR2 containing the amino acid sequence of SEQ ID NO: 35, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 37; or comprises VL CDR1 containing the amino acid sequence of SEQ ID NO: 44, VL CDR2 containing the amino acid sequence of SEQ ID NO: 45, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 37.

[0009] One aspect of the present invention relates to an isolated antibody or fragment thereof that binds to the human PD-L1 protein and comprises a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 52, 54, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 31, 38, 42, 46, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, and 83.

[0010] One aspect of the present invention relates to a heavy chain variable region comprising VH-CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 84, 85, 86, 87, 88, 89, and 90, VH-CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12, 91, 92, 93, 94, 95, 96, 97, and 98, and / or VH-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 14, 99, 100, 101, 102, 103, 104, 105, 106, and 107, and / or SEQ ID NOs: 18, 26, 33, 40 The study targets isolated antibodies or fragments thereof containing VL-CDR1 having an amino acid sequence selected from the group consisting of 44, 108, 109, 110, 111, 112, 113, 114, 115, 116, and 117; VL-CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 28, 35, 45, 118, 119, 120, 121, 122, 123, 124, and 125; and / or VL-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 30, 37, 126, 127, 128, 129, 130, 131, 132, 133, 134, and 135.

[0011] One aspect of the present invention relates to an isolated antibody or fragment thereof that binds to the human PD-L1 protein, wherein the antibody comprises (a) a heavy chain variable region having three CDRs comprising the amino acid sequences GGTFSSYA (SEQ ID NO: 2), IIPIFGTA (SEQ ID NO: 4), and ARGRQMFGAGIDF (SEQ ID NO: 6), and / or a light chain variable region having three CDRs comprising the amino acid sequences SGSIDSNY (SEQ ID NO: 18), EDN (SEQ ID NO: 20), and QSYDSNNRHVI (SEQ ID NO: 22), and (b) the amino acid sequence GYTLS (c) A heavy chain variable region having three CDRs including SHG (SEQ ID NO: 10), ISASHNGHA (SEQ ID NO: 12), and ARVHAALYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs including the amino acid sequences NIGSKG (SEQ ID NO: 26), DDR (SEQ ID NO: 28), and QVWDSGSDHWV (SEQ ID NO: 30), (c) a heavy chain variable region having three CDRs including the amino acid sequences GYTLSSHG (SEQ ID NO: 10), ISASHNGHA (SEQ ID NO: 12), and ARVHAALYYGMDV (SEQ ID NO: 14), (d) A variable region and / or a light chain variable region having three CDRs containing the amino acid sequences NIGDKG (SEQ ID NO: 33), DDS (SEQ ID NO: 35), and QVWDSSSDHWV (SEQ ID NO: 37), respectively; (d) A heavy chain variable region having three CDRs containing the amino acid sequences GYTLSSHG (SEQ ID NO: 10), ISAHNGHA (SEQ ID NO: 12), and ARVHAALYYGMDV (SEQ ID NO: 14), respectively; and / or a light chain variable region having three CDRs containing the amino acid sequences NIGNKG (SEQ ID NO: 40), DDS (SEQ ID NO: 35), and QVWDSSSDHWV (SEQ ID NO: 37), respectively. (e) a light chain variable region having three CDRs including 37), (e) a heavy chain variable region having three CDRs including the amino acid sequences GYTLSSHG (SEQ ID NO: 10), ISAHNGHA (SEQ ID NO: 12), and ARVHAALYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs including the amino acid sequences NIGGKG (SEQ ID NO: 44), DDY (SEQ ID NO: 45), and QVWDSSSDHWV (SEQ ID NO: 37), (f) an amino acid sequence GYTLSSHG (SEQ ID NO: 10), ISAHNGHA (SEQ ID NO: 12),(g) A heavy chain variable region having three CDRs including ARVHAALYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs including the amino acid sequences NIESRS (SEQ ID NO: 108), DDT (SEQ ID NO: 118), and QVWDSSGDLWV (SEQ ID NO: 126), respectively; (g) A heavy chain variable region having three CDRs including the amino acid sequences GYTLSSHG (SEQ ID NO: 10), ISAHNGHA (SEQ ID NO: 12), and ARVHAALYYGMDV (SEQ ID NO: 14), and / or the amino acid sequence NIGSK (h) A light chain variable region having three CDRs including G (SEQ ID NO: 26), DDS (SEQ ID NO: 35), and QVWDSSSDHWV (SEQ ID NO: 37), (h) A heavy chain variable region having three CDRs including the amino acid sequences GYTLSSHG (SEQ ID NO: 10), ISAHNGHA (SEQ ID NO: 12), and ARVHAALYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs including the amino acid sequences NIGSKS (SEQ ID NO: 109), DDS (SEQ ID NO: 35), and QVWDSSSDHWV (SEQ ID NO: 37), (i) Each of the amino (j) A heavy chain variable region having three CDRs including the acid sequences GYTLSSHG (SEQ ID NO: 10), ISAHNGHA (SEQ ID NO: 12), and ARVHAALYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs including the amino acid sequences NIGSKG (SEQ ID NO: 26), DDS (SEQ ID NO: 35), and QVWDSSSDHWV (SEQ ID NO: 37), respectively, (j) three CDRs including the amino acid sequences DFAFSSAW (SEQ ID NO: 84), IKSKTDGETT (SEQ ID NO: 91), and TTGGLGLVYPYYNYIDV (SEQ ID NO: 99), respectively. A heavy chain variable region having one CDR, and / or a light chain variable region having three CDRs containing the amino acid sequences SSNIGSNY (SEQ ID NO: 110), RNN (SEQ ID NO: 119), and AAWDDSLNGLV (SEQ ID NO: 127), respectively; (k) A heavy chain variable region having three CDRs containing the amino acid sequences GYTFTSYG (SEQ ID NO: 85), TSPHNGLT (SEQ ID NO: 92), and AKVHPVFSYALDV (SEQ ID NO: 100), respectively; and / or a heavy chain variable region having three CDRs containing the amino acid sequences SGSIASNY (SEQ ID NO: 111), EDN (SEQ ID NO: 20), respectively.(l) a light chain variable region having three CDRs including QSYDGITVI (SEQ ID NO: 128), (i) a heavy chain variable region having three CDRs including the amino acid sequences GGTFSRYA (SEQ ID NO: 86), IIPIFGRA (SEQ ID NO: 93), and AEGAFNSLAI (SEQ ID NO: 101), and / or a light chain variable region having three CDRs including the amino acid sequences SGSIASNY (SEQ ID NO: 111), ADN (SEQ ID NO: 120), and QSYDSSNHWV (SEQ ID NO: 129), (m) each having the amino acid sequence GYTLSSHG (SEQ ID NO: 1 (0) A heavy chain variable region having three CDRs including ISASHNGHA (SEQ ID NO: 12) and ARVHAALYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs including the amino acid sequences NIGSKS (SEQ ID NO: 109), DDS (SEQ ID NO: 35), and QVWDSSSDHWV (SEQ ID NO: 37), respectively; (n) A heavy chain variable region having three CDRs including the amino acid sequences GYTFTSYG (SEQ ID NO: 85), ISAYNGHA (SEQ ID NO: 94), and ARVHAALYYGMDV (SEQ ID NO: 14), and / or (o) A light chain variable region having three CDRs containing the amino acid sequences NIGSKG (SEQ ID NO: 26), DDS (SEQ ID NO: 35), and QVWDSRSDHWV (SEQ ID NO: 130), (o) a heavy chain variable region having three CDRs containing the amino acid sequences GGTFSSYA (SEQ ID NO: 87), IIPIFGTA (SEQ ID NO: 95), and ARDGSGYDSAGMDD (SEQ ID NO: 102), and / or the amino acid sequences RSNIGSNY (SEQ ID NO: 112), SNN (SEQ ID NO: 121), and AVWDDSLSGVV (SEQ ID NO: 131) (p) A light chain variable region having three CDRs, each containing the amino acid sequences GFTFSSYA (SEQ ID NO: 88), ISYDGSNK (SEQ ID NO: 96), and ARGFGGPDY (SEQ ID NO: 103), and / or a light chain variable region having three CDRs containing the amino acid sequences SGINVGTYR (SEQ ID NO: 113), YKSDSDK (SEQ ID NO: 122), and MIWHSSAYV (SEQ ID NO: 132), each containing the amino acid sequences GYTFSSYG (SEQ ID NO: 89), ISAHNGHA (SEQ ID NO: 12),(r) A heavy chain variable region having three CDRs including ARVHGALYYGMDV (SEQ ID NO: 104), and / or a light chain variable region having three CDRs including the amino acid sequences NIGGKS (SEQ ID NO: 114), DDR (SEQ ID NO: 28), and QVWDSSSDHWV (SEQ ID NO: 37), respectively, (r) a heavy chain variable region having three CDRs including the amino acid sequences GYTLSSHG (SEQ ID NO: 10), ISAHNGHA (SEQ ID NO: 12), and ARVHAALYYGMDV (SEQ ID NO: 14), respectively. A light chain variable region having three CDRs, each containing the amino acid sequences NIGSKG (SEQ ID NO: 26), DDR (SEQ ID NO: 28), and QVWDSSSDHWV (SEQ ID NO: 37), (s) a heavy chain variable region having three CDRs, each containing the amino acid sequences GGTFSSYA (SEQ ID NO: 87), IIPILGIA (SEQ ID NO: 97), and ASGSIVGAAYAFDI (SEQ ID NO: 105), and / or each containing the amino acid sequences NIGGRV (SEQ ID NO: 115), DDT ( (t) A light chain variable region having three CDRs including (sequence number 123) and QVWDSRSDHPV (sequence number 133), (t) a heavy chain variable region having three CDRs including the amino acid sequences GFTFSSYS (sequence number 90), IISDGSAT (sequence number 98), and ARDRSEGGFDP (sequence number 106), and / or three C The study targets isolated antibodies or fragments comprising a light chain variable region having a DR, or (u) a heavy chain variable region having three CDRs containing the amino acid sequences GGTFSRYA (SEQ ID NO: 86), IIPIFGRA (SEQ ID NO: 93), and AEGAFNSLAI (SEQ ID NO: 107), and / or a light chain variable region having three CDRs containing the amino acid sequences SGSIASHF (SEQ ID NO: 117), GDD (SEQ ID NO: 125), and QSYDSSNHVV (SEQ ID NO: 135).

[0012] One aspect of the present invention relates to an isolated monoclonal antibody or antigen-binding fragment that binds to PD-L1, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 8, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 24.

[0013] One aspect of the present invention relates to an isolated monoclonal antibody or antigen-binding fragment that binds to PD-L1, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 16, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 31.

[0014] One aspect of the present invention relates to an isolated monoclonal antibody or antigen-binding fragment that binds to PD-L1, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 16, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 38.

[0015] One aspect of the present invention relates to an isolated monoclonal antibody or antigen-binding fragment that binds to PD-L1, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 16, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 42.

[0016] One aspect of the present invention relates to an isolated monoclonal antibody or antigen-binding fragment that binds to PD-L1, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 16, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 46.

[0017] One aspect of the present invention is an isolated monoclonal antibody or its antigen-binding fragment that binds to PD-L1, comprising a heavy chain, a light chain, or a combination thereof, (a) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 52, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 53. (b) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 54, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 55. (c) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 56, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 57. (d) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 16, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 59. (e) The heavy chain contains an amino acid sequence that is approximately 95% identical to SEQ ID NO: 60, and the light chain contains an amino acid sequence that is approximately 95% identical to SEQ ID NO: 61. (f) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 62, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 63. (g) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 64, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 65. (h) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 66, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 67. (i) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 68, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 69. (j) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 70, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 71. (k) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 72, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 73. (l) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 74, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 75. The (m) heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 76, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 77. (n) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 78, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 79. (o) The heavy chain contains an amino acid sequence that is approximately 95% identical to SEQ ID NO: 80, and the light chain contains an amino acid sequence that is approximately 95% identical to SEQ ID NO: 81, or (p) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 82, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 83. The target is isolated monoclonal antibodies or their antigen-binding fragments.

[0018] One aspect of the present invention is an isolated monoclonal antibody or antigen-binding fragment thereof that binds to PD-L1, comprising a heavy chain, a light chain, or a combination thereof, wherein the antibody or fragment is (a) V having sequence number 8 H V having amino acid sequence and SEQ ID NO: 24 L amino acid sequence, (b) V having sequence number 16 H V having amino acid sequence and SEQ ID NO: 31 L amino acid sequence, (c) V having sequence number 16 H V having amino acid sequence and SEQ ID NO: 38 L amino acid sequence, (d) V having sequence number 16 H V having amino acid sequence and SEQ ID NO: 42 L amino acid sequence, (e) V having sequence number 16 H V having amino acid sequence and SEQ ID NO: 46 L amino acid sequence, (f) V having sequence number 52 H V having amino acid sequence and SEQ ID NO: 53 L amino acid sequence, (g) V having sequence number 54 H V having amino acid sequence and SEQ ID NO: 55 L amino acid sequence, (h) V having SEQ ID NO: 56 H V having an amino acid sequence and SEQ ID NO: 57 L amino acid sequence, (i) V having SEQ ID NO: 16 H V having an amino acid sequence and SEQ ID NO: 59 L amino acid sequence, (j) V having SEQ ID NO: 60 H V having an amino acid sequence and SEQ ID NO: 61 L amino acid sequence, (k) V having SEQ ID NO: 62 H V having an amino acid sequence and SEQ ID NO: 63 L amino acid sequence, (l) V having SEQ ID NO: 64 H V having an amino acid sequence and SEQ ID NO: 65 L amino acid sequence, [[ID=...]] (m) V having SEQ ID NO: 66 H V having an amino acid sequence and SEQ ID NO: 67 L amino acid sequence, (n) V having SEQ ID NO: 68 H V having an amino acid sequence and SEQ ID NO: 69 L amino acid sequence, (o) V having SEQ ID NO: 70 H V having an amino acid sequence and SEQ ID NO: 71 L amino acid sequence, (p) V having SEQ ID NO: 72 H V having an amino acid sequence and SEQ ID NO: 73 L amino acid sequence, (q) V having SEQ ID NO: 74 H V having an amino acid sequence and SEQ ID NO: 75 L amino acid sequence, (r) V having SEQ ID NO: 76 H V having an amino acid sequence and SEQ ID NO: 77 L amino acid sequence, (s) V having SEQ ID NO: H [[ID=...]] V having an amino acid sequence and SEQ ID NO: 79 L amino acid sequence, (t) V having SEQ ID NO: 80 HV having the amino acid sequence and SEQ ID NO: 81 L an amino acid sequence, or (u) V having SEQ ID NO: 82 H V having the amino acid sequence and SEQ ID NO: 83 L an amino acid sequence directed to an isolated monoclonal antibody or an antigen-binding fragment thereof that comprises

[0019] Aspects of the invention are directed to an isolated bispecific antibody comprising a fragment of an antibody described herein and a second antigen-binding fragment that is specific for a molecule on an immune cell. In some embodiments, the molecule on the immune cell is selected from the group consisting of B7H3, B7H4, CD27, CD28, CD40, CD40L, CD47, CD122, CTLA-4, GITR, GITRL, ICOS, ICOS L, LAG-3, LIGHT, OX-40, OX40L, PD-1, TIM3, 4-1BB, TIGIT, VISTA, HEVM, BTLA, and KIR. In some embodiments, each of the fragment and the second fragment is independently selected from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody. In further embodiments, the isolated bispecific antibody further comprises an Fc fragment.

[0020] Aspects of the invention are directed to a nucleic acid encoding an antibody described herein.

[0021] Aspects of the invention are directed to a vector comprising a nucleic acid encoding an antibody described herein.

[0022] Aspects of the invention are directed to a cell comprising a vector comprising a nucleic acid encoding a bispecific antibody described herein.

[0023] Aspects of the invention are directed to a nucleic acid encoding a bispecific antibody described herein.

[0024] Aspects of the invention are directed to a vector comprising a nucleic acid encoding a bispecific antibody described herein.

[0025] Aspects of the present invention relate to cells comprising a vector containing a nucleic acid encoding the bispecific antibody described herein.

[0026] Aspects of the present invention relate to pharmaceutical compositions comprising antibodies or fragments described herein, as well as pharmaceutically acceptable carriers or excipients. In some embodiments, the pharmaceutical composition further comprises at least one additional therapeutic substance. In other embodiments, the therapeutic substance is a toxin, radiolabeled, siRNA, small molecule, or cytokine.

[0027] Aspects of the present invention relate to pharmaceutical compositions comprising the bispecific antibodies described herein and pharmaceutically acceptable carriers or excipients. In some embodiments, the pharmaceutical composition further comprises at least one additional therapeutic substance. In other embodiments, the therapeutic substance is a toxin, radiolabeled, siRNA, small molecule, or cytokine.

[0028] Aspects of the present invention relate to isolated cells containing one or more polynucleotides encoding the antibody or fragment thereof as described herein.

[0029] Aspects of the present invention relate to isolated cells containing one or more polynucleotides encoding the bispecific antibodies described herein.

[0030] Aspects of the present invention relate to a kit comprising at least one antibody as described herein; a syringe, needle, or applicator for administering at least one antibody to a composition subject; and instructions for use.

[0031] One aspect of the present invention relates to a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an intracellular signaling domain, a transmembrane domain, and an extracellular domain, the extracellular domain being an isolated monoclonal antibody or its antigen-binding fragment that binds to the human programmed death ligand 1 (PD-L1) protein, and the monoclonal antibody or fragment comprising a heavy chain, a light chain, or a combination thereof. In some embodiments, the heavy chain is G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 47), G-(X1)-T-(X2)-(X 13 X 14 )-(X3X4)(Sequence No. 205), G-(X1)-TF-(X 13 X 14 CDR1 containing )-Y-(X4)(Sequence ID 206), I-(X8X9X 10 X 11 )-G-(X 12 )-A (Sequence ID 51) or II-(X 15 )-IFG-(X 16 CDR2 containing )-A (SEQ ID NO: 207), and / or CDR3 containing ARGRQMFGAGIDF (SEQ ID NO: 6), ARVHAALYYGMDV (SEQ ID NO: 14), TTGGLGLVYPYYNYIDV (SEQ ID NO: 99), AKVHPVFSYALDV (SEQ ID NO: 100), AEGAFNSLAI (SEQ ID NO: 101), ARDGSGYDSAGMDD (SEQ ID NO: 102), ARGFGGPDY (SEQ ID NO: 103), ARVHGALYYGMDV (SEQ ID NO: 104), ASGSIVGAAYAFDI (SEQ ID NO: 105), ARDRSEGGFDP (SEQ ID NO: 106), or AEGAFNSLAI (SEQ ID NO: 107). In another embodiment, the light chain contains SGSIDSNY (SEQ ID NO: 18), S-(X 17 X 18 )I-(X 19 )-SNY (sequence number 208), or NIG-(X5)-K-(X 20 CDR1 containing (SEQ ID NO: 48), EDN (SEQ ID NO: 20), (X 21 )-DN(Sequence ID 209), (X 22The CDR2 comprises )-NN (SEQ ID NO: 210) or DD-X6 (SEQ ID NO: 49), and / or CDR3 comprises QSYDSNNRHVI (SEQ ID NO: 22), QVWDS-(X7)-SDHWV (SEQ ID NO: 50), QVWDSSGDLWV (SEQ ID NO: 126), AAWDDSLNGLV (SEQ ID NO: 127), QSYDGITVI (SEQ ID NO: 128), QSYDSSNHWV (SEQ ID NO: 129), AVWDDSLSGVV (SEQ ID NO: 131), MIWHSSAYV (SEQ ID NO: 132), NSRDISDNQWQWI (SEQ ID NO: 134), or QSYDSSNHVV (SEQ ID NO: 135). In some embodiments, the antibody for CAR is a fully human antibody or is humanized. In other embodiments, the antibody for CAR is monospecific, bispecific, or multispecific. In further embodiments, the antibody for CAR is a single-chain antibody. In one embodiment, X1, X2, X3, or X4 is a nonpolar amino acid residue. In another embodiment, X1, X2, X 3、 Alternatively, X4 is glycine (G), tyrosine (Y), phenylalanine (F), leucine (L), or alanine (A). In some embodiments, X1, X2, or X4 are hydrophobic amino acid residues, for example, X1, X2, or X4 are glycine (G), leucine (L), or alanine (A). In some embodiments, X3 is a hydrophilic polar amino acid residue. In one embodiment, X3 is histidine (H). In some embodiments, X1 is phenylalanine (F), glycine (G), or tyrosine (Y). In further embodiments, X2 is phenylalanine (F) or leucine (L). In other embodiments, X3 is histidine (H) or tyrosine (Y). In yet another embodiment, X4 is serine (S), glycine (G), or alanine (A). In one embodiment, X8, X9, X 10 , or X 11 These are nonpolar hydrophobic amino acid residues. In some embodiments, X8, X9, X 10 , or X 11 isoleucine (I), proline (P), alanine (A), or phenylalanine (F). In other embodiments, X8, X 10, or X 12 These are polar hydrophilic amino acid residues. In yet another embodiment, X8, X 10 , or X 12 X8 is histidine (H), serine (S), asparagine (N), or threonine (T). In one embodiment, X8 is alanine (A), isoleucine (I), or serine (S). In one embodiment, X9 is tyrosine (Y), serine (S), proline (P), or alanine (A). In one embodiment, X 10 is tyrosine (Y), aspartic acid (D), isoleucine (I), or histidine (H). In one embodiment, X 11 is glycine (G), leucine (L), asparagine (N), or phenylalanine (F). In one embodiment, X 12 X5 is isoleucine (I), arginine (R), threonine (T), or histidine (H). In some embodiments, X5 is a nonpolar hydrophobic amino acid residue. In one embodiment, X5 is glycine (G). In other embodiments, X5 is a polar hydrophilic amino acid residue. In one embodiment, X5 is serine (S), asparagine (N), or aspartic acid (D). In further embodiments, X6 is a nonpolar amino acid residue. In one embodiment, X6 is tyrosine (Y). In some embodiments, X6 is a polar hydrophilic amino acid residue. In one embodiment, X6 is threonine (T), serine (S), or arginine (R). In some embodiments, X7, X 15 , X 16 , X 17 , X 19 , X 20 , or X 21 These are nonpolar hydrophobic amino acid residues. In one embodiment, X7, X 17 , or X 20 This is glycine (G). In other embodiments, X7, X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , or X 21These are polar hydrophilic amino acid residues. In one embodiment, X7, X 14 , or X 21 is serine (S) or arginine (R). In one embodiment, X 13 is serine (S) or threonine (T). In one embodiment, X 15 is proline (P). In one embodiment, X 15 , X 17 , or X 20 is serine(S). In one embodiment, X 16 is threonine (T) or arginine (R). In one embodiment, X 16 isoleucine(I). In one embodiment, X 18 is serine (S) or asparagine (N). In one embodiment, X 19 is glycine (G) or alanine (A). In one embodiment, X 19 is aspartic acid (D). In one embodiment, X 21 is alanine (A). In one embodiment, X 21 The protein is glutamic acid (E). In some embodiments, the transmembrane domain further includes a stalk region located between the extracellular domain and the transmembrane domain. In other embodiments, the transmembrane domain includes CD28. In some embodiments, the CAR further includes one or more additional costimulatory molecules located between the transmembrane domain and the intracellular signaling domain. In further embodiments, the costimulatory molecules are CD28, 4-1BB, ICOS, or OX40. In some embodiments, the intracellular signaling domain includes a CD3 zeta chain. In other embodiments, the antibody of the CAR is Fab or scFV.

[0032] Aspects of the present invention relate to nucleic acids encoding CARs as described herein. In some embodiments, the nucleic acid encoding a CAR further comprises a nucleic acid encoding a polypeptide located after an intracellular signaling domain. In some embodiments, the polypeptide is an antibody or a cytokine. In other embodiments, the antibody is scFV.

[0033] Aspects of the present invention relate to nucleic acids encoding CARs. In one embodiment, the CAR comprises an intracellular signaling domain, a transmembrane domain, and an extracellular domain, and further comprises a nucleic acid encoding a polypeptide located after the intracellular signaling domain, wherein the polypeptide comprises an isolated monoclonal antibody or its antigen-binding fragment that binds to the human programmed death ligand 1 (PD-L1) protein, and the monoclonal antibody or its fragment comprises a heavy chain, a light chain, or a combination thereof. In some embodiments, the heavy chain is G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 47), G-(X1)-T-(X2)-(X 13 X 14 )-(X3X4)(Sequence No. 205), G-(X1)-TF-(X 13 X 14 CDR1 containing )-Y-(X4)(Sequence ID 206), I-(X8X9X 10 X 11 )-G-(X 12 )-A (Sequence ID 51) or II-(X 15 )-IFG-(X 16 CDR2 containing )-A (SEQ ID NO: 207), and / or CDR3 containing ARGRQMFGAGIDF (SEQ ID NO: 6), ARVHAALYYGMDV (SEQ ID NO: 14), TTGGLGLVYPYYNYIDV (SEQ ID NO: 99), AKVHPVFSYALDV (SEQ ID NO: 100), AEGAFNSLAI (SEQ ID NO: 101), ARDGSGYDSAGMDD (SEQ ID NO: 102), ARGFGGPDY (SEQ ID NO: 103), ARVHGALYYGMDV (SEQ ID NO: 104), ASGSIVGAAYAFDI (SEQ ID NO: 105), ARDRSEGGFDP (SEQ ID NO: 106), or AEGAFNSLAI (SEQ ID NO: 107). In another embodiment, the light chain contains SGSIDSNY (SEQ ID NO: 18), S-(X 17 X 18 )I-(X 19 )-SNY (sequence number 208), or NIG-(X5)-K-(X 20 CDR1 containing (SEQ ID NO: 48), EDN (SEQ ID NO: 20), (X 21 )-DN(Sequence ID 209), (X 22The CDR2 includes )-NN (SEQ ID NO: 210) or DD-X6 (SEQ ID NO: 49), and / or the CDR3 includes QSYDSNNRHVI (SEQ ID NO: 22), QVWDS-(X7)-SDHWV (SEQ ID NO: 50), QVWDSSGDLWV (SEQ ID NO: 126), AAWDDSLNGLV (SEQ ID NO: 127), QSYDGITVI (SEQ ID NO: 128), QSYDSSNHWV (SEQ ID NO: 129), AVWDDSLSGVV (SEQ ID NO: 131), MIWHSSAYV (SEQ ID NO: 132), NSRDISDNQWQWI (SEQ ID NO: 134), or QSYDSSNHVV (SEQ ID NO: 135). In some embodiments, the antibody for CAR is a fully human antibody or is humanized. In other embodiments, the antibody for CAR is monospecific, bispecific, or multispecific. In further embodiments, the antibody for CAR is a single-chain antibody. In this embodiment, X1, X2, X3, or X4 are nonpolar amino acid residues. In other embodiments, X1, X2, X 3、 Alternatively, X4 is glycine (G), tyrosine (Y), phenylalanine (F), leucine (L), or alanine (A). In some embodiments, X1, X2, or X4 are hydrophobic amino acid residues, for example, X1, X2, or X4 are glycine (G), leucine (L), or alanine (A). In some embodiments, X3 is a hydrophilic polar amino acid residue. In one embodiment, X3 is histidine (H). In some embodiments, X1 is phenylalanine (F), glycine (G), or tyrosine (Y). In further embodiments, X2 is phenylalanine (F) or leucine (L). In other embodiments, X3 is histidine (H) or tyrosine (Y). In yet another embodiment, X4 is serine (S), glycine (G), or alanine (A). In one embodiment, X8, X9, X 10 , or X 11 These are nonpolar hydrophobic amino acid residues. In some embodiments, X8, X9, X 10 , or X 11 isoleucine (I), proline (P), alanine (A), or phenylalanine (F). In other embodiments, X8, X 10 , or X12 These are polar hydrophilic amino acid residues. In yet another embodiment, X8, X 10 , or X 12 X8 is histidine (H), serine (S), asparagine (N), or threonine (T). In one embodiment, X8 is alanine (A), isoleucine (I), or serine (S). In one embodiment, X9 is tyrosine (Y), serine (S), proline (P), or alanine (A). In one embodiment, X 10 is tyrosine (Y), aspartic acid (D), isoleucine (I), or histidine (H). In one embodiment, X 11 is glycine (G), leucine (L), asparagine (N), or phenylalanine (F). In one embodiment, X 12 X5 is isoleucine (I), arginine (R), threonine (T), or histidine (H). In some embodiments, X5 is a nonpolar hydrophobic amino acid residue. In one embodiment, X5 is glycine (G). In other embodiments, X5 is a polar hydrophilic amino acid residue. In one embodiment, X5 is serine (S), asparagine (N), or aspartic acid (D). In further embodiments, X6 is a nonpolar amino acid residue. In one embodiment, X6 is tyrosine (Y). In some embodiments, X6 is a polar hydrophilic amino acid residue. In one embodiment, X6 is threonine (T), serine (S), or arginine (R). In some embodiments, X7, X 15 , X 16 , X 17 , X 19 , X 20 , or X 21 These are nonpolar hydrophobic amino acid residues. In one embodiment, X7, X 17 , or X 20 This is glycine (G). In other embodiments, X7, X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , or X 21These are polar hydrophilic amino acid residues. In one embodiment, X7, X 14 , or X 21 is serine (S) or arginine (R). In one embodiment, X 13 is serine (S) or threonine (T). In one embodiment, X 15 is proline (P). In one embodiment, X 15 , X 17 , or X 20 is serine(S). In one embodiment, X 16 is threonine (T) or arginine (R). In one embodiment, X 16 isoleucine(I). In one embodiment, X 18 is serine (S) or asparagine (N). In one embodiment, X 19 is glycine (G) or alanine (A). In one embodiment, X 19 is aspartic acid (D). In one embodiment, X 21 is alanine (A). In one embodiment, X 21 The protein is glutamic acid (E). In some embodiments, the transmembrane domain further includes a stalk region located between the extracellular domain and the transmembrane domain. In other embodiments, the transmembrane domain includes CD28. In some embodiments, the CAR further includes one or more additional costimulatory molecules located between the transmembrane domain and the intracellular signaling domain. In further embodiments, the costimulatory molecules are CD28, 4-1BB, ICOS, or OX40. In some embodiments, the intracellular signaling domain includes a CD3 zeta chain. In other embodiments, the antibody of the CAR is Fab or scFV.

[0034] Aspects of the present invention relate to vectors containing nucleic acids encoding CARs as described herein.

[0035] Aspects of the present invention relate to cells hosting a vector containing a nucleic acid encoding a CAR as described herein.

[0036] Aspects of the present invention relate to genetically engineered cells expressing CARS as described herein. In one embodiment, the cells express and retain the chimeric antigen receptor described herein on their cell surface membrane. In some embodiments, the cells are T cells or NK cells. In further embodiments, the T cells are CD4 + or CD8 + In other embodiments, genetically modified cells are CD4 + and CD8 cells + This includes a mixed population of the following:

[0037] Aspects of the present invention relate to genetically engineered cells that express and retain a chimeric antigen receptor on the cell surface membrane, further engineered to express and secrete a polypeptide, wherein the polypeptide is an isolated monoclonal antibody or its antigen-binding fragment that binds to the human programmed death ligand 1 (PD-L1) protein, and the monoclonal antibody or its fragment comprises a heavy chain, a light chain, or a combination thereof. In some embodiments, the heavy chain is G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 47), G-(X1)-T-(X2)-(X 13 X 14 )-(X3X4)(Sequence No. 205), G-(X1)-TF-(X 13 X 14 CDR1 containing )-Y-(X4)(Sequence ID 206), I-(X8X9X 10 X 11 )-G-(X 12 )-A (Sequence ID 51) or II-(X 15 )-IFG-(X 16CDR2 containing )-A (SEQ ID NO: 207), and / or CDR3 containing ARGRQMFGAGIDF (SEQ ID NO: 6), ARVHAALYYGMDV (SEQ ID NO: 14), TTGGLGLVYPYYNYIDV (SEQ ID NO: 99), AKVHPVFSYALDV (SEQ ID NO: 100), AEGAFNSLAI (SEQ ID NO: 101), ARDGSGYDSAGMDD (SEQ ID NO: 102), ARGFGGPDY (SEQ ID NO: 103), ARVHGALYYGMDV (SEQ ID NO: 104), ASGSIVGAAYAFDI (SEQ ID NO: 105), ARDRSEGGFDP (SEQ ID NO: 106), or AEGAFNSLAI (SEQ ID NO: 107). In another embodiment, the light chain contains SGSIDSNY (SEQ ID NO: 18), S-(X 17 X 18 )I-(X 19 )-SNY (sequence number 208), or NIG-(X5)-K-(X 20 CDR1 containing (SEQ ID NO: 48), EDN (SEQ ID NO: 20), (X 21 )-DN(Sequence ID 209), (X 22 The CDR2 includes )-NN (SEQ ID NO: 210) or DD-X6 (SEQ ID NO: 49), and / or the CDR3 includes QSYDSNNRHVI (SEQ ID NO: 22), QVWDS-(X7)-SDHWV (SEQ ID NO: 50), QVWDSSGDLWV (SEQ ID NO: 126), AAWDDSLNGLV (SEQ ID NO: 127), QSYDGITVI (SEQ ID NO: 128), QSYDSSNHWV (SEQ ID NO: 129), AVWDDSLSGVV (SEQ ID NO: 131), MIWHSSAYV (SEQ ID NO: 132), NSRDISDNQWQWI (SEQ ID NO: 134), or QSYDSSNHVV (SEQ ID NO: 135). In some embodiments, the antibody for CAR is a fully human antibody or is humanized. In other embodiments, the antibody for CAR is monospecific, bispecific, or multispecific. In further embodiments, the antibody for CAR is a single-chain antibody. In this embodiment, X1, X2, X3, or X4 are nonpolar amino acid residues. In other embodiments, X1, X2, X 3、Alternatively, X4 is glycine (G), tyrosine (Y), phenylalanine (F), leucine (L), or alanine (A). In some embodiments, X1, X2, or X4 are hydrophobic amino acid residues, for example, X1, X2, or X4 are glycine (G), leucine (L), or alanine (A). In some embodiments, X3 is a hydrophilic polar amino acid residue. In one embodiment, X3 is histidine (H). In some embodiments, X1 is phenylalanine (F), glycine (G), or tyrosine (Y). In further embodiments, X2 is phenylalanine (F) or leucine (L). In other embodiments, X3 is histidine (H) or tyrosine (Y). In yet another embodiment, X4 is serine (S), glycine (G), or alanine (A). In one embodiment, X8, X9, X 10 , or X 11 These are nonpolar hydrophobic amino acid residues. In some embodiments, X8, X9, X 10 , or X 11 isoleucine (I), proline (P), alanine (A), or phenylalanine (F). In other embodiments, X8, X 10 , or X 12 These are polar hydrophilic amino acid residues. In yet another embodiment, X8, X 10 , or X 12 X8 is histidine (H), serine (S), asparagine (N), or threonine (T). In one embodiment, X8 is alanine (A), isoleucine (I), or serine (S). In one embodiment, X9 is tyrosine (Y), serine (S), proline (P), or alanine (A). In one embodiment, X 10 is tyrosine (Y), aspartic acid (D), isoleucine (I), or histidine (H). In one embodiment, X 11 is glycine (G), leucine (L), asparagine (N), or phenylalanine (F). In one embodiment, X 12X5 is isoleucine (I), arginine (R), threonine (T), or histidine (H). In some embodiments, X5 is a nonpolar hydrophobic amino acid residue. In one embodiment, X5 is glycine (G). In other embodiments, X5 is a polar hydrophilic amino acid residue. In one embodiment, X5 is serine (S), asparagine (N), or aspartic acid (D). In further embodiments, X6 is a nonpolar amino acid residue. In one embodiment, X6 is tyrosine (Y). In some embodiments, X6 is a polar hydrophilic amino acid residue. In one embodiment, X6 is threonine (T), serine (S), or arginine (R). In some embodiments, X7, X 15 , X 16 , X 17 , X 19 , X 20 , or X 21 These are nonpolar hydrophobic amino acid residues. In one embodiment, X7, X 17 , or X 20 This is glycine (G). In other embodiments, X7, X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , or X 21 These are polar hydrophilic amino acid residues. In one embodiment, X7, X 14 , or X 21 is serine (S) or arginine (R). In one embodiment, X 13 is serine (S) or threonine (T). In one embodiment, X 15 is proline (P). In one embodiment, X 15 , X 17 , or X 20 is serine(S). In one embodiment, X 16 is threonine (T) or arginine (R). In one embodiment, X 16 isoleucine(I). In one embodiment, X 18 is serine (S) or asparagine (N). In one embodiment, X 19is glycine (G) or alanine (A). In one embodiment, X 19 is aspartic acid (D). In one embodiment, X 21 is alanine (A). In one embodiment, X 21 This is glutamic acid (E).

[0038] One aspect of the present invention relates to a method for treating cancer in a subject. In some embodiments, the method comprises administering to a subject in need of such treatment a therapeutically effective amount of a composition comprising an antibody described herein, a bispecific antibody described herein, a pharmaceutical composition described herein, or a CAR composition described herein. In further embodiments, the method further comprises administering to the subject a chemotherapeutic agent.

[0039] Other objects and advantages of the present invention will be readily apparent from the following description. [Brief explanation of the drawing]

[0040] [Figure 1] A schematic diagram of bispecific GITR-PDL1 light chain fusion is shown. [Figure 2-1] Figure 2 is a graph showing the FACS plot and binding curve of the PD-L1 antibody. [Figure 2-2] See the explanation in Figure 2-1. [Figure 3-1] Figure 3 shows the FACS binding curve of 293T cells stably expressing PD-L1. The antibody was detected via an anti-hFc secondary antibody. [Figure 3-2] See the explanation in Figure 3-1. [Figure 4] This is a schematic diagram showing the kinetic measurements of the aPDL1 antibody (top image). Based on a previous series of competing matrices, a representative clone was used in the final matrix (bottom image). [Figure 5] This graph shows the negative background binding of anti-PDL1 scFv-Fcs to 293T cells. [Figure 6]This graph shows the results of a mixed lymphocyte reaction (MLR) assay to test the biological activity of anti-PDL1 clones. IFNγ was detected by ELISA as a measure of T cell activation. Development of the ELISA plate shows that several clones are comparable to atez. [Figure 7] This is a bar graph showing the results of MLR using αPD-L1 antibody. [Figure 8] This bar graph shows the results of MLR using αPD-L1 antibody (150 nM). [Figure 9] This bar graph shows the results of MLR using αPD-L1 antibody (150 nM). [Figure 10-1] Figure 10 is a schematic diagram of the variable region heavy chain germline alignment (amino acid sequence). [Figure 10-2] See the explanation in Figure 10-1. [Modes for carrying out the invention]

[0041] Detailed description of the invention Abbreviations and definitions A detailed description of one or more preferred embodiments is provided herein. However, it is understood that the invention can be embodied in a variety of forms. Accordingly, the specific details disclosed herein should not be construed as limitations, but rather as representative grounds for the claims and for teaching those skilled in the art to use the invention in any suitable way.

[0042] The singular forms “a,” “an,” and “the” include multiple references unless the context explicitly indicates otherwise. The use of the words “a” or “an” in the claims and / or specification with the term “includes” may mean “one,” but also coincides with the meanings of “one or more,” “at least one,” and “one or more.”

[0043] Whenever any of the phrases "for example," "etc.," or "including" are used herein, it is understood that they are always accompanied by the phrase "without limitation" unless otherwise explicitly stated. Similarly, "for example," "exemplary," etc., are understood to be non-limiting.

[0044] The term "substantially" allows for deviations from descriptive terms that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term "substantially," even if the term is not explicitly listed.

[0045] Terms such as “comprising,” “including,” “having,” and “involving” (as well as “comprises,” “includes,” “has,” and “involves”) are used interchangeably and have the same meaning. Specifically, each term is defined in accordance with the general U.S. Patent Law definition of “comprising,” and is therefore interpreted as an open term meaning “at least the following,” and also as not excluding any additional features, limitations, aspects, etc. Thus, for example, “a process comprising steps a, b, and c” means that the process comprises at least steps a, b, and c. Whenever the terms “a” or “an” are used, they are understood to mean “one or more,” unless such an interpretation is meaningless in the context.

[0046] As used herein, the term “approximately” means roughly, roughly, about, or within that range. When the term “approximately” is used with a numerical range, it modifies that range by extending the upper and lower boundaries of the stated numerical value. Generally, the term “approximately” is used herein to qualify numerical values ​​that are above and below a stated value by a 20 percent above or below (higher or lower) variance.

[0047] PD-L1 Programmed T cell death 1 (PD-1) is a transmembrane protein present on the surface of T cells. When it binds to programmed T cell death ligand 1 (PD-L1) on tumor cells, it results in the suppression of T cell activity and a reduction in T cell-mediated cytotoxicity. Therefore, PD-1 and PD-L1 are immune down regulators or "off switches" for immune checkpoints.

[0048] The immune system must maintain a balance between an effective response to eliminate pathogens and the maintenance of resistance to prevent autoimmune diseases. T cells are central to maintaining this balance, and their proper regulation is primarily regulated by molecules of the B7-CD28 family. The interaction between members of the B7 family, which function as ligands, and members of the CD28 family, which function as receptors, provides important positive signals that initiate, enhance, and maintain T cell responses, as well as important negative signals that limit, terminate, and / or attenuate T cell responses as needed. PD-1 is a member of the CD28 family.

[0049] The binding between PD-L1 and PD-1 significantly influences the regulation of T cell responses. Specifically, the PD-L1 / PD-1 interaction inhibits T cell proliferation, as well as the production of effector cytokines that mediate T cell activity and immune responses, such as IL-2 and IFN-γ. This negative regulatory function is crucial for preventing T cell-mediated autoimmunity and immunopathology. However, the PD-1 / PD-L1 axis has also been shown to be involved in T cell depletion, thereby inhibiting T cell responses and adversely impacting the host. Long-term or chronic antigen stimulation of T cells can induce a negative immunological feedback mechanism, thereby inhibiting antigen-specific responses and potentially leading to immune evasion of pathogens. T cell depletion may also progress to the physical deletion of antigen-specific T cells themselves. The expression of PD-1 in T cells is upregulated in response to chronic antigen stimulation, and its binding to PD-L1 blocks effector function in both CD4+ (T helper cells) and CD8+ (cytotoxic T lymphocytes or CTLs) T cells, thus indicating that the PD-1 / PD-L1 interaction is involved in the induction of T cell depletion.

[0050] Recent studies have shown that some chronic viral infections and cancers have developed immune evasion tactics that specifically utilize the PD-1 / PD-L1 axis by causing T cell depletion via PD-1 / PD-L1. Many human tumor cells and tumor-associated antigen-presenting cells express high levels of PD-L1, suggesting that tumors induce T cell depletion to evade anti-tumor immune responses. For example, during chronic HIV infection, HIV-specific CD8+ T cells are functionally impaired, with reduced ability to produce cytokines and effector molecules, and reduced proliferative capacity. Studies have shown that PD-1 is highly expressed in HIV-specific CD8+ T cells of HIV-infected individuals, suggesting therapeutic potential in the treatment of HIV infection and AIDS patients by blocking the PD-1 / PD-L1 pathway. In summary, agents that block the PD-1 / PD-L1 pathway offer novel therapeutic approaches for various cancers, HIV infection, and / or other diseases and conditions associated with T cell depletion. Therefore, there is an urgent need for agents that can block or prevent the interaction between PD-1 and PD-L1.

[0051] Overexpression of PD-L1 has been detected in various cancers. For example, in breast cancer, PD-L1 overexpression is associated with a high-risk prognostic factor. In renal cell carcinoma, PD-L1 is upregulated, and increased PD-1 expression is also observed in tumor-infiltrating leukocytes. Anti-PD-L1 and anti-PD-1 antibodies have shown some clinical efficacy in Phase I trials for renal cell carcinoma. Therapeutic substances that can bind to PD-1 or PD-L1 may be useful in specifically targeting tumor cells. Agents that can block PD-1 / PD-L1 interactions may be even more useful in treating cancers that evade anti-tumor T cell activity by inducing T cell depletion. The use of such agents alone or in combination with other anticancer agents may enable effective targeting of tumor cells that overexpress PD-L1, thereby enhancing anti-tumor T cell activity and thereby boosting the immune response against the targeted tumor cells.

[0052] PD-1 and PD-L1 can also be upregulated by T cells after chronic antigen stimulation, for example, by chronic infections. During chronic HIV infection, HIV-specific CD8+ T cells are functionally impaired, with reduced ability to produce cytokines and effector molecules, and decreased proliferative capacity. PD-1 is highly expressed in HIV-specific CD8+ T cells of HIV-infected individuals. Therefore, blocking this pathway may enhance the ability of HIV-specific T cells to proliferate and produce cytokines in response to stimulation by HIV peptides, thereby enhancing the immune response to HIV. Other chronic infections, such as chronic viral infections, bacterial infections, and parasitic infections, may also benefit from the use of PD-1 / PD-L1 blockers.

[0053] Aspects of the present invention provide isolated monoclonal antibodies specific to PDL-1. Where used herein in relation to cells, DNA, or RNA, the term “isolated” refers to a molecule isolated from other DNA or RNA present in a natural source of macromolecules. The term “isolated” may also refer to a nucleic acid or peptide that is substantially free from cellular material, viral material, or culture medium, if produced by recombinant DNA technology, or from chemical precursors or other chemicals, if chemically synthesized. For example, “isolated nucleic acid” may include nucleic acid fragments that do not exist naturally as fragments and would not be found in their natural state. “Isolated” may also refer to cells or polypeptides isolated from other cellular proteins or tissues. Isolated polypeptides can include both purified polypeptides and recombinant polypeptides. The isolated antibodies were identified through the use of a 27 billion human single-chain antibody (scFv) phage display library by using PDL-1 as the library selection target. These antibodies represent a novel class of monoclonal antibodies against PD-L1.

[0054] Five unique recombinant monoclonal PD-L1 antibodies are described herein. These include 40mut, 50-6B6.1mut, 50-6B6.2, 50-7B3, and 50-5B9. “Recombinant” in relation to polypeptides (such as antibodies) or polynucleotides may refer to forms of polypeptides or polynucleotides that do not exist in nature, and non-limiting examples of such recombinant forms can be created by combining polynucleotides or polypeptides that do not normally coexist.

[0055] The nucleic acid and amino acid sequences of the monoclonal PD-L1 antibody are provided below; the amino acid sequences of the heavy and light chain complementarity-determining regions (CDRs) of the PD-L1 antibody are provided below. The filename is JPEG2026048871000002.jpg5157.

[0056] (Table 1) Ab 40mut variable region amino acid sequence JPEG2026048871000003.jpg59169

[0057] (Table 2B) Ab 50-6B6.1 mut Variable region amino acid sequence JPEG2026048871000004.jpg65169

[0058] (Table 3B) Ab 50-6B6.2 Variable region amino acid sequence JPEG2026048871000005.jpg65169

[0059] (Table 4B) Ab 50-6B6.2 Variable region amino acid sequence JPEG2026048871000006.jpg65169

[0060] (Table 5B) Ab 50-5B9 Variable Region Amino Acid Sequence JPEG2026048871000007.jpg65169

[0061] (Table 6) Ab 14C61 Variable Region Amino Acid Sequence JPEG2026048871000008.jpg66169

[0062] (Table 7) Ab 1A2 Variable Region Amino Acid Sequence JPEG2026048871000009.jpg65169

[0063] (Table 8) Ab 1A3 Variable Region Amino Acid Sequence JPEG2026048871000010.jpg66169

[0064] (Table 9) Ab 1A6 Variable Region Amino Acid Sequence JPEG2026048871000011.jpg65169

[0065] (Table 10) Ab 1B4 Variable Region Amino Acid Sequence JPEG2026048871000012.jpg65169

[0066] (Table 11) Ab 1C1 variable region amino acid sequence JPEG2026048871000013.jpg65169

[0067] (Table 12) Ab 1C4 variable region amino acid sequence JPEG2026048871000014.jpg65169

[0068] (Table 13) Ab 1C6 variable region amino acid sequence JPEG2026048871000015.jpg65169

[0069] (Table 14) Ab 1D1 Variable Region Amino Acid Sequence JPEG2026048871000016.jpg66169

[0070] (Table 15) Ab 1D2 Variable Region Amino Acid Sequence JPEG2026048871000017.jpg65169

[0071] (Table 16) Ab 1D4 Variable Region Amino Acid Sequence JPEG2026048871000018.jpg72169

[0072] (Table 17) Ab 1E1 Variable Region Amino Acid Sequence JPEG2026048871000019.jpg65169

[0073] (Table 18) Ab 1F1 variable region amino acid sequence JPEG2026048871000020.jpg65169

[0074] (Table 19) Ab 1G1 variable region amino acid sequence JPEG2026048871000021.jpg66169

[0075] (Table 20) Ab 1H2 variable region amino acid sequence JPEG2026048871000022.jpg65169

[0076] (Table 21) Ab 1H5 variable region amino acid sequence JPEG2026048871000023.jpg65169

[0077] The amino acid sequences of the heavy and light chain complementarity-determining regions of the PDL-1 antibody are shown in Tables 6A and 6B below.

[0078] (Table 6A) Heavy chain of PDL-1 antibody (V H Complementarity Determination Region (CDR) of ) JPEG2026048871000024.jpg245150

[0079] (Table 6B) Light chain of PDL-1 antibody (V L Complementarity Determination Region (CDR) of ) JPEG2026048871000025.jpg245148

[0080] The amino acid sequences of the heavy and light chain framework regions of the PDL-1 antibody are shown in Tables 7A and 7B below.

[0081] (Table 7A) Heavy chain of PDL-1 antibody (V H ) Framework domain (FR) JPEG2026048871000026.jpg88165JPEG2026048871000027.jpg250169JPEG2026048871000028.jpg166167

[0082] (Table 7B) Heavy chain of PDL-1 antibody (V L ) Framework domain (FR) JPEG2026048871000029.jpg35169JPEG2026048871000030.jpg243169JPEG2026048871000031.jpg230169

[0083] The PD-L1 antibodies described herein bind to PD-L1. In one embodiment, the PD-L1 antibody has high affinity and high specificity for PD-L1. Some embodiments also feature antibodies that have a certain percentage of identity or similarity to the amino acid or nucleotide sequence of the anti-PD-L1 antibody described herein. For example, “homologousity” or “identity” or “similarity” refers to sequence similarity between two peptides or two nucleic acid molecules. Homologousity can be determined by comparing the positions of each sequence, which may be aligned for comparison purposes. If the positions of the sequences being compared are occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. For example, an antibody may have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher amino acid sequence identity when compared to a specific region or the full length of any one of the anti-PD-L1 antibodies described herein. The sequence identity or similarity of the nucleic acids and proteins of the present invention can be determined by sequence comparison and / or alignment using methods known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology, or by software programs known in the art. For example, the sequence identity or similarity percentage of the nucleic acids and proteins of the present invention can be determined using sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

[0084] As used herein, “polypeptide” can encompass a single “polypeptide” as well as multiple “polypeptides,” and refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any one or more chains of two or more amino acids and does not refer to a specific length of the product. Thus, any other term used to refer to a peptide, dipeptide, tripeptide, oligopeptide, “protein,” “amino acid chain,” or a chain of two or more amino acids may, as herein, refer to a “polypeptide,” and the term “polypeptide” may be used in place of or interchangeably with any of these terms. “Polypeptide” may also refer to post-expression modified products of a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification with amino acids that do not exist in nature. Polypeptides may originate from natural biological sources or be produced by recombinant technology and do not necessarily have to be translated from a specific nucleic acid sequence. They may be produced by any method, including chemical synthesis. With respect to amino acid sequences, those skilled in the art will readily recognize that individual substitutions, deletions, or additions to nucleic acids, peptides, polypeptides, or protein sequences that modify, add, delete, or substitute a single amino acid or a small percentage of amino acids in the encoded sequence are collectively referred to herein as “conservatively modified variants.” In some embodiments, the modification results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the art.

[0085] For example, a "conservative amino acid substitution" is one in which an amino acid residue is substituted with an amino acid residue having a similar side chain. In this art, families of amino acid residues having similar side chains are defined as basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues in immunoglobulin polypeptides are preferably substituted with other amino acid residues derived from the same side chain family. In another embodiment, the amino acid chain can be substituted with structurally similar chains that differ in the order and / or composition of their side chain family members.

[0086] antibody As used herein, “antibody” or “antigen-binding polypeptide” may refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody may be a whole antibody, any antigen-binding fragment, or a single chain thereof. For example, an “antibody” may include any protein or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule having biological activity to bind to an antigen. Non-limiting examples include the complementarity-determining region (CDR) of a heavy or light chain or its ligand-binding portion, the variable region of a heavy or light chain, the constant region of a heavy or light chain, the framework (FR) region, or any portion thereof, or at least a portion of a binding protein. As used herein, the term “antibody” may refer to an immunoglobulin molecule and an immunoglobulin (Ig) molecule, i.e., the immunoactive portion of a molecule containing an antigen-binding site that specifically binds to (immunely reacts with) an antigen. “Specifically binding” or “immunely reacting” means that the antibody reacts with one or more antigenicity-determining sites of a desired antigen and not with other polypeptides.

[0087] As used herein, the terms “antibody fragment” or “antigen-binding fragment” refer to F (ab’)2 F (ab)2 F ab ', F abThis refers to a portion of an antibody, such as Fv, scFv, etc. Regardless of its structure, an antibody fragment binds to the same antigen recognized by the complete antibody. The term "antibody fragment" can also encompass aptamers (such as Spiegelmer), minibodies, and diabodies. The term "antibody fragment" can also encompass any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen and forming a complex. The antibodies, antigen-binding polypeptides, variants, or derivatives described herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', F(ab')2, Fd, Fvs, single-chain Fv(scFv), single-chain antibodies, dAb (domain antibodies), minibodies, disulfide-binding Fv(sdFv), fragments containing any of the VL or VH domains, fragments generated by Fab expression libraries, and anti-idiotype (anti-Id) antibodies.

[0088] "Single-chain variable fragment" or "scFv" refers to the heavy chain (V) of immunoglobulins. H ) and light chain (V L This refers to a fusion protein of the variable region of ). A single-chain Fv ("scFv") polypeptide molecule is a covalently linked VH:VL heterodimer, which can be expressed from a gene fusion containing VH and VL coding genes linked by a peptide coding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). In some embodiments, the region is linked by a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility and serine or threonine for solubility, in which case V H The N-terminus and V LThe C-terminus can be linked to the C-terminus, or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker. Numerous methods have been described for identifying the chemical structure to convert naturally aggregated but chemically separated light and heavy polypeptide chains from the antibody V region into scFv molecules that will fold into a three-dimensional structure substantially similar to the structure of the antigen-binding site. See, for example, U.S. Patents 5,091,513, 5,892,019, 5,132,405, and 4,946,778, which incorporate their entirety by reference, respectively.

[0089] Very large naive human scFv libraries have been constructed and can be constructed to provide a large source of antibody genes rearranged for numerous target molecules. Smaller libraries can be constructed from individuals with infectious diseases to isolate disease-specific antibodies. (See Barbas et al., Proc. Natl. Acad. Sci. USA 89:9339-43 (1992), Zebedee et al, Proc. Natl. Acad. Sci. USA 89:3 175-79 (1992)).

[0090] Antibody molecules obtained from humans are classified into five classes of immunoglobulins: IgG, IgM, IgA, IgE, and IgD, which differ from one another in the properties of the heavy chains present in the molecules. Those skilled in the art will understand that the heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and that there are several subclasses within these (e.g., γ1-γ4). Certain classes also have subclasses, such as IgG1, IgG2, IgG3, and IgG4, as well as others. Subclasses (isotypes) of immunoglobulins, such as IgG1, IgG2, IgG3, IgG4, and IgG5, are well-characterized and are known to provide functional specificity. In the case of IgG, a standard immunoglobulin molecule contains two identical light-chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy-chain polypeptides with a molecular weight of 53,000-70,000. The four chains are typically linked by disulfide bonds in a "Y" shape, with the light chain surrounding the heavy chain, starting from the opening of the "Y" and continuing to the variable region. The immunoglobulin or antibody molecules described herein may be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0091] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be bound to either a kappa or lambda light chain. Generally, light and heavy chains are covalently linked to each other, and when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the "tails" of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. In heavy chains, the amino acid sequence extends from the N-terminus of the Y-branched ends to the C-terminus at the bottom of each chain.

[0092] Both the light and heavy chains are divided into structural and functional homology regions. The terms “constant” and “variable” are used in a functional sense. The variable domains (VL and VH) of both the light and heavy chain portions determine antigen recognition and specificity. Conversely, the constant domains (CL, as well as CH1, CH2, or CH3) of the light and heavy chains confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, and complement binding. The term “antigen-binding site” or “binding region” can refer to the portion of the immunoglobulin molecule involved in antigen binding. Antigen-binding sites are formed by amino acid residues in the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly distinct segments within the V regions of the heavy and light chains, called “hypervariable regions,” are inserted between more conserved adjacent segments known as “framework regions” or “FR.” Thus, the term “FR” refers to the naturally occurring amino acid sequences between and adjacent to the hypervariable regions of immunoglobulins. In antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the antigen it binds to, and the three hypervariable regions of the heavy chain and the three hypervariable regions of the light chain are called "complementarity-determining regions" or "CDRs". The VH and VL regions, including the CDRs and framework (FR), of the PD-1 antibody are shown in Tables 1A to 15B.

[0093] The six CDRs present in each antigen-binding domain are short, discontinuous sequences of amino acids that specifically align to form the antigen-binding domain when the antibody takes its three-dimensional configuration in an aqueous environment. The remaining amino acids of the antigen-binding domain, the FR region, exhibit less intermolecular variation. The framework region primarily conforms to a β-sheet structure, with the CDRs linking together to form loops and, in some cases, forming part of the β-sheet structure. The framework region functions to form a scaffold for aligning the CDRs in the correct orientation through non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs provides a complementary surface to the epitope on the antigen in the immune response, facilitating the non-covalent binding of the antibody to the congeneral epitope. The amino acids containing the CDR and framework regions, respectively, can be readily identified by those skilled in the art for the heavy chain or light chain variable region, since they have been previously identified (see “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., USD Department of Health and Human Services, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0094] Where there are two or more definitions for a term used and / or permitted in the art, the definitions used herein are intended to encompass all such meanings unless explicitly stated otherwise. A specific example is the use of the term “complementarity-determining region” (“CDR”) to describe non-adjacent antigen-binding sites found within the variable regions of both heavy-chain and light-chain polypeptides. This particular region is described by Kabat et al., USDept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and Chothia et al., J.Mol.Biol.196:901-917 (1987), which are incorporated herein by reference in their entirety. The definitions of CDR by Kabat and Chothia include overlaps or subsets of amino acid residues when compared to one another. Nevertheless, it is intended that applying either definition to refer to the CDR of an antibody or its variant is also within the scope of the terms defined and used herein. For comparison, the appropriate amino acid residues encompassing the CDR as defined by each of the above references are listed in the table below. The exact residue numbers containing a particular CDR vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues constitute a specific CDR simply by knowing the variable region amino acid sequence of the antibody. JPEG2026048871000032.jpg65160

[0095] Kabat et al. defined a numbering system for variable domain sequences applicable to any antibody. Those skilled in the art can clearly assign this “Kabat numbering” system to any variable domain sequence, without relying on other experimental data of the sequence itself. As used herein, “Kabat numbering” refers to the numbering system described in Kabat et al., USDept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0096] In addition to the table above, the Kabat numbering system describes the CDR region as follows: CDR-H1 begins around amino acid 31 (i.e., about 9 residues after the first cysteine ​​residue), contains about 5-7 amino acids, and ends with the following tryptophan residue. CDR-H2 begins 15 residues after the end of CDR-H1, contains about 16-19 amino acids, and ends with the following arginine or lysine residue. CDR-H3 begins about 33 amino acid residues after the end of CDR-H2, contains 3-25 amino acids, and ends with the sequence WGXG (where X is any amino acid). CDR-L1 begins around residue 24 (i.e., following the cysteine ​​residue), contains about 10-17 residues, and ends with the following tryptophan residue. CDR-L2 begins about 16 residues after the end of CDR-L1 and contains about 7 residues. CDR-L3 begins approximately 33 residues after the end of CDR-L2 (i.e., following a cysteine ​​residue), contains approximately 7 to 11 residues, and ends with the sequence F or WGXG (where X is any amino acid).

[0097] As used herein, the term “epitope” may include any protein determinant that can specifically bind to an immunoglobulin, scFv, or T cell receptor. The variable region allows an antibody to selectively recognize and specifically bind to an epitope on an antigen. For example, a combination of the VL and VH domains of an antibody, or a subset of complementarity-determining regions (CDRs), forms a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites located at the ends of each arm of the Y. Epitope determinants typically consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural and specific charge characteristics. For example, antibodies can be produced against the N-terminal or C-terminal peptide of a polypeptide. More specifically, the antigen-binding site is defined by three CDRs on the VH and VL chains, respectively (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In one embodiment, the antibody is PD-L1 (Genbank accession number NP_054862; 290 amino acid residue length) containing the amino acid sequence of SEQ ID NO: 204: The file JPEG2026048871000033.jpg22169 can be targeted.

[0098] As used herein, the terms “immunological binding” and “immunological binding properties” may refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. The strength, or affinity, of an immunological binding interaction is determined by the dissociation constant (K) of the interaction. d ) can be expressed as a smaller K d This represents greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, whose rates depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that equally affect the rates in both directions. Thus, both "on rate constants" (K) on) and "off-speed constant" (K off ) can be determined by calculating the concentration and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). K off / K on The ratio of these parameters cancels out all parameters unrelated to affinity, and the dissociation constant K D This is equivalent to (see Davies et al. (1990) Annual Rev Biochem 59:439-473 in general). The antibodies of the present invention have been measured by kinetic assays such as radioligand binding assays, or by similar assays known to those skilled in the art, such as BIAcore or Octet (BLI), and have an equilibrium binding constant (K D ) can specifically bind to the PD-1 epitope when its ratio is ≤1 μM, ≤10 μM, ≤10 nM, ≤10 pM, or ≤100 pM to about 1 pM. For example, in some embodiments, K D This is between approximately 1E-12M and 1E-11M. D In some embodiments, K D This is between approximately 1E-11M and 1E-10M. D In some embodiments, K D This is between approximately 1E-10M and 1E-9M. D In some embodiments, K D The K is approximately between 1E-9M and 1E-8M. D In some embodiments, K D This is between approximately 1E-8M and 1E-7M. D In some embodiments, K D This is between approximately 1E-7M and 1E-6M. D For example, in some embodiments, K D It is approximately 1E-12M, and in other embodiments, K D It is approximately 1E-11M. In some embodiments, K D It is approximately 1E-10M, and in other embodiments, K D In some embodiments, K D It is approximately 1E-8M, and in other embodiments, KD In some embodiments, K D It is approximately 1E-6M, and in other embodiments, K D It is approximately 1E-5M. In some embodiments, for example, K D It is approximately 3E-11M, and in other embodiments, K D It is approximately 3E-12M. In some embodiments, K D Its length is approximately 6E-11M. "Specifically binding" or "specific to" can refer to an antibody that binds to an epitope via its antigen-binding domain, and that the binding involves some complementarity between the antigen-binding domain and the epitope. For example, an antibody is said to "specifically bind" to an epitope if it binds to that epitope via its antigen-binding domain more easily than it would to bind to a random, unrelated epitope.

[0099] For example, PD-L1 antibodies can be monovalent or bivalent and can be single-stranded or double-stranded. Functionally, the binding affinity of PD-L1 antibodies is 10 -5 M~10 -12 It is within the range of M. For example, the binding affinity of the PD-L1 antibody is 10 -6 M~10 -12 M, 10 -7 M~10 -12 M, 10 -8 M~10 -12 M, 10 -9 M~10 -12 M, 10 -5 M~10 -11 M, 10 -6 M~10 -11 M, 10 -7 M~10 -11 M, 10 -8 M~10 -11 M, 10 -9 M~10 -11 M, 10 -10 M~10 -11 M, 10 -5 M~10 -10 M, 10 -6 M~10 -10 M, 10 -7 M~10 -10M, 10 -8 M~10 -10 M, 10 -9 M~10 -10 M, 10 -5 M~10 -9 M, 10 -6 M~10 -9 M, 10 -7 M~10 -9 M, 10 -8 M~10 -9 M, 10 -5 M~10 -8 M, 10 -6 M~10 -8 M, 10 -7 M~10 -8 M, 10 -5 M~10 -7 M, 10 -6 M~10 -7 M, or 10 -5 M~10 -6 It is M.

[0100] The PD-L1 protein of the present invention, or its derivatives, fragments, analogs, homologs, or homologous molecular species, can be used as an immunogen in the production of antibodies that immunologically specifically bind to these protein components, for example, amino acid residues including SEQ ID NO: 204. The PD-L1 protein, or its derivatives, fragments, analogs, homologs, or homologous molecular species coupled to proteoliposomes, can be used as an immunogen in the production of antibodies that immunologically specifically bind to these protein components.

[0101] Those skilled in the art will recognize that, without excessive experimentation, it is possible to determine whether a human monoclonal antibody has the same specificity as the human monoclonal antibody of the present invention by determining whether the former prevents the latter from binding to PD-L1. If the human monoclonal antibody being tested shows reduced binding by the human monoclonal antibody of the present invention and competes with the human monoclonal antibody of the present invention, then these two monoclonal antibodies are likely to bind to the same or closely related epitopes.

[0102] Another method for determining whether a human monoclonal antibody has the specificity of the human monoclonal antibody of the present invention is to pre-incubate the human monoclonal antibody of the present invention with a PD-L1 protein that normally reacts with it, then add the human monoclonal antibody to be tested, and determine whether the human monoclonal antibody to be tested is inhibited in its ability to bind to PD-L1. If the human monoclonal antibody to be tested is inhibited, it likely has the same or functionally equivalent epitope specificity as the monoclonal antibody of the present invention. Screening of the human monoclonal antibody of the present invention can also be carried out by utilizing PD-L1 and determining whether the monoclonal antibody to be tested can neutralize PD-L1.

[0103] Various procedures known within the Art can be used to produce polyclonal or monoclonal antibodies directed against the proteins of the present invention, or against their derivatives, fragments, analogues, homologs, or homologous molecular species. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0104] Antibodies can be purified by well-known techniques such as affinity chromatography using protein A or protein G, which primarily provide the IgG fraction of immunoserum. Subsequently, or alternatively, immunospecific antibodies can be purified by immunoaffinity chromatography by immobilizing the specific antigen or epitope that is the target of the desired immunoglobulin onto a column. The purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0105] As used herein, the terms “monoclonal antibody,” “mAb,” “Mab,” or “monoclonal antibody composition” may refer to a group of antibody molecules containing only one species of antibody molecule, consisting of a specific light chain gene product and a specific heavy chain gene product. In particular, the complementarity-determining region (CDR) of a monoclonal antibody is identical in all molecules of the group. A MAb contains an antigen-binding site that can react immunologically with an antigen epitope characterized by a specific binding affinity to it.

[0106] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In hybridoma methods, mice, hamsters, or other suitable host animals are typically immunized with an immunizer to induce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizer. Alternatively, lymphocytes can be immunized in vitro.

[0107] The immunizing agent may include protein antigens, their fragments, or fusion proteins. For example, peripheral blood lymphocytes may be used if human-derived cells are desired, or spleen cells or lymph node cells may be used if a non-human mammalian source is desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). The immortalized cell line may be transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. For example, rat or mouse myeloma cell lines may be used. The hybridoma cells may be cultured in a suitable medium containing one or more substances that inhibit the proliferation or survival of non-fusioned immortalized cells. For example, if parent cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium typically contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), and these substances prevent the growth of HGPRT-deficient cells.

[0108] Useful immortalized cell lines are those that efficiently fuse, maintain stable high levels of antibody expression by selected antibody-producing cells, and are sensitive to culture media such as HAT medium. Examples of immortalized cell lines include mouse myeloma lines available from the Salk Institute Cell Distribution Center (San Diego, California) and the American Type Culture Collection (Manassas, Virginia). Human myeloma and mouse-human heterozygous myeloma cell lines have also been described for the production of human monoclonal antibodies. (See Kozbor, J. Immunol, 133:3001 (1984), Brodeur et al, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

[0109] Next, the culture medium in which hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies against the antigen. For example, the binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosolvent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis as described in Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, for therapeutic applications of monoclonal antibodies, it is important to identify antibodies that have high specificity and high binding affinity to the target antigen.

[0110] After the desired hybridoma cells are identified, clones can be subcloned using limiting dilution procedures and grown using standard methods (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986), pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle Medium and RPMI-1640 Medium. Alternatively, hybridoma cells can be grown in vivo as ascites in mammals.

[0111] Monoclonal antibodies secreted by subclones can be isolated or purified from culture media or ascites fluid by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0112] Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567 (which is incorporated herein by reference in its entirety). The DNA encoding the monoclonal antibodies of the present invention can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the mouse antibody). Hybridoma cells of the present invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into host cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, to obtain the synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, for example, by substituting the coding sequences of human heavy and light chain constant domains for homologous mouse sequences (see U.S. Patent No. 4,816,567, Morrison, Nature 368,812-13 (1994)) or by covalently bonding all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can be used in place of the constant domain of the antibody of the present invention, or in place of the variable domain of one antigen-binding site of the antibody of the present invention, to produce a chimeric bivalent antibody.

[0113] Fully human antibodies are antibody molecules in which the entire sequence of both the light and heavy chains, including the CDR, is derived from human genes. Such antibodies are referred to herein as “humanized antibodies” or “fully human antibodies.” Human monoclonal antibodies can be prepared using trioma techniques, human B-cell hybridoma techniques (see Kozbor, et al, 1983 Immunol Today 4:72), and EBV hybridoma techniques for producing human monoclonal antibodies (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies can be used and produced by using human hybridomas (see Cote, et al, 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells with Epstein-Barr virus in vitro (Cole, et al., 1985 In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96).

[0114] Humanized antibodies can be antibodies derived from non-human species (such as mice), but their light and heavy chain protein sequences are modified to increase similarity to antibody variants produced in humans. Humanized antibodies are antibody molecules derived from non-human antibodies that bind to a desired antigen, having one or more complementarity-determining regions (CDRs) derived from non-human species and a framework region derived from a human immunoglobulin molecule. In many cases, framework residues in the human framework region are substituted with corresponding residues from the CDR donor antibody, thereby altering, and preferably improving, antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interaction between the CDR and framework residues to identify framework residues important for antigen binding, and by comparing sequences to identify abnormal framework residues at specific positions. (See, for example, Queen et al., USPat. No. 5, 585, 089 and Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entirety.) For example, the non-human portion of an antibody (such as the CDR of the light and / or heavy chain) can bind to the target antigen.

[0115] Antibodies can be humanized using various techniques known in the art, such as CDR transplantation (EP239,400, PCT International Publication No. 91 / 09967, U.S. Patent Nos. 5,225,539, 5,530,101 and 5,585,089), veneering or resurfacing (EP592,106, EP519,596, Padlan, Molecular Immunology 28(4 / 5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814(1994); Roguska et al., Proc. Natl. Sci. USA 91:969-973(1994)), and chain shuffling (U.S. Patent No. 5,565,332, which is incorporated in its entirety by reference). Humanization (also known as reshaping or CDR grafting) is a well-established technique known to those skilled in the art for reducing the immunogenicity of monoclonal antibodies (mAbs) derived from heterologous sources (usually rodents) and improving the activation of the human immune system (see, for example, Hou S, Li B, Wang L, Qian W, Zhang D, Hong X, Wang H, Guo Y (July 2008) "Humanization of an anti-CD34 monoclonal antibody by complementarity-determining region grafting based on computer-assisted molecular modeling" J Biochem. 144(1):115-20). Antibodies can be humanized by methods known in the art, such as CDR transplantation. See also Safdari et al., (2013) Biotechnol Genet Eng Rev.; 29:175-86. Furthermore, humanized antibodies can be produced in transgenic plants as an inexpensive alternative to existing mammalian systems. For example, the transgenic plants could be tobacco plants, namely Nicotiana benthamiana and Nicotiana tabaccum. The antibodies are purified from the leaves of the plants.Stable transformation of plants can be achieved using Agrobacterium tumefaciens or the particle gun method. For example, a nucleic acid expression vector containing at least heavy and light chain sequences is expressed via transformation in a bacterial culture, i.e., A. tumefaciens strain BLA4404. Plant infiltration can be achieved by injection. Soluble leaf extracts can be prepared by grinding leaf tissue in a mortar and centrifugation. Antibody isolation and purification can be readily carried out by many methods known to those skilled in the art. Other methods of antibody production in plants are described, for example, in Fischer et al., Vaccine, 2003, 21:820-5, and Ko et al., Current Topics in Microbiology and Immunology, Vol.332, 2009, pp.55-78. Accordingly, the present invention further provides any cells or plants comprising a vector encoding or producing the antibodies of the present invention.

[0116] Human monoclonal antibodies, such as fully human and humanized antibodies, can be prepared using trioma technology, human B-cell hybridoma technology (see Kozbor, et al, 1983 Immunol Today 4:72), and EBV hybridoma technology that produces human monoclonal antibodies (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies can be used and produced by using human hybridomas (see Cote, et al, 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells with Epstein-Barr virus in vitro (Cole, et al., 1985 In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96).

[0117] In addition, human antibodies can also be produced using other techniques, including phage display libraries (see Hoogenboom and Winter, J.Mol.Biol, 227:381 (1991), Marks et al., J.Mol.Biol, 222:581 (1991)). Similarly, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, such as mice in which the endogenous immunoglobulin gene is partially or completely inactivated. After the challenge, human antibody production is observed, which is very similar in all aspects to that seen in humans, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patents No. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, as well as in Marks et al., Bio / Technology 10,779-783 (1992), Lonberg et al., Nature 368,856-859 (1994), Morrison, Nature 368,812-13 (1994), Fishwild et al., Nature Biotechnology 14,845-51 (1996), Neuberger, Nature Biotechnology 14,826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13. It is described in 65-93 (1995).

[0118] Human antibodies can also be produced using transgenic non-human animals that are modified to produce fully human antibodies rather than endogenous antibodies in response to antigenic challenge. (See PCT International Publication 94 / 02602 and U.S. Patent No. 6,673,986). Endogenous genes encoding heavy and light chain immunoglobulin chains in the non-human host are neutralized, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host genome. Human genes are incorporated, for example, using a yeast artificial chromosome containing the required human DNA segment. Animals that provide all the desired modifications are then obtained as offspring by mating intermediate transgenic animals that have fewer complements than the complete complements of the modifications. A non-limiting example of such a non-human animal is the mouse, called Xenomouse™ as disclosed in PCT Publications WO96 / 33735 and WO96 / 34096. This animal produces B cells that secrete fully human immunoglobulins. Antibodies can be obtained, for example, as preparations of polyclonal antibodies, directly from animals after immunization with the immunogen of interest, or alternatively, from immortalized B cells of animal origin, such as hybridomas that produce monoclonal antibodies. In addition, genes encoding immunoglobulins with human variable regions can be recovered and expressed to directly obtain antibodies, or further modified to obtain antibody analogs, such as single-stranded Fv(scFv) molecules.

[0119] Therefore, such techniques can be used to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technique for producing human antibodies, see Lonberg and Huszar Int. Rev. Immunol. 73:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies, and protocols for producing such antibodies, see, for example, PCT Publications WO98 / 24893, WO96 / 34096, 96 / 33735, U.S. Patents 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporated herein by reference in their entirety. In addition, companies such as Creative BioLabs (Shirley, NY) can offer services to provide human antibodies against selected antigens using technologies similar to those described above.

[0120] An example of a method for producing a non-human host, exemplified as a mouse lacking expression of endogenous immunoglobulin heavy chains, is disclosed in U.S. Patent No. 5,939,598. This can be obtained by a method comprising: deleting a J-segment gene from at least one endogenous heavy chain locus in embryonic stem cells to prevent locus rearrangement and the formation of a transcript of the rearranged immunoglobulin heavy chain locus, wherein the deletion is carried out by a targeted vector containing a gene encoding a selectable marker; and producing a transgenic mouse from embryonic stem cells, wherein its somatic and germ cells contain a gene encoding a selectable marker.

[0121] One method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. This method involves introducing an expression vector containing a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing both a heavy chain and a light chain.

[0122] Further improvements to this procedure include methods for identifying clinically relevant immunogenic epitopes and corresponding methods for selecting antibodies that bind immunospecifically to these relevant epitopes with high affinity, as disclosed in PCT Publication WO99 / 53049.

[0123] The antibody of interest can also be expressed by a vector containing a DNA segment encoding the single-chain antibody described herein.

[0124] These vectors may include liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, etc. Vectors may also include chemical conjugates such as those described in WO93 / 64701, having a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid binding moiety (e.g., polylysine); viral vectors (e.g., DNA or RNA viral vectors); fusion proteins such as those described in PCT / US95 / 02140 (WO95 / 22618), which contain a targeting moiety (e.g., an antibody specific to a target cell) and a nucleic acid binding moiety (e.g., protamine); plasmids; phages; and viral vectors. Vectors may be chromosomal, non-chromosomal, or synthetic. Retroviral vectors may also be used, such as Moloney's mouse leukemia virus.

[0125] DNA virus vectors may also be used, including poxvirus vectors such as orthopox or avipox vectors, herpesvirus vectors such as herpes simplex virus type I (HSV) vectors (see Geller, A.I. et al, J. Neurochem, 64:487 (1995), Lim, F., et al, in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995), Geller, A.I. et al, Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993), Geller, A.I., et al, Proc Natl. Acad. Sci USA 87:1149 (1990), Adenovirus Vectors (LeGal LaSalle et al, Science, 259:988 (1993), Davidson, et al, Nat. Genet 3:219 (1993), Yang, et al, J. Virol. 69:2004 (1995) and Adeno-associated Virus Vectors (Kaplitt, M.G.. et al, Nat. Genet. 8:148 (1994)).

[0126] Poxvirus vectors introduce genes into the cytoplasm of cells. Avipoxvirus vectors result in only short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors can be used to introduce nucleic acids into nerve cells. Adenovirus vectors result in shorter-term expression (about 2 months) than adeno-associated virus (about 4 months), and thus shorter than HSV vectors. The particular vector chosen will depend on the target cell and the condition being treated. Introduction can be by standard techniques such as infection, transfection, transduction, or transformation. Examples of modes of gene transfer include, for example, naked DNA, CaP04 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and virus vectors.

[0127] Vectors can be used to essentially target any desired target cell. For example, stereotactic injection can be used to direct a vector (e.g., adenovirus, HSV) to a desired location. In addition, particles can be delivered by intracerebroventricular (icv) injection using a minipump infusion system such as the SynchroMed Infusion System. Methods based on bulk flow called convection have also been shown to be effective in delivering large molecules to extended regions of the brain and may be useful for delivering vectors to target cells. (See Bobo et al, Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994), Morrison et al, Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used include catheter, intravenous, parenteral, intraperitoneal, and subcutaneous injection, as well as oral or other known routes of administration.

[0128] These vectors can be used in a variety of ways, for example, to express large amounts of antibodies that can be used to detect the presence of PD-L1 in a sample. Antibodies can also be used to attempt to bind and disrupt PD-L1 activity. In one embodiment, the antibodies of the invention are full-length antibodies that include an Fc region similar to the wild-type Fc region that binds to Fc receptors.

[0129] The technique can be adapted for the production of single-chain antibodies specific for the antigenic proteins of the invention (see, e.g., U.S. Patent No. 4,946,778). In addition, the method ab can be adapted for the construction of expression libraries (see, e.g., Huse, et al, 1989 Science 246:1275-1281) to produce monoclonal F abThis can enable rapid and effective identification of fragments. Antibody fragments containing idiotypes against protein antigens can be produced by techniques known in the art, but are not limited to, (i) F produced by pepsin digestion of antibody molecules. (ab’)2 Fragment, (ii)F (ab’)2 F is produced by reducing the disulfide bridges of the fragments. ab (iii) F produced by treatment of the fragment, antibody molecule with papain and a reducing agent. ab Fragments, and (iv)F v Includes fragments.

[0130] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies consist of two covalently bound antibodies. Such antibodies enable, for example, the targeting of immune system cells to undesirable cells (see U.S. Patent No. 4,676,980) and enable the treatment of HIV infection (see PCT Publications WO91 / 00360 and WO92 / 20373). Antibodies are intended to be able to be prepared in vitro using known methods in the field of protein synthesis chemistry, such as those using crosslinking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of reagents suitable for this purpose include iminothiolates and methyl-4-mercaptobutyrimidates, as well as those disclosed, for example, in U.S. Patent No. 4,676,980.

[0131] The antibodies of the present invention can be modified with respect to effector function, for example, to enhance the efficacy of the antibody in the treatment of cancer. For example, a cysteine ​​residue can be introduced into the Fc region, thereby enabling the formation of interchain disulfide bonds in this region. The homodimeric antibodies thus produced may have improved internalization ability and / or increased complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). (See Caron et al, J. Exp Med., 176:1 191-195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, antibodies having a double Fc region, thereby possessing enhanced complement lysis and ADCC ability, can be manipulated. (See Stevenson et al, Anti-Cancer Drug Design, 3:219-230 (1989)).

[0132] In certain embodiments, the antibodies of the present invention may include Fc variants containing amino acid substitutions that modify the antigen-independent effector function of the antibody, particularly the circulating half-life of the antibody. Such antibodies, when compared to antibodies lacking these substitutions, exhibit either increased or decreased binding to FcRn, and therefore have increased or decreased serum half-lives, respectively. Fc variants with improved affinity for FcRn are expected to have a longer serum half-life, and such molecules have useful applications in methods of treating mammals where a longer half-life of the administered antibody is desirable, for example, to treat chronic diseases or disorders. In contrast, Fc variants with reduced FcRn binding affinity are expected to have a shorter half-life, and such molecules are also useful, for example, for administration to mammals where a shortened circulating time may be advantageous, such as for in vivo diagnostic imaging, or in situations where the starting antibody has toxic side effects if it remains in circulation for a long period of time. Fc variants with reduced FcRn binding affinity are less likely to cross the placenta and are therefore useful in the treatment of diseases or disorders in pregnant women. In addition, other applications where reduced FcRn binding affinity may be desirable include applications where localization to the brain, kidneys, and / or liver is desirable. In one embodiment, an Fc variant-containing antibody may exhibit reduced transport from the vascular system across the renal glomerular epithelium. In another embodiment, an Fc variant-containing antibody may exhibit reduced transport from the brain across the blood-brain barrier (BBB) ​​into the vascular space. In one embodiment, an antibody with modified FcRn binding contains an Fc domain having one or more amino acid substitutions within the “FcRn binding loop” of the Fc domain. The FcRn binding loop consists of amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions that modify FcRn binding activity are disclosed in PCT Publication WO05 / 047327, which is incorporated herein by reference. In certain exemplary embodiments, the antibody or fragment thereof of the present invention comprises an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering).

[0133] In some embodiments, mutations are introduced into the constant region of an mAb so as to alter the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the mAb. For example, the mutation is an LALA mutation in the CH2 domain. In one embodiment, the antibody (e.g., a human mAb, or a bispecific Ab) contains a mutation on one scFv unit of a heterodimeric mAb that reduces ADCC activity. In another embodiment, the mAb contains mutations on both strands of a heterodimeric mAb that completely eliminate ADCC activity. For example, a mutation introduced into one or both scFv units of an mAb is an LALA mutation in the CH2 domain. These mAbs with variable ADCC activity can be optimized so that the mAb exhibits maximum selective killing toward cells expressing one antigen recognized by the mAb, but minimum killing toward a second antigen recognized by the mAb.

[0134] In other embodiments, antibodies for use in the diagnostic and therapeutic methods described herein have a constant region, such as the heavy chain constant region of IgG1 or IgG4, which is modified to reduce or eliminate glycosylation. For example, the antibodies of the present invention may also include Fc variants that include amino acid substitutions that modify the glycosylation of the antibody. For example, in Fc variants, glycosylation (e.g., N-linked or O-linked glycosylation) can be reduced. In some embodiments, the Fc variant has reduced glycosylation of an N-linked glycan commonly found at amino acid position 297 (EU numbering). In another embodiment, the antibody has amino acid substitutions near or within a glycosylation motif, such as an N-linked glycosylation motif containing the amino acid sequence NXT or NXS. In a specific embodiment, the antibody includes an Fc variant having an amino acid substitution at amino acid position 228 or 299 (EU numbering). In a more specific embodiment, the antibody includes the IgG1 or IgG4 constant region containing S228P and T299A mutations (EU numbering).

[0135] Exemplary amino acid substitutions that reduce or alter glycosylation are disclosed in PCT Publication WO05 / 018572, incorporated herein by reference. In some embodiments, the antibody or fragment of the present invention is modified to eliminate glycosylation. Such an antibody or fragment may be referred to as an "agly" antibody or fragment (e.g., an "agly" antibody). Not bound by theory, an "agly" antibody or fragment may have an improved safety and stability profile in vivo. An exemplary agly antibody or fragment includes a deglycosylated Fc region of an IgG4 antibody that lacks Fc effector function, thereby eliminating the possibility of Fc-mediated toxicity to normal living tissues and cells expressing PD-L1. In yet other embodiments, the antibody or fragment of the present invention includes a modified glycan. For example, the antibody may have a reduced number of fucose residues on the N-glycan at Asn297 in the Fc region, i.e., it is defucosylated. In another embodiment, the antibody may have a modified number of sialic acid residues on the N-glycan at Asn297 in the Fc region.

[0136] The present invention also covers cytotoxic agents such as toxins (e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or immunoconjugates (the latter being radioconjugates) comprising antibodies conjugated to radioisotopes.

[0137] Enzymatically active toxins and their fragments that can be used include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modexin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, geronin, mitogenin, restrictoctocin, phenomycin, enomycin, and trichothecenes. Various radionuclides are available for the production of radioconjugated antibodies. Non-limiting examples include: 212 Bi, 131 I, 131 In, 90 Y, and 186 Re is one example.

[0138] Antibody and cytotoxic agent conjugates are prepared using various bifunctional protein conjugates such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutarelaldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., triene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al, Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radioactive nucleotides to antibodies. (See PCT Publication WO94 / 11026 and U.S. Patent No. 5,736,137).

[0139] Those skilled in the art will recognize that a wide variety of possible parts can be conjugated to the resulting antibody or other molecules of the present invention. (See, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, JMCruse and RELewis, Jr(eds), Carger Press, New York, (1989), the full contents of which are incorporated herein by reference).

[0140] Binding can be achieved by any chemical reaction that will bind the two molecules, insofar as the antibody and the other part retain their respective activities. This binding can include many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordination bonding, and complex formation. In one embodiment, the binding is covalent. Covalent bonding can be achieved by direct condensation of existing side chains or by the incorporation of external crosslinking molecules. Many divalent or polyvalent binding agents are useful for binding protein molecules, such as the antibody of the present invention, to other molecules. For example, typical binding agents can include organic compounds such as thioesters, carbodiimides, succinimides, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to cover all classes of binding agents known in the art, but rather to be an example of more general binding agents. (See Killen and Lindstrom, Jour.Immun.133:1335-2549 (1984), Jansen et al., Immunological Reviews 62:185-216 (1982), and Vitetta et al, Science 238:1098 (1987)). Non-limiting examples of linkers are described in the literature. (For example, see Ramakrishnan, S. et al., Cancer Res.44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Patent No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives conjugated to antibodies by oligopeptide linkers.Non-limiting examples of useful linkers that can be used with the antibodies of the present invention include: (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pridinedithio)-toluene (Pierce Chem. Co., Cat. (21558G)), (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamide]hexanoate (Pierce Chem. Co., catalog no. 21651G), (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propionamide]hexanoate (Pierce Chem. Co., catalog no. 2165-G), and (v) sulfo-NHS (hydroxysulfosuccinimidide conjugated to EDC: Pierce Examples include Chem.Co., catalog number 24510.

[0141] The linkers described herein contain components with different attributes, resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylate salts. NHS-ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, linker SMPT can contain sterically hindered disulfide bonds to form conjugates with improved stability. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in fewer available conjugates. In particular, sulfo-NHS can enhance the stability of carbodimide bonds. When carbodimide bonds (such as EDC) are used in combination with sulfo-NHS, they form esters that are more resistant to hydrolysis than carbodimide bond reactions alone.

[0142] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies are prepared by methods known in the art, such as those described in Epstein et al, Proc. Natl. Acad. Sci. USA, 82:3688 (1985), Hwang et al, Proc. Natl. Acad. Sci. USA, 77:4030 (1980), and U.S. Patents No. 4,485,045 and No. 4,544,545. Liposomes with extended circulation time are disclosed in U.S. Patent No. 5,013,556.

[0143] Useful liposomes, not limited to those described above, can be produced by reverse-phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivativeized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of a defined pore size to produce liposomes having a desired diameter. The Fab' fragment of the antibody of the present invention can be conjugated into liposomes via a disulfide exchange reaction, as described in Martin et al, J. Biol. Chem., 257:286-288 (1982).

[0144] multispecific antibodies A multispecific antibody is an antibody capable of recognizing two or more different antigens. For example, a bispecific antibody (bsAb) is an antibody containing two variable domains or scFv units so that the resulting antibody recognizes two different antigens. For example, a triplicate antibody (tsAb) is an antibody containing two variable domains or scFv units so that the resulting antibody recognizes three different antigens. The present invention provides multispecific antibodies, such as a bispecific antibody that recognizes PD-L1 and a second antigen. For example, PD-L1 is an immune checkpoint molecule and also a tumor antigen. As a tumor antigen targeting molecule, a bispecific antibody can be generated by combining an antibody or antigen-binding fragment specific to PD-L1 with a second antigen-binding fragment specific to immune cells. In some embodiments, the immune cells are selected from the group consisting of T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils, and mast cells. Molecules on immune cells that can be targeted include, but are not limited to, CD3, CD16, CD19, CD28, and CD64. Other non-limiting examples include PD-1, CTLA-4, LAG-3 (also known as CD223), CD28, CD122, 4-1BB (also known as CD137), TIM3, OX-40 or OX40L, CD40 or CD40L, LIGHT, ICOS / ICOSL, GITR / GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA (also known as CD272), killer cell immunoglobulin-like receptors (KIRs), and CD47.Exemplary second antigens include tumor-associated antigens (e.g., LINGO1, EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, and tenascin), cytokines (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT, and GITRL), and cell surface receptors. Bispecific antibodies in various formats are also provided herein. In some embodiments, each of the anti-PD-L1 fragment and the second fragment is independently selected from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody. In some embodiments, the bispecific antibody further comprises an Fc fragment. The bispecific antibodies of the present invention comprise combinations of heavy and light chains of PD-L1 antibodies, or scFvs, as disclosed herein.

[0145] <​​​​​​​​​​​​​​​​​​JPEG2026048871000036.jpg197167

[0149] (Table 12B) Ab#E1-3H7 constant region amino acid sequence - wild-type IgG1 monomer JPEG2026048871000037.jpg17169JPEG2026048871000038.jpg98169

[0150] (Table 13A) Ab#E1-3H7 constant region nucleic acid sequence - IgG1 LALA-aPDL-1 40mut JPEG2026048871000039.jpg193169

[0151] (Table 13B) Ab#E1-3H7 constant region amino acid sequence - IgG1 LALA-aPDL-1 40mut JPEG2026048871000040.jpg109167

[0152] (Table 14A) Ab#E1-3H7 constant region nucleic acid sequence - IgG1 LALA-aPD-L1 50-6B6.1 mut JPEG2026048871000041.jpg204167

[0153] (Table 14B) Ab#E1-3H7 constant region amino acid sequence - IgG1 LALA-aPD-L1 50-6B6.1 mut JPEG2026048871000042.jpg109167

[0154] (Table 15A) Ab#E1-3H7 constant region nucleic acid sequence - IgG1 LALA-aPD-L1 50-6B6.2 JPEG2026048871000043.jpg202165

[0155] (Table 15B) Ab#E1-3H7 Constant Region Amino Acid Sequence - IgG1 LALA-aPD-L1 50-6B6.2 JPEG2026048871000044.jpg109167

[0156] (Table 16A) Ab#E1-3H7 constant region nucleic acid sequence - IgG1 LALA-aPD-L1 50-7B3 JPEG2026048871000045.jpg212169

[0157] (Table 16B) Ab#E1-3H7 constant region amino acid sequence - IgG1 LALA-aPD-L1 50-7B3 JPEG2026048871000046.jpg117169

[0158] (Table 17A) Ab#E1-3H7 constant region nucleic acid sequence-IgG1 LALA-aPD-L1 50-5B9 JPEG2026048871000047.jpg202165

[0159] (Table 17B) Ab#E1-3H7 constant region amino acid sequence - IgG1 LALA-aPD-L1 50-5B9 JPEG2026048871000048.jpg109167

[0160] The multispecific antibodies of the present invention (e.g., bispecific and tripspecific antibodies) can be constructed using methods known in the art. In some embodiments, the bispecific antibody is a single polypeptide in which two scFv fragments are linked by a long linker polypeptide of sufficient length to allow intramolecular association between two scFv units to form an antibody. In other embodiments, the bispecific antibody is two or more polypeptides linked by covalent or non-covalent bonds. In some embodiments, the amino acid linker (GGGGSGGGGS; "(G4S)2") which can be used with the scFv fusion constructs described herein may be produced with a longer G4S linker to improve flexibility. For example, the linker can be "(G4S)3" (e.g., GGGGSGGGGSGGGGS), "(G4S)4" (e.g., GGGGSGGGGSGGGGSGGGGS), "(G4S)5" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGS), "(G4S)6" (e.g., GGGGSGGGGSGGGGGSGGGGGSGGGGGS), "(G4S)7" (e.g., GGGGSGGGGSGGGGGSGGGGGSGGGGGSGGGGS), and so on. For example, the use of the (G4S)5 linker may provide greater flexibility and improved expression. In some embodiments, the linker can also be (GS) n (GGS) n (GGGS) n (GGSG) n (GGSGG) n , or (GGGGS) n This may be the case, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Non-limiting examples of linkers known to those skilled in the art that can be used are described in U.S. Patent No. 9,708,412, U.S. Patent Application Publications 2018 / 0134789 and 2020 / 0148771, and PCT Publication WO2019 / 051122 (the entire contents of each of these are incorporated by reference).

[0161] In another embodiment, multispecific antibodies (e.g., bispecific and trispecific antibodies) can be constructed using the “knob-into-hole” method (Ridgway et al., Protein Eng 7:617-621 (1996)). In this method, Ig heavy chains of two different variable domains are reduced to selectively cleave the heavy chain pairing while retaining the heavy-light chain pairing. Two heavy-light chain heterodimers that recognize two different antigens are mixed to facilitate heteroligation pairing mediated through an engineered “knob-into-hole” in the CH3 domain.

[0162] In another embodiment, multispecific antibodies (e.g., bispecific and triplicate antibodies) can be constructed by exchanging heavy-light chain dimers from two or more different antibodies to produce hybrid antibodies, where the first heavy-light chain dimer recognizes PD-L1 and the second heavy-light chain dimer recognizes a second antigen. In some embodiments, bispecific antibodies can be constructed by exchanging heavy-light chain dimers from two or more different antibodies to produce hybrid antibodies, where the first heavy-light chain dimer recognizes a second antigen and the second heavy-light chain dimer recognizes PD-L1. The mechanism of heavy-light chain dimerization is analogous to the formation of human IgG4, which also functions as a bispecific molecule. Dimerization of IgG heavy chains is driven by intramolecular forces, such as the pairing of the CH3 domain of each heavy chain with disulfide crosslinks. The presence of a specific amino acid (R409) in the CH3 domain has been shown to facilitate dimerization and the construction of the IgG4 molecule. Heavy chain pairing is further stabilized by inter-heavy chain disulfide crosslinking in the hinge region of the antibody. Specifically, in IgG4, the hinge region contains the amino acid sequence Cys-Pro-Ser-Cys at amino acids 226-230 (compared to the stable IgG1 hinge region containing the sequence Cys-Pro-Pro-Cys). This sequence difference at serine position 229 is related to IgG4's tendency to form intrachain disulfides in the hinge region (Van der Neut Kolfschoten, M. et al, 2007, Science 317:1554-1557 and Labrijn, AF et al, 2011, Journal of Immunol 187:3238-3246).

[0163] Therefore, the bispecific antibodies of the present invention can be constructed by introducing the R409 residue in the CH3 domain and the Cys-Pro-Ser-Cys sequence in the hinge region of the antibody that recognizes PD-L1 or a second antigen, thereby exchanging the heavy-light dimers to produce an antibody molecule having one heavy-light dimer that recognizes PD-L1 and a second heavy-light dimer that recognizes a second antigen, the second antigen being any antigen disclosed herein. Known IgG4 molecules can also be modified so that the heavy and light chains recognize PD-L1 or a second antigen, as disclosed herein. The use of this method for constructing the bispecific antibodies of the present invention may be beneficial due to the unique characteristics of the IgG4 molecule, in which the Fc region differs from other IgG subtypes in that it has poor interaction with effector systems of the immune response, such as complement and Fc receptors, expressed by certain leukocytes. This unique property makes these IgG4-based bispecific antibodies attractive for therapeutic applications where the antibody needs to bind to a target and functionally alter the target-related signaling pathway, but without inducing effector activity.

[0164] In some embodiments, mutations are introduced into the constant region of bsAb so as to alter the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of bsAb. For example, the mutation is an LALA mutation in the CH2 domain. In one embodiment, bsAb contains a mutation on one scFv unit of heterodimer bsAb that reduces ADCC activity. In another embodiment, bsAb contains mutations on both strands of heterodimer bsAb that completely eliminate ADCC activity. For example, a mutation introduced into one or both scFv units of bsAb is an LALA mutation in the CH2 domain. These bsAbs with variable ADCC activity can be optimized so that bsAb exhibits maximum selective killing toward cells expressing one antigen recognized by bsAb, but minimum killing toward a second antigen recognized by bsAb.

[0165] The bispecific antibodies disclosed herein can be effectively used to treat chronic infections, diseases, or medical conditions, such as cancer.

[0166] Use of antibodies against PD-L1 The antibodies of the present invention, which specifically bind to the PD-L1 protein or fragments thereof, can be administered for the treatment of PD-L1-related diseases or disorders. “PD-L1-related diseases or disorders” include conditions and / or symptoms associated with conditions characterized by elevated levels of PD-L1 and / or activation of PD-L1-mediated cellular signaling pathways. Exemplary PD-L1-related diseases or disorders include, but are not limited to, cancer and autoimmune diseases.

[0167] Antibodies of the present invention, such as bispecific, polyclonal, monoclonal, humanized, and fully human antibodies, can be used as therapeutic agents. Such agents would generally be used to treat or prevent cancer in a subject, to improve vaccine efficiency, or to enhance the natural immune response. Antibody preparations, for example, those having high specificity and high affinity for their target antigen, are administered to a subject and are generally thought to produce effects due to binding to the target. Antibody administration can inactivate, inhibit, or interfere with the activity of the PD-L1 protein.

[0168] The antibody of the present invention, which specifically binds to the PD-L1 protein or a fragment thereof, can be administered in the form of a pharmaceutical composition for the treatment of cancer. Principles and precautions involved in the preparation of therapeutic pharmaceutical compositions containing the antibody, as well as guidance in the selection of components, are provided, for example, in Remington: The Science And Practice Of Pharmacy 20th ed. (Alfonso R. Gennaro, et al, editors) Mack Pub. Co., Easton, Pa., 2000, Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994, and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0169] The specific dosage and treatment plan for a particular patient depends on various factors, including the specific antibody used, its variant or derivative, the patient's age, weight, general health status, sex, and diet, as well as the timing of administration, excretion frequency, concomitant drug use, and the severity of the specific disease being treated. The determination of such factors by healthcare professionals is within the scope of the skills of those skilled in the art. The dosage is also considered to depend on the individual patient being treated, the route of administration, the type of formulation, the properties of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined by pharmacokinetic principles well known in the art.

[0170] The therapeutically effective dose of the antibody of the present invention can be the amount necessary to achieve the therapeutic objective. As described above, the therapeutic objective may be the binding interaction between the antibody and its target antigen, which in certain cases interferes with the function of the target. The amount to be administered further depends on the binding affinity of the antibody to its specific antigen and on the rate at which the administered antibody is depleted from the free volume of the other subject to which it is administered. The dose of the antigen-binding polypeptide described herein administered to a subject (e.g., a patient) is typically 0.1 mg / kg to 100 mg / kg patient body weight, 0.1 mg / kg to 20 mg / kg patient body weight, or 1 mg / kg to 10 mg / kg patient body weight. Human antibodies have a longer half-life in the human body than antibodies from other species due to the immune response to exogenous polypeptides. Therefore, it is often possible to administer human antibodies in lower doses and at lower frequencies. Furthermore, the dose and frequency of administration of the antibodies of this disclosure can be reduced by enhancing antibody uptake and penetration into tissues (e.g., the brain) through modifications such as lipidization. The general range for therapeutically effective administration of the antibody or antibody fragment of the present invention may, as a non-limiting example, be about 0.1 mg / kg body weight to about 50 mg / kg body weight. The general frequency of administration may be, for example, in the range of twice a day to once a week.

[0171] When antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the variable region sequence of the antibody, a peptide molecule that retains the ability to bind to the target protein sequence can be designed. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. (See, for example, Marasco et al, Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)). The formulation may also contain two or more active compounds necessary for the specific indication being treated, preferably those having complementary activities that do not adversely affect each other. Alternatively or additionally, the composition may include agents that enhance its function, such as cytotoxic agents, cytokines (e.g., IL-15), chemotherapeutic agents, or growth inhibitors. Such molecules are appropriately present in combination in amounts effective for the intended purpose.

[0172] The active ingredient can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions, respectively.

[0173] Preparations used for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filtration membrane.

[0174] Sustained-release preparations can be prepared. Preferred examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, the matrices in the form of molded articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT (an injectable microsphere consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyrate. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow for molecular release over more than 100 days, while certain hydrogels release proteins over shorter periods.

[0175] The antibody according to the present invention can be used as an agent for detecting the presence of PD-L1 (or its protein fragments) in a sample. For example, the antibody may contain a detectable label. The antibody may be polyclonal or monoclonal. A intact antibody or its fragment (e.g., F ab , scFv, or F (ab)2) can be used. With respect to probes or antibodies, the term “labeled” can include direct labeling of the probe or antibody by binding (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include the detection of a primary antibody using a fluorescently labeled secondary antibody, and the end labeling of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin. The term “biological sample” can include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and biological fluids present within the subject. Therefore, the use of the term “biological sample” can include blood, and fractions or components of blood, including serum, plasma, or lymph. That is, the detection methods of the present invention can be used to detect analytes mRNA, proteins, or genomic DNA in biological samples in vitro and in vivo. For example, in vitro techniques for the detection of analytes mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detecting analyte proteins include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting analyte genomic DNA include Southern hybridization.

[0176] Procedures for performing immunoassays are described, for example, in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, JRCrowther (Ed.), Human Press, Totowa, NJ, 1995; “Immunoassay”, E. Diamandis and T. Christophorus, Academic Press, Inc., San Diego, CA, 1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detecting analyte proteins involve introducing labeled anti-analyte protein antibodies into the target. For example, the antibody may be labeled with a radiomarker whose presence and location in the target can be detected by standard imaging techniques.

[0177] Antibodies against the PD-L1 protein (or fragments thereof) can be used in methods known in the art related to the localization and / or quantification of the PD-L1 protein (e.g., in measuring the level of PD-L1 protein in a suitable physiological sample, in diagnostic methods, or in protein imaging). In certain embodiments, antibodies containing an antigen-binding domain derived from the antibody and specific to the PD-L1 protein, or its derivatives, fragments, analogs, or homologs, are used as pharmaceutically active compounds (hereinafter referred to as "therapeutic agents").

[0178] The PD-L1 polypeptide can be isolated using the PD-L1 protein-specific antibody of the present invention by standard techniques such as immunoaffinity assay, chromatography, or immunoprecipitation. Antibodies against the PD-L1 protein (or fragments thereof) can be used diagnostically, for example, to monitor protein levels in tissues as part of a clinical trial procedure, or to determine the effectiveness of a given treatment regimen.

[0179] Detection can be facilitated by binding (i.e., physically linking) antibodies to detectable substances. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Suitable enzyme examples include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dancylcloride, or phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; suitable radioactive materials include 125 I, 131 I, 35 S, 32 P or 3 H can be mentioned.

[0180] The antibodies or active substances (also referred to herein as “active compounds”) of the present invention, as well as their derivatives, fragments, analogs, and homologs, can be incorporated into pharmaceutically suitable compositions. Such compositions typically comprise the antibody or active substance and a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier” can include any solvent, dispersion medium, coating, antimicrobial and antifungal agent, isotonic agent, and absorption retarder suitable for pharmaceutically administered use. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the art, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, physiological saline, Ringer’s solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixative oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, its use in a composition is intended. Auxiliary active compounds can also be incorporated into the composition.

[0181] The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be sealed in ampoules, disposable syringes, or glass or plastic multi-dose vials.

[0182] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In embodiments, the composition is sterile and fluid enough to allow easy passage through an injection needle. It may be stable under manufacturing and storage conditions and can be preserved against microbial contamination such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents in the composition, such as sugars and polyhydric alcohols, such as mannitol, sorbitol, and sodium chloride. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents in the composition, such as aluminum monostearate and gelatin.

[0183] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent having, as needed, one or a combination of the components listed above, followed by filtration sterilization. For example, a dispersion can be prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion solvent and other necessary components listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and lyophilization, which yield powders of the active component and any additional desired components from the previously sterile filtered solution.

[0184] Oral compositions include an inert diluent or an edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, in which case the compound in the fluid carrier is applied orally, swirled in the mouth, and either spat out or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds of similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or sterol; flow enhancers such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavorings such as peppermint, methyl salicylate, or orange flavor.

[0185] For administration by inhalation, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas like carbon dioxide, or a nebulizer.

[0186] Systemic administration may also be by mucosal or percutaneous means. For mucosal or percutaneous administration, a penetrating agent suitable for the barrier to penetration is used in the formulation. Such penetrating agents are commonly known in the art and include, for example, for mucosal administration, cleansing agents, bile salts, and fusidic acid derivatives. Mucosal administration can be achieved through the use of nasal sprays or suppositories. For percutaneous administration, the active compound is formulated into ointments, plasters, gels, or creams, which are commonly known in the art.

[0187] The compounds can also be prepared in the form of suppositories (e.g., those having a conventional suppository base such as cocoa butter and other glycerides) or retained enemas for rectal delivery.

[0188] In one embodiment, the active compound is prepared on a carrier that will protect the compound from rapid elimination from the body, such as a controlled-release formulation, which includes implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes targeting cells infected with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0189] For ease of administration and uniformity of dosage, oral or parenteral compositions can be formulated in dose unit forms. As used herein, dose unit forms refer to physically distinct units suitable as a single dose for the subject being treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in conjunction with the required pharmaceutical carrier. The specifications of the dose unit forms of the present invention are determined and directly depend on the specific characteristics of the active compound, the particular therapeutic effect to be achieved, and the limitations inherent in the techniques for formulating such active compounds for the treatment of an individual.

[0190] Pharmaceutical compositions may be included in containers, packs, or dispensers along with administration instructions.

[0191] Treatment method As used herein, the terms “to treat” or “treatment” refer to both therapeutic actions and preventive or protective measures aimed at preventing or slowing (mitigating) undesirable physiological changes or impairments, such as the progression of cancer. Beneficial or desirable clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, stable (i.e., non-worsening) state of disease, delay or stabilization of disease progression, improvement or relief of the condition, and remission (partial or total), whether detectable or not. “Treatment” means extending survival time compared to the survival rate expected without treatment. Those who require treatment include those who already have a disease or impairment, as well as those who are susceptible to a disease or impairment, or who need to prevent a disease or impairment.

[0192] The present invention provides both prophylactic and therapeutic methods for treating subjects at risk (or susceptible) to cancer or other cell proliferation-related diseases or disorders. Such diseases or disorders include, but are not limited to, diseases or disorders associated with the abnormal expression of PD-L1. For example, the method is used to treat, prevent, or alleviate the signs of cancer. In one embodiment, the method is used to treat, prevent, or alleviate the symptoms of a solid tumor. Non-limiting examples of other tumors that can be treated by embodiments thereof include lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, or stomach cancer. Furthermore, the method of the present invention can be used to treat hematological cancers such as leukemia and lymphoma. Alternatively, the method can be used to treat, prevent, or alleviate the symptoms of metastatic cancer.

[0193] Accordingly, in one embodiment, the present invention provides a method for preventing, treating, or alleviating symptomatic cancer or proliferative disorders or impairments in a patient by administering the patient a monoclonal antibody, scFv antibody, or bispecific antibody of the present invention. For example, an anti-PD-L1 antibody can be administered in a therapeutically effective dose.

[0194] Individuals at risk of cancer or cell proliferation-related disorders or impairments may include patients with a family history of cancer or those exposed to substances known or suspected to cause cancer. Preventive medication may be administered before the onset of cancerous signs, either to prevent the disease or, alternatively, to delay its progression.

[0195] In another embodiment, tumor cell growth is inhibited by contacting cells with the anti-PD-L1 antibody of the present invention. The cells can be any cells that express PD-L1.

[0196] The present invention further provides both prophylactic and therapeutic methods for treating subjects at risk (or susceptible) to chronic viral, bacterial, or parasitic infections. The present invention also provides therapeutic methods for both prophylactic and therapeutic methods for treating subjects at risk of developing T-cell depletion-related diseases, disorders, or symptoms. The present invention also provides therapeutic methods for both prophylactic and therapeutic methods for treating subjects at risk of developing T-cell depletion-related diseases, disorders, or symptoms. Such diseases or disorders include, but are not limited to, HIV, AIDS, and chronic bacterial, viral, or parasitic infections. Other such chronic infections include, for example, those caused by hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus 1 (HSV-1), H. pylori, or Toxoplasma gondii.

[0197] Methods for increasing or enhancing an immune response to an antigen are also included in the present invention. The immune response is increased or enhanced by administering to a subject a monoclonal antibody, scFv antibody, or bispecific antibody of the present invention. The immune response is enhanced, for example, by enhancing antigen-specific T effector function. The antigen is a virus (e.g., HIV), a bacterium, a parasite, or a tumor antigen. The immune response is a spontaneous immune response. A spontaneous immune response means an immune response that is a result of an infection. The infection is a chronic infection. An increase or enhancement of the immune response to an antigen can be measured by many methods known in the art. For example, the immune response can be measured by measuring any one of the following: T cell activity, T cell proliferation, T cell activation, effector cytokine production, and T cell transcription profile. Alternatively, the immune response is a response induced by vaccination.

[0198] Therefore, in another embodiment, the present invention provides a method for increasing vaccine efficiency by administering the monoclonal antibody or scFv antibody and vaccine of the present invention to a subject. The antibody and vaccine are administered sequentially or simultaneously. The vaccine is an oncological vaccine, a bacterial vaccine, or a viral vaccine.

[0199] Combination method The compositions of the present invention described herein can be administered in combination with chemotherapeutic agents. Chemotherapy agents that can be administered with the compositions described herein include antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); anti-estrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, phloxuridine, interferon alpha-2b, glutamic acid, pricamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, and sulfate). Examples include, but are not limited to, vincristine; hormones (e.g., medroxyprogesterone, estramustine sodium phosphate, ethinylestradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianicene, and testolactone); nitrogen mustard derivatives (e.g., mephalen, colambucil, mechloretamine (nitrogen mustard), and thiotepa); steroids and combinations (e.g., betamethasone sodium phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0200] In additional embodiments, the compositions of the present invention described herein may be administered in combination with cytokines. Cytokines that may be administered together with the compositions include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0201] In additional embodiments, the compositions described herein may be administered in combination with other therapeutic or prophylactic regimens, such as radiotherapy.

[0202] In some embodiments, the compositions described herein may be administered in combination with other immunotherapeutic agents. Non-limiting examples of immunotherapeutic agents include simtuzumab, avagovomab, adecatumumab, aftuzumab, alemtuzumab, altumomab, amatsuximab, anatumomab, alsitumomab, bavituximab, bectomomab, bevacizumab, vibatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, sitatuzumab, xixtumumab, cribatuzumab, conatumumab, daratumumab, doroditumab, and zuri. Gotumab, ducizimab, detumomab, desetuzumab, dalotuzumab, eclomeximab, elotuzumab, encituximab, erzmaxomab, etalacizumab, falletuzumab, ficratuzumab, phyditumumab, frambotuzumab, futuximab, ganitumumab, gemtuzumab, girentuzumab, grembatumumab, ibritumomab, igobomab, imagatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab Iratumumab, rabetuzumab, lexatumumab, lintuzumab, rorbotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitsumomab, moxetumomab, nalnatumumab, naptumomab, nesitumumab, nimotuzumab, nofetumomab, okalatuzumab, ofatumumab, oraratumumab, onarutuzumab, oporutuzumab, olegobomab, panitumumab, pulsatuzumab, patrizumab, pemtumomab, Examples include pertuzumab, pintumomab, pritumumab, lacotumomab, radretumumab, rilotumumab, rituximab, lobatumumab, satumomab, sibrotuzumab, siltuximab, solitomab, takatuzumab, tapritumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotzumab, ubrituximab, bertuzumab, borsetuzumab, botumumab, saltumumab, CC49, and 3F8.

[0203] The present invention provides a method for treating cancer in a patient by administering two antibodies that bind to the same epitope of the PD-L1 protein, or alternatively, to two different epitopes of the PD-1 protein. Alternatively, cancer can be treated by administering a first antibody that binds to PD-L1 and a second antibody that binds to a protein other than PD-L1. In other embodiments, cancer can be treated by administering a bispecific antibody that binds to both PD-L1 and a protein other than PD-L1. For example, other proteins other than PD-L1 include, but are not limited to, GITR. For example, other proteins other than PD-L1 may be tumor-associated antigens, or other proteins other than PD-L1 may be cytokines.

[0204] In some embodiments, the present invention provides administering an anti-PD-L1 antibody alone or in combination with additional antibodies that recognize another protein other than PD-L1, along with cells capable of achieving or enhancing an immune response. For example, these cells may be peripheral blood mononuclear cells (PBMCs), or any cell type found in PBMCs, such as cytotoxic T cells, macrophages, and natural killer (NK) cells.

[0205] Furthermore, the present invention provides the administration of antibodies and antineoplastic agents that bind to the PD-L1 protein, as well as other therapeutic agents comprising biomolecules such as small molecules, growth factors, cytokines or peptides, peptide mimes, peptoids, polynucleotides, lipid-derived mediators, low-molecular-weight bioamines, hormones, neuropeptides, and proteases. Small molecules include, but are not limited to, inorganic and small organic molecules. Suitable growth factors or cytokines include IL-2, GM-CSF, IL-12, and TNF-alpha. Small molecule libraries are known in the art. (See Lam, Anticancer Drug Des., 12:145, 1997).

[0206] Chimeric antigen receptor (CAR) T cell therapy Cell therapies, such as chimeric antigen receptor (CAR) T-cell therapy, are also provided herein. CAR T-cell therapy redirects a patient's T cells to kill tumor cells through exogenous expression of a CAR. The CAR may be a transmembrane fusion protein that links the antigen-recognition domain of an antibody to the intracellular signaling domain of a T cell receptor and co-receptor. Suitable cells that can be contacted with the anti-PD-L1 antibody of the present invention (or manipulated to express the anti-PD-L1 antibody as described herein) can be used. Solid tumors present unique challenges for CAR-T therapy. Unlike hematological malignancies, tumor-associated target proteins are overexpressed between tumors and healthy tissues, resulting in on-target / off-tumor T-cell killing in healthy tissues. Furthermore, immunosuppression in the tumor microenvironment (TME) limits the activation of CAR-T cells toward tumor killing. Through such contact or manipulation, the cells can then be introduced into a cancer patient in need of treatment. Cancer patients may have any of the types of cancer disclosed herein. The cells (e.g., T cells) may be, but are not limited to, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof. Exemplary CARS useful in embodiments of the present invention include, for example, those disclosed in PCT / US2015 / 067225 and PCT / US2019 / 022272, each of which is incorporated herein by reference in whole.

[0207] In one embodiment, the PD-L1 antibody discussed herein can be used in the construction of a multispecific antibody or as a payload for CAR-T cells. For example, in one embodiment, the anti-PD-L1 antibody discussed herein can be used for targeting CARS (i.e., as a targeting moiety). In another embodiment, the anti-PD-L1 antibody discussed herein can be used as a targeting moiety, and different PD-L1 antibodies targeting different epitopes can be used as payloads. In yet another embodiment, the payload can be an immunomodulatory antibody payload. In some embodiments, the PD-L1 antibody described herein can be used as a targeting moiety in CARs (e.g., PD-L1 + It can be used as a secretory checkpoint blocking antibody to kill tumor cells or to reverse T cell depletion.

[0208] For example, embodiments of the present invention include a chimeric antigen receptor (CAR) comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain. In embodiments, the extracellular domain is an isolated monoclonal antibody or its antigen-binding fragment that binds to the human programmed death ligand 1 (PD-L1) protein. For example, the monoclonal antibody or its fragment comprises a heavy chain, a light chain, or a combination thereof, where the heavy chain is G-(X1)-T-(X2)-SS-(X3X4)(SEQ ID NO: 47), G-(X1)-T-(X2)-(X 13 X 14 )-(X3X4)(Sequence No. 205), G-(X1)-TF-(X 13 X 14 CDR1 containing )-Y-(X4)(Sequence ID 206), I-(X8X9X 10 X 11 )-G-(X 12 )-A (Sequence No. 51), or II-(X 15 )-IFG-(X 16CDR2 containing )-A (SEQ ID NO: 207), and / or CDR3 containing ARGRQMFGAGIDF (SEQ ID NO: 6) or ARVHAALYYGMDV (SEQ ID NO: 14), TTGGLGLVYPYYNYIDV (SEQ ID NO: 99), AKVHPVFSYALDV (SEQ ID NO: 100), AEEGAFNSLAI (SEQ ID NO: 101), ARDGSGYDSAGMDD (SEQ ID NO: 102), ARGFGGPDY (SEQ ID NO: 103), ARVHGALYYGMDV (SEQ ID NO: 104), ASGSIVGAAYAFDI (SEQ ID NO: 105), ARDRSEGGFDP (SEQ ID NO: 106), or AEEGAFNSLAI (SEQ ID NO: 107), the light chain is S-(X 17 X 18 )I-(X 19 )-SNY (Sequence No. 208) or NIG-(X5)-K-(X 20 CDR1 containing (Sequence ID 48), (X 21 )-DN(Sequence ID 209), (X 22 Includes CDR2 containing )-NN (sequence number 210) or DD-X6 (sequence number 49), and / or CDR3 containing QSYDSNNRHVI (sequence number 22), QVWDS-(X7)-SDHWV (sequence number 50), QVWDSSGDLWV (sequence number 126), AAWDDSLNGLV (sequence number 127), QSYDGITVI (sequence number 128), QSYDSSNHWV (sequence number 129), AVWDDSLSGVV (sequence number 131), MIWHSSAYV (sequence number 132), NSRDISDNQWQWI (sequence number 134), or QSYDSSNHVV (sequence number 135).

[0209] The CAR according to the present invention may comprise at least one transmembrane polypeptide comprising at least one extracellular ligand-binding domain and at least one transmembrane polypeptide comprising at least one intracellular signaling domain, such that the polypeptides assemble together to form a chimeric antigen receptor.

[0210] As used herein, the term “extracellular ligand-binding domain” is defined as an oligonucleotide or polypeptide capable of binding to a ligand. For example, a domain may be able to interact with cell surface molecules. For instance, an extracellular ligand-binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state.

[0211] In one embodiment, the extracellular ligand-binding domain may include an antigen-binding domain derived from an antibody against the target antigen. For example, the target may be PD-L1. Therefore, the CAR may be specific to PD-L1. In one embodiment, the extracellular ligand-binding domain is a single-chain antibody fragment (scFv) containing light chain (VL) and heavy chain (VH) variable fragments of a target antigen-specific monoclonal antibody conjugated by a flexible linker. For example, the scFv antibody is specific to PD-L1. However, it is understood that binding domains other than scFv, such as, for example, a single-domain antibody fragment or receptor ligand from a camelid, an antibody-binding domain, an antibody hypervariable loop, or a CDR, can also be used for predefined targeting of lymphocytes.

[0212] In embodiments, the transmembrane domain includes a stalk region between the extracellular ligand-binding domain and the transmembrane domain. The term "stalk region" can mean any oligo or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, the stalk region is used to provide greater flexibility and accessibility to the extracellular ligand-binding domain. The stalk region can contain up to 300 amino acids, such as 10 to 100 amino acids. In embodiments, the stalk region contains 25 to 50 amino acids. The stalk region may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, or CD28, or from all or part of the antibody constant region. Alternatively, the stalk region may be a synthetic sequence corresponding to a naturally occurring stalk sequence, or it may be a completely synthetic stalk sequence. In preferred embodiments, the stalk region is part of a human CD8 alpha chain.

[0213] In the embodiment, the transmembrane domain may include CD28.

[0214] The signaling domain or intracellular signaling domain of the CAR of the present invention is involved in intracellular signaling after binding of the extracellular ligand-binding domain to its target, resulting in the activation of immune cells and immune responses. In other words, the signaling domain is involved in the activation of at least one of the normal effector functions of the immune cells on which the CAR expresses. For example, the effector function of a T cell may be cytolytic activity, including cytokine secretion, or helper activity. Therefore, the term “signal conversion domain” can refer to a portion of a protein that converts effector signaling function signals and instructs cells to perform specific functions.

[0215] The signal conversion domain can include two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation and those that act antigen-independently to provide secondary or co-stimulatory signals. The primary cytoplasmic signaling sequence can include a signaling motif known as an immune receptor-activated tyrosine motif, which is an ITAM. ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that function as binding sites for syk / zap70 class tyrosine kinases. Examples of ITAMs used in the present invention, in non-limiting examples, may include those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In preferred embodiments, the signaling domain of the CAR can include a CD3 zeta signaling domain or an intraplasmic domain of an Fc epsilon RI beta or gamma chain. In another preferred embodiment, signaling is provided by CD3 zeta along with co-stimulation provided by CD28 and tumor necrosis factor receptors (TNFr) (such as 4-1BB or OX40).

[0216] In some embodiments, the intracellular signaling domain of the CAR of the present invention includes a co-stimulatory signaling molecule. In some embodiments, the intracellular signaling domain contains two, three, four, or more co-stimulatory molecules in tandem. The co-stimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand necessary for an efficient immune response.

[0217] A "costimulatory ligand" can refer to a molecule on an antigen-presenting cell that specifically binds to a co-stimulatory molecule on a T cell, thereby providing a signal that mediates T cell responses, including but not limited to proliferative activation and differentiation, in addition to the primary signal provided by, for example, the binding of peptide-carrying MHC molecules to the TCR / CD3 complex. Examples of costimulatory ligands include CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecules (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, Toll ligand receptor, and ligands that specifically bind to B7-H3 in particular. Costimulatory ligands may include, but are not limited to, agonists or antibodies that bind to them. Costimulatory ligands also include ligands and antibodies that specifically bind to, among other things, costimulatory molecules present on T cells, such as, but are not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0218] A “costimulatory molecule” can refer to a congenital binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, including but not limited to proliferation. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors. Examples of costimulatory molecules include ligands that specifically bind to CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0219] In some embodiments, the selection of CD28 as the co-stimulatory domain of the CAR may be based on the fact that CD28 CARs induce an active proliferation response and enhance effector function, while 4-1BB-based CARs induce more progressive T cell accumulation, which may offset their lower immediate efficacy. In one embodiment, CD28 is replaced by 41BB in the CAR construct.

[0220] In another specific embodiment, the signaling domain is a TNFR-related factor 2 (TRAF2) binding motif, which is the cytoplasmic tail of a co-stimulatory TNFR member family. The cytoplasmic tail of a co-stimulatory TNFR family member contains a TRAF2 binding motif consisting of a major conserved motif (P / S / A)X(Q / E)E) or a minor motif (PXQXXD), where X is any amino acid. TRAF proteins are recruited to the intracellular tails of many TNFRs in response to receptor trimerization.

[0221] A distinguishing feature of suitable multiple transmembrane polypeptides is their ability to be expressed on the surface of immune cells, particularly lymphocytes or natural killer (NK) cells, and to interact together to induce a cellular response of immune cells against predefined target cells. Different transmembrane polypeptides of the CARs of the present invention, including extracellular ligand-binding domains and / or signaling domains, interact together to participate in signaling after binding to target ligands and induce an immune response. The transmembrane domains may be derived from either natural or synthetic sources. The transmembrane domains may be derived from any membrane-bound or transmembrane protein.

[0222] As used herein, the term “part” may refer to any subset of a molecule, i.e., a shorter peptide. Alternatively, an amino acid sequence functional variant of a polypeptide may be prepared by mutations in the DNA encoding the polypeptide. Such variants or functional variants include, for example, deletions, insertions, or substitutions of residues in the amino acid sequence. Any combination of deletions, insertions, and substitutions may be performed to arrive at the final construct, provided that the final construct has the desired activity and, in particular, exhibits specific anti-target cellular immune activity. The functionality of the CARs of the present invention in host cells is detectable by assays suitable for demonstrating the signaling ability of the CAR when a specific target is bound. Such assays are available to those skilled in the art. For example, such assays may enable the detection of signaling pathways triggered upon target binding, such as assays involving the measurement of increased calcium ion release, intracellular tyrosine phosphorylation, inositol phosphate turnover, or the production of interleukin (IL) 2, interferon γ, GM-CSF, IL-3, and IL-4 resulting therefrom.

[0223] Cells that express CAR Embodiments of the present invention include cells expressing a CAR (i.e., CART). The cells may be of any kind, including immune cells capable of expressing a CAR for cancer treatment, or bacterial cells possessing an expression vector encoding a CAR. Where used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All these terms also include their offspring, which are any subsequent generations. It is understood that not all offspring may be identical due to planned or accidental mutations. In the context of expressing heterologous nucleic acid sequences, “host cell” refers to a eukaryotic cell capable of replicating the vector and / or expressing the heterologous gene encoded by the vector. Host cells can and have been used as recipients of vectors. Host cells may be “transfected” or “transformed,” which refers to the process by which an exogenous nucleic acid is introduced into or transferred to a host cell. Transformed cells include primary subject cells and their offspring. As used herein, the terms “manipulated” and “recombinant” cells or host cells are intended to refer to cells into which an exogenous nucleic acid sequence, such as a vector, has been introduced. Recombinant cells can therefore be distinguished from naturally occurring cells that do not contain the nucleic acid introduced by recombination. In embodiments of the present invention, the host cells are T cells, including cytotoxic T cells (TCs, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), and NK cells and NKT cells are also included in the present invention.

[0224] Some vectors may utilize regulatory sequences that enable replication and / or expression in both prokaryotic and eukaryotic cells. Those skilled in the art will further understand the conditions under which all of the above host cells can be incubated and maintained, and the conditions under which the vectors can be replicated. Furthermore, techniques and conditions that enable the large-scale production of vectors, as well as the production of nucleic acids encoded by the vectors and their homologous polypeptides, proteins, or peptides, are also understood and known.

[0225] Cells can be autologous cells, syngeneic cells, allogeneic cells, and in some cases, heterogeneic cells.

[0226] In many situations, it may be desirable to be able to kill modified CTLs when treatment termination is desired, when cells become neoplastic, in studies where the absence of cells after their presence is of interest, or in other events. For this purpose, it may be possible to provide the expression of specific gene products, such as inducible suicide genes, that can kill modified cells under controlled conditions.

[0227] Armed CART The present invention further includes CARTs modified to secrete one or more polypeptides. Armed CARTs have the advantage of simultaneously secreting polypeptides at a target site, such as a tumor site. The polypeptides may be, for example, antibodies or cytokines. For example, the antibodies are PD-L1 specific, such as the antibodies and fragments described herein. In other embodiments, the secreted antibodies may be antibodies specific to CAIX, GITR, PD-L2, PD-1, or CCR4 (see, for example, the sequences described in PCT publication number WO2016 / 1009085, in which the entire application is incorporated by reference).

[0228] Armed CARTs can be constructed by including a nucleic acid encoding a target secreted polypeptide following an intracellular signaling domain. In embodiments, an internal ribosome entry site (IRES) is present, positioned between the intracellular signaling domain and the target polypeptide. Those skilled in the art will understand that by using multiple IRES sequences in tandem, two or more polypeptides can be expressed.

[0229] In this embodiment, CART cells can be maintained using cytokines such as IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.

[0230] Cytokines that share the γc receptor, such as IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, are important for the development and maintenance of memory T cells. Among them, IL-21 promotes a less differentiated phenotype associated with the enrichment of tumor-specific CD8 T cells, and increases the antitumor effect in mouse melanoma models compared to IL-2 or IL-15.

[0231] In certain embodiments, CART cells are maintained with IL-21.

[0232] Introducing the construct to CTL The expression vector encoding the CAR may be introduced as one or more DNA molecules or constructs, which may contain at least one marker that allows for the selection of host cells containing the constructs.

[0233] The constructs can be prepared by conventional methods, and the genes and regulatory regions can be isolated, ligated, cloned into a suitable cloning host, and analyzed by restriction, sequencing, or other convenient means. In particular, PCR can be used to isolate individual fragments containing all or part of the functional units, and one or more mutations can be introduced using "primer repair," ligation, in vitro mutagenesis, etc., as appropriate. Once completed and demonstrated to have the appropriate sequence, the construct can then be introduced into CTLs by any convenient means. The constructs may be incorporated and packaged for infection or transduction into cells, such as non-replicating defective viral genomes, adenoviruses, adeno-associated viruses (AAVs), herpes simplex virus (HSV), or retroviral vectors or lentiviral vectors. The constructs may optionally contain viral sequences for transfection. Alternatively, the constructs may be introduced by fusion, electroporation, bioristic methods, transfection, lipofection, etc. Host cells can be grown and expanded in a culture before the construct is introduced, and then the construct can be introduced and appropriately processed to incorporate it. Next, the cells are augmented and screened for markers present in the construct. Various markers that can be successfully used include HPRT, neomycin resistance, thymidine kinase, and hygromycin resistance.

[0234] In some cases, if it is desired that a construct be incorporated into a specific locus, it may have a target site for homologous recombination. For example, an endogenous gene can be knocked out and replaced with the gene encoded by the construct (at the same locus or elsewhere) using materials and methods known in the art for homologous recombination. For homologous recombination, either an OMEGA or O-vector can be used. See, for example, Thomas and Capecchi, Cell (1987) 51, 503-512; Mansour, et al., Nature (1988) 336, 348-352; and Joyner, et al., Nature (1989) 338, 153-156.

[0235] The constructs can be introduced as a single DNA molecule encoding at least one CAR and, optionally, another gene, or as different DNA molecules having one or more genes. Other genes may include, for example, genes encoding therapeutic molecules or suicide genes. The constructs may be introduced simultaneously or sequentially, each having the same or different markers.

[0236] Vectors containing useful elements such as bacterial or yeast replication origins, selectable and / or amplified markers, and promoter / enhancer elements for expression in prokaryotes or eukaryotes, which can be used for preparing constructor DNA stocks and performing transfections, are well known in the art and many are commercially available.

[0237] How to use CAR-expressing cells The cells described herein can be used to treat cancer or other cell proliferation-related diseases or disorders. Such diseases or disorders include, but are not limited to, diseases or disorders related to the abnormal expression of PD-L1. In another embodiment, the isolated cells according to the present invention can be used to manufacture pharmaceuticals for treating cancer or other cell proliferation-related diseases or disorders. Such diseases or disorders include, but are not limited to, diseases or disorders related to the abnormal expression of PD-L1.

[0238] The embodiments described herein depend on a method for treating a patient in need of treatment, the method comprising at least one of the steps of (a) providing chimeric antigen receptor cells according to the present invention, and (b) administering the cells to the patient.

[0239] The treatment may result in remission, cure, or prevention. It may be part of autoimmunotherapy or allogeneic immunotherapy. "Auto" means that the cells, cell lines, or cell populations used to treat the patient originate from the patient or from a human leukocyte antigen (HLA)-matched donor. "Allogeneic" means that the cells or cell populations used to treat the patient originate from a donor rather than from the patient.

[0240] The present invention is particularly well suited to allogeneic immunotherapy, insofar as it allows for the transformation of T cells, typically obtained from a donor, into non-alloreactive cells. This can be performed under standard protocols and replicated as many times as needed. The resulting modified T cells can be pooled and administered to one or more patients, making them available as a "ready-made" therapeutic product.

[0241] Cancers that can be treated with the antibodies or CAR compositions described herein include non-angiogenic or substantially non-angiogenic tumors, as well as angiogenic tumors. Cancers may include non-solid tumors (e.g., hematological malignancies, e.g., leukemia and lymphoma) or solid tumors. Types of cancers that can be treated with the CARs of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, e.g., sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included. For example, cancers for which checkpoint inhibition is a standard treatment for several malignancies (referred to herein as “checkpoint inhibitor cancers”) can be treated with the antibodies and / or CAR compositions described herein. Checkpoint inhibitor cancers include, but are not limited to, melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), renal cell carcinoma (RCC), chronic lymphocytic leukemia (CLL; such as B-cell CLL or T-cell CLL), classical Hodgkin lymphoma (cHL), head and neck squamous cell carcinoma (HNSCC), colorectal cancer (CRC), gastric cancer, hepatocellular carcinoma (HCC), primary mediastinal large B-cell lymphoma (PMLBCL), bladder cancer, urothelial carcinoma, endometrial cancer, cervical cancer, breast cancer (e.g., triple-negative breast cancer), Merkel cell carcinoma (MCC), and adult and pediatric solid tumors with high microsatellite instability (MSI-H) or DNA mismatch repair deficiency (dMMR) (doi:10.1016 / j.csbj.2019.03.006). The treatments described herein may also include other cancers for which checkpoint inhibitor therapy is under investigation. While we do not wish to be bound by theory, RCC and B-CLL mouse models could be used for treatment in CAR T factories, which are models relevant to human diseases.

[0242] For example, treatment may be antibody and / or CAR-T therapy in combination with one or more therapies for cancer selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, and radiotherapy.

[0243] According to embodiments of the present invention, the treatment can be administered to patients receiving immunosuppressive therapy. In fact, the present invention may rely on cells or cell populations that have been made resistant to at least one immunosuppressant due to inactivation of genes encoding receptors for such immunosuppressants. In this embodiment, the immunosuppressive therapy should help in the selection and proliferation of T cells according to the present invention within the patient.

[0244] In further embodiments, the cell composition of the present invention is administered to a patient in combination with (e.g., before, simultaneously with, or after) a T-cell ablative therapy using any of the following: bone marrow transplantation, a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or an antibody such as OKT3 or CAM PATH. In another embodiment, the cell composition of the present invention is administered after a B-cell ablative therapy using an agent that reacts with CD20, such as rituxan. For example, in one embodiment, a subject may receive standard treatment with high-dose chemotherapy and then undergo peripheral blood stem cell transplantation. In a specific embodiment, after transplantation, the subject receives an infusion of the augmented immune cells of the present invention. In further embodiments, the augmented cells are administered before or after surgery. The modified cells obtained by any of the methods described herein can be used in specific embodiments of the present invention for treating patients in need of treatment for host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD). Therefore, the scope of the present invention includes methods for treating patients in need of treatment for host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD), comprising administering to the patient an effective amount of modified cells containing inactivated TCR alpha and / or TCR beta genes.

[0245] Cell administration The present invention is suitable for allogeneic immunotherapy, insofar as it allows for the transformation of T cells, typically obtained from a donor, into non-alloreactive cells. This can be performed under a standard protocol and replicated as many times as needed. The resulting modified T cells can be pooled and administered to one or more patients, making them available as a "ready-made" therapeutic product.

[0246] Depending on the properties of the cells, they can be introduced into a host organism, such as a mammal, in a wide variety of ways. In certain embodiments, cells can be introduced into a tumor site, while in alternative embodiments, cells home to or are modified to home to cancer. The number of cells used depends on various circumstances, the purpose of introduction, the lifespan of the cells, the protocol used, such as the number of doses, the cell's ability to proliferate, and the stability of the recombinant construct. The cells may be applied as a dispersion and are usually injected into or near the site of interest. The cells may be in a physiologically acceptable medium.

[0247] In some embodiments, cells are encapsulated to inhibit immune recognition and then positioned at the tumor site.

[0248] Cells can be administered as desired. Various protocols can be used depending on the desired response, administration method, cell lifespan, and the number of cells present. The number of administrations depends, at least in part, on the factors mentioned above.

[0249] The administration of cells or cell populations according to the present invention can be carried out by any convenient method, including aerosol inhalation, injection, ingestion, blood transfusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodulately, intramedullarily, intramuscularly, intravenously, or intralymphatically, or intraperitoneally. In one embodiment, the cell composition of the present invention is preferably administered by intravenous injection.

[0250] The administration of cells or cell populations is 10 per kg of body weight. 4 ~10 9 individual cells, for example, 105 ~10 6 The administration can consist of cells / kg body weight, encompassing all integer values ​​of cell counts within these ranges. Cells or cell populations may be administered in one or more doses. In another embodiment, the effective amount of cells is administered as a single dose. In yet another embodiment, the effective amount of cells is administered in two or more doses over a period of time. The timing of administration is within the discretion of the attending physician and depends on the patient's clinical condition. Cells or cell populations can be obtained from any source, such as a blood bank or donor. While individual needs vary, determining the optimal range of effective doses of a given cell type for a particular disease or condition is within the scope of this art. Effective dose means the amount that provides a therapeutic or preventive benefit. The dose administered will depend on the recipient's age, health condition and weight, the type of concurrent treatment, if any, the frequency of treatment, and the nature of the desired effect.

[0251] It should be understood that the system is affected by many variables that can change over time and under different circumstances, such as the rate of loss of cellular activity as a result of cell loss or the expression activity of individual cells, including the cellular response to the ligand, expression efficiency, and, where appropriate, secretion levels, the activity of the expression product, and the specific needs of the patient. Therefore, for each individual patient, even if there are pluripotent stem cells that can be administered to the entire population, it is expected that each patient will be monitored for their appropriate individual dosage, and such practice of monitoring patients is routine in the art.

[0252] Nucleic acid-based expression systems The CAR of the present invention can be expressed from an expression vector. Recombination techniques for producing such expression vectors are well known in the art.

[0253] The term “vector” can refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell into which it can replicate. A nucleic acid sequence can be “exogenous,” meaning that it is either foreign to the cell into which the vector is introduced, or the sequence is homologous to an intracellular sequence but located in a position within the host cell nucleic acid where the sequence is not normally found. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (such as YACs). Those skilled in the art will have sufficient equipment to construct vectors using standard recombination techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both incorporated herein by reference).

[0254] The term “expression vector” can refer to any type of gene construct containing nucleic acids that encode transcriptionable RNA. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of “regulatory sequences” that refer to nucleic acid sequences necessary for the transcription and possibly translation of the operably linked coding sequence in a particular host cell. In addition to regulatory sequences that govern transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that perform other functions, as described below.

[0255] A “promoter” can refer to a regulatory sequence, which is a region of a nucleic acid sequence whose transcription initiation and rate are controlled. It may include a genetic element to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind to initiate specific transcription of the nucleic acid sequence. The terms “operatably positioned,” “operatably linked,” “controlled,” and “transcriptionally controlled” mean that the promoter is in the correct functional location and / or orientation relative to the nucleic acid sequence to control the transcription initiation and / or expression of that sequence.

[0256] A promoter may contain a sequence that functions to position the start site for RNA synthesis. The best-known example of this is the TATA box, but in some promoters without a TATA box, such as the promoter of the mammalian terminal deoxynucleotidyltransferase gene or the SV40 late gene, a separate element covering the start site itself helps to fix the start location. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in a region 30-110 bp upstream of the start site, but some promoters have been shown to also contain functional elements downstream of the start site. To place the coding sequence under the "control" of the promoter, the 5' end of the transcription start site in the transcription reading frame is positioned "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates the transcription of the DNA and promotes the expression of the encoded RNA.

[0257] In many cases, the spacing between promoter elements is flexible, so that the promoter's function is maintained even if the elements are reversed or moved relative to each other. In the tk promoter, the spacing between promoter elements can be widened to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to be able to function cooperatively or independently to activate transcription. Promoters may or may not be used in combination with "enhancers," which refer to cis-acting regulatory elements involved in the transcriptional activation of nucleic acid sequences.

[0258] A promoter may be naturally associated with a nucleic acid sequence, as can be obtained by isolating a 5-prime non-coding sequence located upstream of the coding segment and / or exon. Such a promoter may be called “endogenous.” Similarly, an enhancer may be naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages may be gained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the nucleic acid sequence in its natural environment. Recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with the nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from other viruses, or from prokaryotic or eukaryotic cells, and promoters or enhancers that are “not naturally occurring,” i.e., different elements of different transcriptional regulatory regions, and / or mutations that alter expression. For example, the most commonly used promoters in recombinant DNA construction include the lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to synthetically generating promoter and enhancer nucleic acid sequences, sequences may also be generated using recombinant cloning and / or nucleic acid amplification techniques, including PCR™, in relation to the compositions disclosed herein (see U.S. Patents 4,683,202 and 5,928,906, respectively, incorporated herein by reference). Furthermore, regulatory sequences directing the transcription and / or expression of sequences within non-nuclear organelles such as mitochondria and chloroplasts are also considered to be usable.

[0259] Naturally, it will be important to use promoters and / or enhancers that effectively guide the expression of DNA segments in selected organelles, cell types, tissues, organs, or organisms for expression. Those skilled in the art of molecular biology are generally familiar with the use of promoter, enhancer, and cell type combinations for protein expression (see, for example, Sambrook et al. 1989, incorporated herein by reference). The promoter used may be useful under appropriate conditions that direct high-level expression of the introduced DNA segment, such as constitutive, tissue-specific, inducible, and / or favorable for the large-scale production of recombinant proteins and / or peptides. Promoters may be heterogeneous or endogenous.

[0260] Furthermore, expression can also be promoted using any combination of promoter / enhancer. The use of T3, T7, or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from specific bacterial promoters if the appropriate bacterial polymerase is provided as part of the delivery complex or as an additional gene expression construct.

[0261] Assays for characterizing the identity of tissue-specific promoters or elements, as well as their activity, are well known to those skilled in the art.

[0262] Efficient translation of code sequences may also require specific start signals. These signals may include ATG start codons or adjacent sequences. It may be necessary to provide exogenous translation control signals, including ATG start codons. Those skilled in the art will be able to easily determine this and provide the necessary signals.

[0263] In certain embodiments of the present invention, internal ribosome entry site (IRES) elements are used to construct multiple genes or polycistronic messages, which may be used in the present invention.

[0264] Vectors can contain multiple cloning sites (MCS), which are nucleic acid regions containing multiple restriction enzyme sites, any of which can be used to digest the vector in combination with standard recombination techniques. "Restriction enzyme digestion" refers to the catalytic cleavage of nucleic acid molecules by enzymes that function only at specific locations on the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is well understood by those skilled in the art. Often, restriction enzymes that cleave within the MCS are used to linearize or fragment the vector, allowing foreign sequences to be ligated into the vector. "Ligation" refers to the process of forming a phosphodiester bond between two nucleic acid fragments, which may or may not be adjacent to each other. Techniques involving restriction enzymes and ligation reactions are well known to those skilled in the art of recombination techniques.

[0265] Splicing sites, termination signals, replication origins, and selectable markers can also be used.

[0266] In certain embodiments, plasmid vectors can be used to transform host cells. Plasmid vectors containing replicons and regulatory sequences derived from a species compatible with the host cell can be used in relation to these hosts. Vectors typically possess replication sites, as well as marking sequences that can provide phenotypic selection in transformed cells. In a non-limiting example, Escherichia coli (E. coli) is often transformed using derivatives of pBR322, a plasmid derived from the E. coli species. Since pBR322 contains genes for ampicillin and tetracycline resistance, transformed cells can be easily identified. The pBR plasmid, or other microbial plasmids or phages, must also contain, or be modified to contain, a promoter that the microorganism can use for the expression of its own proteins.

[0267] Furthermore, phage vectors containing replicons and regulatory sequences compatible with host microorganisms can be used as transformation vectors in relation to these hosts. For example, phage-lambda GEM TM.11 can be used in the creation of recombinant phage vectors that can be used to transform host cells such as E. coli LE392.

[0268] Further useful plasmid vectors include the pIN vector (Inouye et al., 1985) and the pGEX vector, which are used to generate glutathione S-transferase (GST) soluble fusion proteins for later purification, isolation, or cleavage. Other suitable fusion proteins include those containing galactosidase, ubiquitin, etc.

[0269] Bacterial host cells containing the expression vector, such as E. coli, are grown in any of many suitable media, e.g., LB. As will be understood by those skilled in the art, recombinant protein expression in a particular vector can be induced by contacting the host cells with a promoter-specific active agent, for example by adding IPTG to the medium, or by switching the incubation to a high temperature. After culturing the bacteria for a further 2 to 24 hours, the cells are harvested by centrifugation and washed to remove any residual medium.

[0270] The ability of certain viruses to infect cells, invade cells, integrate into the host cell genome, and stably and efficiently express viral genes via receptor-mediated endocytosis makes them attractive candidates for the introduction of foreign nucleic acids into cells (e.g., mammalian cells). Components of the present invention may be viral vectors encoding one or more CARs of the present invention. Non-limiting examples of viral vectors that may be used to deliver the nucleic acids of the present invention are described herein.

[0271] Methods for nucleic acid delivery include the use of adenovirus expression vectors. While adenovirus vectors are known to have a low ability to integrate into genomic DNA, this capability is offset by the high gene transfer efficiency achieved by these vectors. “Adenovirus expression vector” means containing a construct that (a) is sufficient to support the packaging of the construct, and (b) is sufficient to ultimately express the tissue or cell-specific construct cloned therein. Knowing that the genetic makeup or adenovirus is a 36kb, linear, double-stranded DNA virus, large fragments of adenovirus DNA can be replaced with foreign sequences of up to 7kb (Grunhaus and Horwitz, 1992).

[0272] Nucleic acids can be introduced into cells using adenovirus-assisted transfection. Increased transfection efficiency has been reported in cell systems using adenovirus-coupled systems (Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno-associated viruses (AAVs) are attractive vector systems for use in the cells of the present invention because of their high integration frequency and ability to infect non-dividing cells, and are therefore useful for gene delivery to mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo. AAVs have a broad infectious host range (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Details relating to the generation and use of rAAV vectors are described in U.S. Patents 5,139,941 and 4,797,368, which are incorporated herein by reference, respectively.

[0273] Retroviruses are useful as delivery vectors due to their ability to integrate their genes into the host genome, introduce large amounts of foreign genetic material, infect a wide range of species and cell types, and package into specific cell lines (Miller, 1992).

[0274] To construct a retroviral vector, nucleic acid (e.g., one encoding the desired sequence) is inserted into the viral genome in place of a specific viral sequence, producing a virus with replication defects. To generate virions, a packaging cell line is constructed that contains the gag, pol, and env genes but lacks the LTR and packaging components (Mann et al., 1983). When a recombinant plasmid containing cDNA, along with the retroviral LTR and packaging sequence, is introduced into a special cell line (e.g., by calcium phosphate precipitation), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture medium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors can infect various types of cells. However, host cell division is required for integration and stable expression (Paskind et al., 1975).

[0275] Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural functions. Lentiviral vectors are well known in the art (see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997, U.S. Patents 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency virus: HIV-1, HIV-2, and simian immunodeficiency virus: SIV. Lentiviral vectors are produced by multiple attenuation of HIV pathogenic genes, for example, by deleting genes env, vif, vpr, vpu, and nef, resulting in biosafe vectors.

[0276] Recombinant lentiviral vectors can infect non-dividing cells and can be used for gene transfer and nucleic acid sequence expression both in vivo and ex vivo. For example, a recombinant lentivirus that can infect non-dividing cells in which a suitable host cell has been transfected with two or more vectors possessing packaging functions, i.e., gag, pol, and env, as well as rev and tat, is described in U.S. Patent No. 5,994,136, which is incorporated herein by reference. Recombinant viruses can be targeted by binding an envelope protein to an antibody or a specific ligand in order to target receptors of a particular cell type. For example, by inserting a sequence of interest (including regulatory regions) into a viral vector along with another gene encoding a ligand for a receptor on a specific target cell, the vector is now target-specific.

[0277] In this invention, other viral vectors can be used as vaccine constructs. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), Sindbis virus, cytomegalovirus, and herpes simplex virus can be used. These provide several attractive features to various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

[0278] In some embodiments, the delivered nucleic acid may be contained within an infectious virus engineered to express a specific binding ligand. Thus, the viral particle specifically binds to a homologous receptor on the target cell, delivering its contents to the cell. A novel approach designed to enable the specific targeting of retroviral vectors was developed based on the chemical modification of retroviruses by chemically adding lactose residues to the viral envelope. This modification enables specific infection of hepatocytes via the sialycoprotein receptor.

[0279] Another approach targeting recombinant retroviruses was designed, using biotinylated antibodies against retroviral envelope proteins and specific cell receptors. The antibodies were conjugated via the biotin component using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated in vitro infection of various human cells harboring these surface antigens by ecotropic viruses (Roux et al., 1989).

[0280] Suitable methods for nucleic acid delivery for cell transfection or transformation are known to those skilled in the art. Such methods include, but are not limited to, ex vivotransfection and direct delivery of DNA by injection. By applying techniques known in the art, cells can be transformed stably or transiently.

[0281] Ex vivo transformation Methods for transfecting eukaryotic cells and tissues extracted from living organisms in vitro are known to those skilled in the art. Therefore, it is conceivable that cells or tissues can be extracted and transfected ex vivo using the nucleic acids of the present invention. In certain embodiments, the transplanted cells or tissues can be placed in a living organism. Preferably, the nucleic acids are expressed in the transplanted cells.

[0282] The present invention kit Any of the compositions described herein may be included in the kit.

[0283] Some components of the kit may be packaged in either an aqueous medium or a lyophilized form. The kit's container means generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the components are placed and, preferably, can be appropriately dispensed. If the kit contains two or more components, the kit also typically includes a second, third, or other additional container into which additional components can be individually placed. However, various combinations of components may be contained in vials. The kits of the present invention also typically include means for tightly sealing and containing the components for commercial sale. Such containers may include injection-molded or blow-molded plastic containers into which the desired vials are held.

[0284] If the components of the kit are provided in one and / or more liquid solutions, the liquid solutions are aqueous solutions, and sterile aqueous solutions are particularly useful. In some cases, the container means itself may be a syringe, pipette, and / or other similar device from which the formulation can be applied to the infected area of ​​the body, injected into an animal, and / or applied to and / or mixed with other components of the kit.

[0285] However, the components of the kit may be supplied as dry powders. If the reagents and / or components are supplied as dry powders, the powders may be reconstituted by the addition of a suitable solvent. It is also assumed that the solvent may be supplied in a separate container. The kit may also include a second container for sterile, pharmaceutically acceptable buffers and / or other diluents.

[0286] In embodiments of the present invention, the cells used in cell therapy are provided in a kit, and in some cases, the cells are essentially the sole component of the kit. The kit may include reagents and materials for producing the desired cells. In certain embodiments, the reagents and materials include primers, nucleotides, suitable buffers or buffer reagents, salts, etc., for amplifying the desired sequence, and in some cases, the reagents include vectors and / or DNA encoding the CARs described herein, and / or modulating elements therefor.

[0287] In certain embodiments, the kit contains one or more instruments suitable for extracting one or more samples from an organism. The instruments may be syringes, scalpels, etc.

[0288] In some cases of the present invention, the kit includes, in addition to embodiments of cell therapy, a second cancer therapy such as chemotherapy, hormone therapy, and / or immunotherapy. The kit can be tailored to the specific cancer of an individual and may include each individual's second cancer therapy.

[0289] Diagnostic assay Anti-PD-L1 antibodies can be used, for example, as part of a clinical trial procedure to determine the effectiveness of a given therapeutic and / or prophylactic regimen, or diagnostically to monitor the development or progression of cancer.

[0290] In some embodiments, for diagnostic purposes, the anti-PD-L1 antibody of the present invention is linked to a detectable portion to provide a method for detecting cancer cells in subjects at risk of or suffering from cancer.

[0291] The detectable portion can be conjugated directly or indirectly, for example, by using a fluorescent secondary antibody. Direct conjugation can be achieved, for example, by standard chemical binding of a fluorophore to the antibody or antibody fragment, or through genetic engineering. Chimeras, or fusion proteins containing an antibody or antibody fragment conjugated to a fluorescent or bioluminescent protein, can be constructed. For example, Casadei, et al, (Proc Natl Acad Sci US A. 1990 Mar; 87(6): 2047-51) describe a method for constructing a vector construct capable of expressing an aequorin and antibody fusion protein gene in mammalian cells.

[0292] As used herein, the term “labeled” with respect to a probe or antibody may include direct labeling of the probe or antibody by binding (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include the detection of a primary antibody using a fluorescently labeled secondary antibody, and the terminal labeling of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin. The term “biological sample” is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and biological fluids present within a subject. That is, cells expressing PD-L1 can be detected in vitro and in vivo in a biological sample using the detection methods of the present invention. For example, in vitro techniques for the detection of PD-L1 include enzyme-linked immunosolvent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. Furthermore, an in vivo technique for the detection of PD-L1 is the introduction of a labeled anti-PD-L1 antibody into the subject. For example, antibodies can be labeled with radioactive markers whose presence and location in a target can be detected by standard imaging techniques.

[0293] In the case of a “targeted” conjugate, i.e., a conjugate containing a targeted moiety—a molecule or feature designed to localize the conjugate within a subject or animal at a specific site(s)—localization can refer to a state where an equilibrium is essentially achieved between the bound “localized” entities and the unbound “free” entities within the subject. The rate at which such equilibrium is achieved depends on the route of administration. For example, a conjugate administered by intravenous injection may achieve localization within minutes of injection. On the other hand, a conjugate administered orally may take several hours to achieve localization. Alternatively, localization can simply refer to the location of the entities within the subject or animal over a selected period after administration. Another example is when localization is achieved when the moiety becomes distributed after administration.

[0294] It is understood that a reasonable estimate of the time required to achieve localization can be made by those skilled in the art. Furthermore, the state of localization as a function of time can be tracked by imaging a detectable portion (e.g., a light-emitting conjugate) according to the method of the present invention, such as with a photodetector device. The “photodetector device” used should be capable of imaging faint light from within a mammal over a reasonable time and should have sufficient sensitivity to construct an image using the signal from such a device.

[0295] If it is possible to use an extremely bright photogenerating portion and / or detect a photogenerating fusion protein localized near the surface of the object or animal being imaged, then "night vision" goggles or standard high-sensitivity video cameras such as Silicon Intensified Tube (SIT) cameras (e.g., from Hammamatsu Photonic Systems, Bridgewater, NJ) can be used. However, more typically, a more sensitive photodetection method is required.

[0296] At extremely low light levels, the photon flux per unit area becomes so low that the imaged scene no longer appears continuous. Instead, it is represented by individual photons distinct from one another, both in time and space. When viewed on a monitor, such an image appears as a series of shimmering points of light, each representing a single detected photon. By accumulating these detected photons over time in a digital image processor, an image can be acquired and constructed. In contrast to conventional cameras where an intensity value is assigned to the signal at each image point, the amplitude of the signal is irrelevant in photon counting imaging. The goal is simply to detect the presence of a signal (photon) and count the occurrence of the signal relative to its location over time.

[0297] At least two types of photodetector devices, described below, can detect individual photons and generate signals that can be analyzed by an image processor. Noise-reducing photodetector devices achieve sensitivity not by amplifying the photon signal, but by reducing the background noise of the photon detector. Noise is reduced primarily by cooling the detector array. Devices include charge-coupled device (CCD) cameras called "back-thinning" cooled CCD cameras. In more sensitive instruments, cooling is achieved using liquid nitrogen, for example, to raise the temperature of the CCD array to about -120°C. "Back-thinning" refers to an ultra-thin backplate that reduces the path length that photons travel until detected, thereby increasing quantum efficiency. A particularly sensitive back-thinning cryogenic CCD camera is the Series 200 camera, "TECH 512," available from Photometries, Ltd. (Tucson, Arizona).

[0298] A “photon amplification device” amplifies photons before they strike the detection screen. This class includes CCD cameras equipped with enhancement tubes, such as microchannel enhancement tubes. Microchannel enhancement tubes typically contain a metal array of channels perpendicular to and extending parallel to the camera’s detection screen. The microchannel array is placed between the sample, object, or animal being imaged and the camera. Most photons entering the array’s channels contact the sides of the channels before exiting. A voltage applied across the array results in the emission of many electrons from each photon collision. Electrons from such collisions exit their channels of origin in a “shotgun” pattern and are detected by the camera.

[0299] By arranging enhanced microchannel arrays in series, even higher sensitivity can be achieved, resulting in electrons generated in the first stage subsequently yielding an amplified signal in the second stage. However, this increase in sensitivity is achieved at the expense of spatial resolution, which decreases with each additional stage of amplification. An exemplary microchannel enhancement tube-based single-photon detection device is the C2400 series, available from Hamamatsu.

[0300] An image processor processes signals generated by a photodetector device that counts photons to construct an image that can be displayed on a monitor or printed on a video printer. Such image processors are typically sold as part of a system that includes the high-sensitivity photon counting camera mentioned above, and are therefore available from the same source. Image processors are usually connected to personal computers such as IBM-compatible PCs or Apple Macintosh (Apple Computer, Cupertino, Calif), which may or may not be included as part of a purchased imaging system. Once the images are in the form of digital files, they can be manipulated and printed by various image processing programs ("ADOBE PHOTOSHOP", Adobe Systems, Mt.View, Calif, etc.).

[0301] In one embodiment, the biological sample contains protein molecules from the subject of test. One preferred biological sample is a peripheral blood leukocyte sample isolated from the subject by conventional means.

[0302] The present invention also includes kits for detecting the presence of PD-L1 or PD-L1-expressing cells in a biological sample. For example, a kit may include a labeled compound or active agent (e.g., anti-PD-L1 scFv or monoclonal antibody) capable of detecting cancer or tumor cells in a biological sample, means for determining the amount of PD-L1 in the sample, and means for comparing the amount of PD-L1 in the sample with a standard. In some embodiments, the standard is a non-cancer cell or its cell extract. The compound or active agent can be packaged in a suitable container. The kit may further include instructions for detecting cancer in a sample using the kit.

[0303] Other Embodiments Although the present invention has been described in detail, the foregoing description is intended to illustrate, and not to limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0304] The present invention is further described in the following examples, which do not limit the scope of the invention as described in the claims. [Examples]

[0305] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary modes of constructing and carrying out the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, and similar results can be obtained using alternative methods; therefore, these examples are for illustrative purposes only.

[0306] Example 1 - Panning of Antibodies The PD-L1 antibodies of the present invention were discovered via PMPL panning. Briefly, to enhance affinity for PD-L1, the heavy chain variable regions of anti-PD-L1 antibodies #42 and #50 were cloned into pFarber lambda and kappa light chain display libraries, respectively, to create the #42-light chain shuffling (LCS) library and the #52 LCS library. Next, each library was panned for four rounds against PD-L1-mouse Fc soluble antigen at decreasing antigen concentrations (three rounds at 1 ug / ml PD-L1-mFc, and the fourth round at 0.5 and 0.1 ug / ml, respectively; tubes were coated with 1 ml). Single colonies were screened against soluble PD-L1-mFc by ELISA. Positive clones were enriched and showed increased affinity after the second panning. If necessary, phages eluted after the third panning were also cloned into yeast display libraries and screened for high-affinity binders via flow cytometry. Anti-PD-L1 antibodies #42 and #50, previously discovered in Dr. Marasco's laboratory, exhibited low affinity for PD-L1 compared to benchmark commercially available antibodies. Knowing that the heavy chains of #42 and #50 primarily contribute to PD-L1 binding specificity, novel single-chain Fv phage display libraries were constructed using a light chain shuffling technique. In this technique, either the #42 or #50 heavy chain variable region is fused with random kappa and lambda light chain variable regions, resulting in #42 LCS and #50 LCS libraries with approximately 2 × 10⁸ diversity, respectively. Panning the novel #42 and #52 LCS libraries at reduced antigen concentrations revealed high-affinity anti-PD-L1 antibodies containing the original #42 or #50 heavy chain and novel light chain sequences. Other antibodies were discovered by panning using naive phage libraries.

[0307] Example 2 - Double Bond Assay For each well of a 96-well plate, 2E5 CHO-GITR cells were washed with MACS buffer and resuspended in 100 μl of MACS buffer. Three-fold serial dilutions of the bispecific GITR-PDL1 Lc fusion were prepared in a separate 96-well plate at a starting concentration of 9 μg / ml. 50 μl of Ab dilution was added to 100 μl of buffer containing the cells to obtain a final starting concentration of 3 μg / ml. Ab dilutions were 3, 1, 0.33, 0.111, 0.037, 0.012, 0.004, and 0.0014. Cells were incubated with Ab at 4°C for 30 minutes, spun down, and washed twice with 250 μl of MACS buffer. After the final wash, cells were resuspended in MACS buffer containing 10 μg / ml PD-L1-rbFc fusion (extracellular domain of PD-L1) and incubated at 4°C for 30 minutes. The cells were washed twice with 250 μl of MACS buffer and resuspended in 100 μl of MACS buffer containing 2 μg / ml of FITC donkey anti-rabbit IgG (minimum x-reactivity) antibody (BioLegend catalog number 406403). The cells were incubated at 4°C for 20 minutes. The cells were washed twice with 250 μl of MACS buffer and resuspended in 200 μl of MACS buffer. The plates were then read using a Fortessa HTS FACS plate reader.

[0308] GITR LC fusion antibodies can simultaneously bind to both GITR (membrane-bound) and PD-L1 (soluble protein) (Figure 2). PD-L1 antibodies (42mut and 50-6B6.1mut) can bind to soluble PD-L1 better as light chain fusions than 50-6B6.2, 50-7B5, and 50-5B9 antibodies.

[0309] Example 3 - Mixed Lymphocyte Response (MLR) Protocol CD14+ monocytes were isolated using Miltenyi CD14+ microbeads. Cells were cultured in Miltenyi Mo-DC medium (pre-prepared medium containing GM-CSF + IL4). After culturing the cells for 5 days, TNF-α (1000 U / ml), IL-1β (5 ng / ml), IL-6 (10 ng / ml), and prostaglandin E2 (PGE2) (1 μM) were added, and the cells were cultured for 2 days to mature the DCs. T cells were isolated on the day of the MLR experiment (using the CD4+ negative selection kit StemCell). 100,000 T cells and 10,000 MoDC cells were used per well for the MLR. Antibodies were added at various concentrations, and the culture medium was incubated for 5 days.

[0310] The supernatant was saved for ELISA screening (e.g., IFNγ).

[0311] MLR atezolizumab-resistant PDL1 ab. One T-cell donor and one DC donor were used.

[0312] Anti-PDL1 abs of the scFv-Fc form were tested against commercially available atezolizumab, anti-PDL1#42 scFv-Fc, and nonspecific ab controls. As shown in Figures 6–9, the addition of atezolizumab and specific anti-PDL1 abs such as anti-PDL1#42 increased cytokine production compared to nonspecific controls.

[0313] equivalent Those skilled in the art will be able to recognize or confirm numerous equivalents to the specific substances and procedures described herein without using anything beyond routine experiments. Such equivalents are considered to fall within the scope of the present invention and are covered by the appended claims.

Claims

1. An isolated monoclonal antibody or its antigen-binding fragment that binds to the human programmed death ligand 1 (PD-L1) protein, comprising a heavy chain, a light chain, or a combination thereof, The aforementioned heavy chain, G-(X １ )-T-(X ２ )-SS-(X ３ X ４ )(SEQ ID NO:47), G-(X １ )-T-(X ２ )-(X １３ X １４ )-(X ３ X ４ )(SEQ ID NO:205), or G-(X １ )-TF-(X １３ X １４ )-Y-(X ４ )(SEQ ID NO:206) comprising CDR1, I - (X ８ X ９ X １０ X １１ )-G-(X １２ )-A (Sequence No. 51), or II-(X １５ )-IFG-(X １６ )-A (Sequence ID: 207) including CDR2, and / or CDR3 containing ARGRQMFGAGIDF (SEQ ID NO: 6), ARVHAALYYGMDV (SEQ ID NO: 14), TTGGLGLVYPYYNYIDV (SEQ ID NO: 99), AKVHPVFSYALDV (SEQ ID NO: 100), AEEGAFNSLAI (SEQ ID NO: 101), ARDGSGYDSAGMDD (SEQ ID NO: 102), ARGFGGGPY (SEQ ID NO: 103), ARVHGALYYYGMDV (SEQ ID NO: 104), ASGSIVGAAYAFDI (SEQ ID NO: 105), ARDRSEGGFDP (SEQ ID NO: 106), or AEEGAFNSLAI (SEQ ID NO: 107). Includes, The aforementioned light chain, S-(X １７ X １８ ) I - (X １９ )-SNY (Sequence No. 208) or NIG-(X ５ )-K-(X ２０ CDR1 containing (Sequence ID 48), (X ２１ )-DN (Sequence ID 209), (X ２２ )-NN (Sequence ID 210), or DD-X ６ CDR2 containing (Sequence ID 49), and / or QSYDSNNRHVI (Sequence ID 22), QVWDS-(X ７ ) - CDR3 containing SDHWV (SEQ ID NO: 50), QVWDSSGDLWV (SEQ ID NO: 126), AAWDDSLNGLV (SEQ ID NO: 127), QSYDGITVI (SEQ ID NO: 128), QSYDSSNHWV (SEQ ID NO: 129), AVWDDSLSGVV (SEQ ID NO: 131), MIWHSSAYV (SEQ ID NO: 132), NSRDISDNQWQWI (SEQ ID NO: 134), or QSYDSSNHVV (SEQ ID NO: 135) The isolated monoclonal antibody or its antigen-binding fragment, comprising the above.

2. The antibody according to claim 1, which is either a fully human antibody or has been humanized.

3. The antibody according to claim 1, which is monospecific, bispecific, or multispecific.

4. The antibody according to claim 1, which is a single-chain antibody.

5. At least 3.3 × 10 －９ The antibody according to claim 1, having binding affinity for M.

6. The antibody or fragment according to claim 1, further comprising a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

7. X １ , X ２ , X ３、 or X ４ The antibody according to claim 1, wherein the residue is a nonpolar amino acid residue.

8. X １ , X ２ , X ３、 or X ４ The antibody according to claim 7, wherein the antibody is glycine (G), tyrosine (Y), phenylalanine (F), leucine (L), or alanine (A).

9. X １ , X ２ , or X ４ The antibody according to claim 1, wherein the residue is a hydrophobic amino acid residue.

10. X １ , X ２、 or X ４ The antibody according to claim 9, wherein the antibody is glycine (G), leucine (L), or alanine (A).

11. X ３ The antibody according to claim 1, wherein the residue is a hydrophilic polar amino acid residue.

12. X ３ The antibody according to claim 11, wherein the antibody is histidine (H).

13. X １ The antibody according to claim 1, wherein the antibody is phenylalanine (F), glycine (G), or tyrosine (Y).

14. X ２ The antibody according to claim 1, wherein the antibody is phenylalanine (F) or leucine (L).

15. X ３ The antibody according to claim 1, wherein the antibody is histidine (H) or tyrosine (Y).

16. X ４ The antibody according to claim 1, wherein the antibody is serine (S), glycine (G), or alanine (A).

17. X ８ , X ９ , X １０ , or X １１ The antibody according to claim 1, wherein the amino acid residue is nonpolar hydrophobic.

18. X ８ , X ９、 X １０ , or X １１ The antibody according to claim 17, wherein the antibody is isoleucine (I), proline (P), alanine (A), or phenylalanine (F).

19. X ８ , X １０、 or X １２ The antibody according to claim 1, wherein the amino acid residue is polar hydrophilic.

20. X ８ , X １０ , or X １２ The antibody according to claim 19, wherein the compound is histidine (H), serine (S), asparagine (N), or threonine (T).

21. X ８ The antibody according to claim 1, wherein the antibody is alanine (A), isoleucine (I), or serine (S).

22. X ９ The antibody according to claim 1, wherein the antibody is proline (P), tyrosine (Y), serine (S), or alanine (A).

23. X １０ The antibody according to claim 1, wherein the antibody is tyrosine (Y), aspartic acid (D), isoleucine (I), or histidine (H).

24. X １１ The antibody according to claim 1, wherein the antibody is glycine (G), leucine (L), asparagine (N), or phenylalanine (F).

25. X １２ The antibody according to claim 1, wherein the antibody is isoleucine (I), arginine (R), threonine (T), or histidine (H).

26. X ５ The antibody according to claim 1, wherein the amino acid residue is nonpolar hydrophobic.

27. X ５ The antibody according to claim 26, wherein the antibody is glycine (G).

28. X ５ The antibody according to claim 1, wherein the amino acid residue is polar hydrophilic.

29. X ５ The antibody according to claim 28, wherein the antibody is serine (S), asparagine (N), or aspartic acid (D).

30. X ６ The antibody according to claim 1, wherein the residue is a nonpolar amino acid residue.

31. X ６ The antibody according to claim 30, wherein the antibody is tyrosine (Y).

32. X ６ The antibody according to claim 1, wherein the amino acid residue is polar hydrophilic.

33. X ６ The antibody according to claim 32, wherein the antibody is serine (S), threonine (T), or arginine (R).

34. X ７ , X １５ , X １６ , X １７ , X １９ , X ２０ , or X ２１ The antibody according to claim 1, wherein the amino acid residue is nonpolar hydrophobic.

35. X ７ , X １７ , or X ２０ The antibody according to claim 34, wherein the antibody is glycine (G).

36. X ７ 、X １３ 、X １４ 、X １５ 、X １６、 X １７ 、X １８ 、X １９ 、 or X ２１ is a polar hydrophilic amino acid residue, the antibody according to claim 1.

37. X ７ , X １４ , or X ２１ The antibody according to claim 36, wherein the antibody is serine (S) or arginine (R).

38. X １３ The antibody according to claim 36, wherein the antibody is serine (S) or threonine (T).

39. X １５ The antibody according to claim 34, wherein the antibody is proline (P).

40. X １５ , X １７ , or X ２０ The antibody according to claim 36, wherein the antibody is serine (S).

41. X １６ The antibody according to claim 36, wherein the antibody is threonine (T) or arginine (R).

42. X １６ The antibody according to claim 34, wherein the antibody is isoleucine (I).

43. X １８ The antibody according to claim 36, wherein the antibody is serine (S) or asparagine (N).

44. X １９ The antibody according to claim 34, wherein the antibody is glycine (G) or alanine (A).

45. X １９ The antibody according to claim 36, wherein the antibody is aspartic acid (D).

46. X ２１ The antibody according to claim 34, wherein the antibody is alanine (A).

47. X ２１ The antibody according to claim 36, wherein the antibody is glutamic acid (E).

48. It binds to the human programmed death ligand 1 (PD-L1) protein, and (a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 2, VH CDR2 containing the amino acid sequence of SEQ ID NO: 4, VH CDR3 containing the amino acid sequence of SEQ ID NO: 6, VL CDR1 containing the amino acid sequence of SEQ ID NO: 18, VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 22, or (b) VH CDR1 containing the amino acid sequence of SEQ ID NO: 10, VH CDR2 containing the amino acid sequence of SEQ ID NO: 12, VH CDR3 containing the amino acid sequence of SEQ ID NO: 14, VL CDR1 containing the amino acid sequence of SEQ ID NO: 48, VL CDR2 containing the amino acid sequence of SEQ ID NO: 49, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 50 Isolated antibodies or fragments thereof, including [the specified substance].

49. X of sequence number 48 ５ The antibody according to claim 48, wherein the amino acid residue is nonpolar hydrophobic.

50. X ５ The antibody according to claim 49, wherein the antibody is glycine (G).

51. X ５ The antibody according to claim 48, wherein the amino acid residue is polar hydrophilic.

52. X ５ The antibody according to claim 51, wherein the antibody is serine (S), asparagine (N), or aspartic acid (D).

53. X of sequence number 49 ６ The antibody according to claim 48, wherein the amino acid residue is nonpolar.

54. X ６ The antibody according to claim 53, wherein the antibody is tyrosine (Y).

55. X ６ The antibody according to claim 48, wherein the amino acid residue is polar hydrophilic.

56. X ６ The antibody according to claim 55, wherein the antibody is serine (S), threonine (T), or arginine (R).

57. X of sequence number 50 ７ The antibody according to claim 48, wherein the amino acid residue is nonpolar hydrophobic.

58. X ７ The antibody according to claim 57, wherein the antibody is glycine (G).

59. X ７ The antibody according to claim 38, wherein the amino acid residue is polar hydrophilic.

60. X ７ The antibody according to claim 59, wherein the antibody is serine (S) or arginine (R).

61. The antibody according to claim 48, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 26, 33, 40, or 44.

62. The antibody according to claim 48, wherein VL CDR2 comprises the amino acid sequence of SEQ ID NO: 28, 35, or 45.

63. The antibody according to claim 48, wherein VL CDR3 comprises the amino acid sequence of SEQ ID NO: 30 or 37.

64. The antibody according to claim 48, wherein the antibody in (b) comprises VL CDR1 containing the amino acid sequence of SEQ ID NO: 26, VL CDR2 containing the amino acid sequence of SEQ ID NO: 28, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 30, or VL CDR1 containing the amino acid sequence of SEQ ID NO: 33, VL CDR2 containing the amino acid sequence of SEQ ID NO: 35, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 37, or VL CDR1 containing the amino acid sequence of SEQ ID NO: 40, VL CDR2 containing the amino acid sequence of SEQ ID NO: 35, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 37, or VL CDR1 containing the amino acid sequence of SEQ ID NO: 44, VL CDR2 containing the amino acid sequence of SEQ ID NO: 45, and VL CDR3 containing the amino acid sequence of SEQ ID NO:

37.

65. It binds to the human PD-L1 protein, and A heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 16, 52, 54, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82, A light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 31, 38, 42, 46, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, and 83, and Isolated antibodies or fragments thereof, including [the specified substance].

66. It binds to the human PD-L1 protein, and VH-CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 84, 85, 86, 87, 88, 89, and 90, VH-CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12, 91, 92, 93, 94, 95, 96, 97, and 98, and / or VH-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 14, 99, 100, 101, 102, 103, 104, 105, 106, and 107. Including heavy chain variable regions, and / or VL-CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 26, 33, 40, 44, 108, 109, 110, 111, 112, 113, 114, 115, 116, and 117, VL-CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 28, 35, 45, 118, 119, 120, 121, 122, 123, 124, and 125, and / or VL-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 30, 37, 126, 127, 128, 129, 130, 131, 132, 133, 134, and 135. Light chain variable region including Isolated antibodies or fragments thereof, including [the specified substance].

67. An isolated antibody or fragment thereof that binds to the human PD-L1 protein, wherein the antibody is (a) A heavy chain variable region having three CDRs, each containing the amino acid sequences GGTFSSYA (SEQ ID NO: 2), IIPIFGTA (SEQ ID NO: 4), and ARGRQMFGAGIDF (SEQ ID NO: 6), and / or a light chain variable region having three CDRs, each containing the amino acid sequences SGSIDSNY (SEQ ID NO: 18), EDN (SEQ ID NO: 20), and QSYDSNNRHVI (SEQ ID NO: 22), (b) A heavy chain variable region having three CDRs, each containing the amino acid sequences GYTLSSHG (SEQ ID NO: 10), ISAHNGHA (SEQ ID NO: 12), and ARVHAALYYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs, each containing the amino acid sequences NIGSKG (SEQ ID NO: 26), DDR (SEQ ID NO: 28), and QVWDSGSDHWV (SEQ ID NO: 30), (c) A heavy chain variable region having three CDRs, each containing the amino acid sequences GYTLSSHG (SEQ ID NO: 10), ISAHNGHA (SEQ ID NO: 12), and ARVHAALYYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs, each containing the amino acid sequences NIGDKG (SEQ ID NO: 33), DDS (SEQ ID NO: 35), and QVWDSSSSDHWV (SEQ ID NO: 37), (d) A heavy chain variable region having three CDRs, each containing the amino acid sequences GYTLSSHG (SEQ ID NO: 10), ISAHNGHA (SEQ ID NO: 12), and ARVHAALYYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs, each containing the amino acid sequences NIGNKG (SEQ ID NO: 40), DDS (SEQ ID NO: 35), and QVWDSSSSDHWV (SEQ ID NO: 37), (e) A heavy chain variable region having three CDRs, each containing the amino acid sequences GYTLSSHG (SEQ ID NO: 10), ISAHNGHA (SEQ ID NO: 12), and ARVHAALYYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs, each containing the amino acid sequences NIGGKG (SEQ ID NO: 44), DDY (SEQ ID NO: 45), and QVWDSSSSDHWV (SEQ ID NO: 37), (f) A heavy chain variable region having three CDRs, each containing the amino acid sequences GYTLSSHG (SEQ ID NO: 10), ISAHNGHA (SEQ ID NO: 12), and ARVHAALYYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs, each containing the amino acid sequences NIESRS (SEQ ID NO: 108), DDT (SEQ ID NO: 118), and QVWDSSGDLWV (SEQ ID NO: 126), (g) A heavy chain variable region having three CDRs, each containing the amino acid sequences GYTLSSHG (SEQ ID NO: 10), ISAHNGHA (SEQ ID NO: 12), and ARVHAALYYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs, each containing the amino acid sequences NIGSKG (SEQ ID NO: 26), DDS (SEQ ID NO: 35), and QVWDSSSSDHWV (SEQ ID NO: 37), (h) A heavy chain variable region having three CDRs, each containing the amino acid sequences GYTLSSHG (SEQ ID NO: 10), ISAHNGHA (SEQ ID NO: 12), and ARVHAALYYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs, each containing the amino acid sequences NIGSKS (SEQ ID NO: 109), DDS (SEQ ID NO: 35), and QVWDSSSSDHWV (SEQ ID NO: 37), (i) A heavy chain variable region having three CDRs, each containing the amino acid sequences GYTLSSHG (SEQ ID NO: 10), ISAHNGHA (SEQ ID NO: 12), and ARVHAALYYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs, each containing the amino acid sequences NIGSKG (SEQ ID NO: 26), DDS (SEQ ID NO: 35), and QVWDSSSSDHWV (SEQ ID NO: 37), (j) A heavy chain variable region having three CDRs, each containing the amino acid sequences DFAFSSAW (SEQ ID NO: 84), IKSKTDGETT (SEQ ID NO: 91), and TTGGLGLVYPYYNYIDV (SEQ ID NO: 99), and / or a light chain variable region having three CDRs, each containing the amino acid sequences SSNIGSNY (SEQ ID NO: 110), RNN (SEQ ID NO: 119), and AAWDDSLNGLV (SEQ ID NO: 127), (k) A heavy chain variable region having three CDRs, each containing the amino acid sequences GYTFTSYG (SEQ ID NO: 85), TSPHNGLT (SEQ ID NO: 92), and AKVHPVFSYALDV (SEQ ID NO: 100), and / or a light chain variable region having three CDRs, each containing the amino acid sequences SGSIASNY (SEQ ID NO: 111), EDN (SEQ ID NO: 20), and QSYDGITVI (SEQ ID NO: 128), (l) A heavy chain variable region having three CDRs, each containing the amino acid sequences GGTFSRYA (SEQ ID NO: 86), IIPIFGRA (SEQ ID NO: 93), and AEEGAFNSLAI (SEQ ID NO: 101), and / or a light chain variable region having three CDRs, each containing the amino acid sequences SGSIASNY (SEQ ID NO: 111), ADN (SEQ ID NO: 120), and QSYDSSNHWV (SEQ ID NO: 129), (m) A heavy chain variable region having three CDRs, each containing the amino acid sequences GYTLSSHG (SEQ ID NO: 10), ISAHNGHA (SEQ ID NO: 12), and ARVHAALYYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs, each containing the amino acid sequences NIGSKS (SEQ ID NO: 109), DDS (SEQ ID NO: 35), and QVWDSSSSDHWV (SEQ ID NO: 37), (n) A heavy chain variable region having three CDRs, each containing the amino acid sequences GYTFTSYG (SEQ ID NO: 85), ISAYNGHA (SEQ ID NO: 94), and ARVHAALYYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs, each containing the amino acid sequences NIGSKG (SEQ ID NO: 26), DDS (SEQ ID NO: 35), and QVWDSRSDHWV (SEQ ID NO: 130), (o) A heavy chain variable region having three CDRs, each containing the amino acid sequences GGTFSSYA (SEQ ID NO: 87), IIPIFGTA (SEQ ID NO: 95), and ARDGSGYDSAGMDD (SEQ ID NO: 102), and / or a light chain variable region having three CDRs, each containing the amino acid sequences RSNIGSNY (SEQ ID NO: 112), SNN (SEQ ID NO: 121), and AVWDDSLSGVV (SEQ ID NO: 131), (p) A heavy chain variable region having three CDRs, each containing the amino acid sequences GTFFSSYA (SEQ ID NO: 88), ISYDGSNK (SEQ ID NO: 96), and ARGFGGGPDY (SEQ ID NO: 103), and / or a light chain variable region having three CDRs, each containing the amino acid sequences SGINVGTYR (SEQ ID NO: 113), YKSDSDK (SEQ ID NO: 122), and MIWHSSAYV (SEQ ID NO: 132), (q) A heavy chain variable region having three CDRs, each containing the amino acid sequences GYTFSSYG (SEQ ID NO: 89), ISAHNGHA (SEQ ID NO: 12), and ARVHGALYYGMDV (SEQ ID NO: 104), and / or a light chain variable region having three CDRs, each containing the amino acid sequences NIGGKS (SEQ ID NO: 114), DDR (SEQ ID NO: 28), and QVWDSSSSDHWV (SEQ ID NO: 37), (r) A heavy chain variable region having three CDRs, each containing the amino acid sequences GYTLSSHG (SEQ ID NO: 10), ISAHNGHA (SEQ ID NO: 12), and ARVHAALYYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs, each containing the amino acid sequences NIGSKG (SEQ ID NO: 26), DDR (SEQ ID NO: 28), and QVWDSSSSDHWV (SEQ ID NO: 37), (s) Heavy chain variable region having three CDRs containing the amino acid sequences GGTFSSYA (SEQ ID NO: 87), IIPILGIA (SEQ ID NO: 97), and ASGSIVGAAAYAFDI (SEQ ID NO: 105), and / or Light chain variable region having three CDRs containing the amino acid sequences NIGGRV (SEQ ID NO: 115), DDT (SEQ ID NO: 123), and QVWDSRSDHPV (SEQ ID NO: 133), (t) A heavy chain variable region having three CDRs, each containing the amino acid sequences GTFFSSYS (SEQ ID NO: 90), IISDGSAT (SEQ ID NO: 98), and ARDRSEGGFDP (SEQ ID NO: 106), and / or a light chain variable region having three CDRs, each containing the amino acid sequences SLRSYY (SEQ ID NO: 116), GKN (SEQ ID NO: 124), and NSRDISDNQWQWI (SEQ ID NO: 134), or (u) Heavy chain variable regions having three CDRs, each containing the amino acid sequences GGTFSRYA (SEQ ID NO: 86), IIPIFGRA (SEQ ID NO: 93), and AEEGAFNSLAI (SEQ ID NO: 107), and / or light chain variable regions having three CDRs, each containing the amino acid sequences SGSIASHF (SEQ ID NO: 117), GDD (SEQ ID NO: 125), and QSYDSSNHVV (SEQ ID NO: 135). The isolated antibody or a fragment thereof, including the above.

68. An isolated monoclonal antibody or antigen-binding fragment that binds to PD-L1, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 8, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:

24.

69. An isolated monoclonal antibody or antigen-binding fragment that binds to PD-L1, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 16, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:

31.

70. An isolated monoclonal antibody or antigen-binding fragment that binds to PD-L1, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 16, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:

38.

71. An isolated monoclonal antibody or antigen-binding fragment that binds to PD-L1, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 16, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:

42.

72. An isolated monoclonal antibody or antigen-binding fragment that binds to PD-L1, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 16, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:

46.

73. An isolated monoclonal antibody or its antigen-binding fragment that binds to PD-L1, comprising a heavy chain, a light chain, or a combination thereof, (a) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 52, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO:

53. (b) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 54, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO:

55. (c) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 56, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO:

57. (d) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 16, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO:

59. (e) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 60, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO:

61. (f) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 62, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO:

63. (g) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 64, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO:

65. (h) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 66, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO:

67. (i) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 68, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO:

69. (j) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 70, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO:

71. (k) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 72, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO:

73. (l) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 74, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO:

75. (m) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 76, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO:

77. (n) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 78, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO:

79. (o) The heavy chain contains an amino acid sequence that is approximately 95% identical to SEQ ID NO: 80, and the light chain contains an amino acid sequence that is approximately 95% identical to SEQ ID NO: 81, or (p) The heavy chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO: 82, and the light chain contains an amino acid sequence that is approximately 95% identical to that of SEQ ID NO:

83. The isolated monoclonal antibody or its antigen-binding fragment.

74. An isolated monoclonal antibody or antigen-binding fragment that binds to PD-L1, comprising a heavy chain, a light chain, or a combination thereof, wherein the antibody or fragment is (a) V having sequence number 8 Ｈ V having amino acid sequence and sequence number 24 Ｌ amino acid sequence, (b) V having sequence number 16 Ｈ V having amino acid sequence and sequence number 31 Ｌ amino acid sequence, (c) V having sequence number 16 Ｈ V having amino acid sequence and sequence number 38 Ｌ amino acid sequence, (d) V having sequence number 16 Ｈ V having amino acid sequence and sequence number 42 Ｌ amino acid sequence, (e) V having sequence number 16 Ｈ V having amino acid sequence and sequence number 46 Ｌ amino acid sequence, (f) V having sequence number 52 Ｈ V having amino acid sequence and sequence number 53 Ｌ amino acid sequence, (g) V having sequence number 54 Ｈ V having amino acid sequence and sequence number 55 Ｌ amino acid sequence, (h) V having sequence number 56 Ｈ V having amino acid sequence and sequence number 57 Ｌ amino acid sequence, (i) V having sequence number 16 Ｈ V having amino acid sequence and sequence number 59 Ｌ amino acid sequence, (j) V having sequence number 60 Ｈ V having amino acid sequence and sequence number 61 Ｌ amino acid sequence, (k) V having sequence number 62 Ｈ V having amino acid sequence and sequence number 63 Ｌ amino acid sequence, (l) V having sequence number 64 Ｈ V having amino acid sequence and sequence number 65 Ｌ amino acid sequence, (m) V having sequence number 66 Ｈ V having amino acid sequence and sequence number 67 Ｌ amino acid sequence, (n) V having sequence number 68 Ｈ V having amino acid sequence and sequence number 69 Ｌ amino acid sequence, (o) V having sequence number 70 Ｈ V having amino acid sequence and sequence number 71 Ｌ amino acid sequence, (p) V having sequence number 72 Ｈ V having amino acid sequence and sequence number 73 Ｌ amino acid sequence, (q) V having sequence number 74 Ｈ V having amino acid sequence and sequence number 75 Ｌ amino acid sequence, (r) V having sequence number 76 Ｈ V having amino acid sequence and SEQ ID NO: 77 Ｌ amino acid sequence, (s) V having sequence number 78 Ｈ V having amino acid sequence and sequence number 79 Ｌ amino acid sequence, (t) V having sequence number 80 Ｈ V having amino acid sequence and sequence number 81 Ｌ amino acid sequence, or (u) V having sequence number 82 Ｈ V having amino acid sequence and sequence number 83 Ｌ amino acid sequence The isolated monoclonal antibody or its antigen-binding fragment, comprising the above.

75. An isolated bispecific antibody comprising a fragment according to claim 1, 48, 65, 66, 67, 68, 69, 70, 71, 72, 73, or 74, and a second antigen-binding fragment having specificity for molecules on immune cells.

76. The bispecific antibody according to claim 75, wherein the molecule is selected from the group consisting of B7H3, B7H4, CD27, CD28, CD40, CD40L, CD47, CD122, CTLA-4, GITR, GITRL, ICOS, ICOSL, LAG-3, LIGHT, OX-40, OX40L, PD-1, TIM3, 4-1BB, TIGIT, VISTA, HEVM, BTLA, and KIR.

77. The bispecific antibody according to claim 75, wherein each of the aforementioned fragment and the second fragment is independently selected from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody.

78. The bispecific antibody according to claim 75, further comprising an Fc fragment.

79. A nucleic acid encoding an antibody according to any one of claims 1 to 74.

80. A nucleic acid encoding a bispecific antibody according to any one of claims 75 to 78.

81. A pharmaceutical composition comprising an antibody or fragment thereof according to any one of claims 1 to 74, and a pharmaceutically acceptable carrier or excipient.

82. The pharmaceutical composition according to claim 81, further comprising at least one additional therapeutic substance.

83. The pharmaceutical composition according to claim 82, wherein the therapeutic substance is a toxin, a radiolabeled substance, siRNA, a small molecule, or a cytokine.

84. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 75 to 78, and a pharmaceutically acceptable carrier or excipient.

85. The pharmaceutical composition according to claim 84, further comprising at least one additional therapeutic substance.

86. The pharmaceutical composition according to claim 85, wherein the therapeutic substance is a toxin, a radiolabeled substance, siRNA, a small molecule, or a cytokine.

87. An isolated cell comprising one or more polynucleotides encoding an antibody or fragment thereof according to any one of claims 1 to 74.

88. An isolated cell comprising one or more polynucleotides encoding a bispecific antibody or a fragment thereof according to any one of claims 75 to 78.

89. A vector comprising the nucleic acid described in claim 79 or 80.

90. A cell comprising the vector according to claim 89.

91. A kit comprising: at least one antibody composition according to claim 81 or 84; a syringe, needle, or applicator for administering the at least one antibody to a subject; and instructions for use.

92. A method for treating cancer in a subject, comprising administering to a subject in need of such treatment a therapeutically effective amount of a composition comprising an antibody according to any one of claims 1 to 74, a bispecific antibody according to any one of claims 75 to 78, a pharmaceutical composition according to claim 81 or 84, or a CAR composition according to any one of claims 95 to 101.

93. The method according to claim 92, further comprising administering a chemotherapeutic agent to the subject.

94. The method according to claim 92, wherein the cancer is a checkpoint inhibitor cancer.

95. A chimeric antigen receptor (CAR) comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain, wherein the extracellular domain is an isolated monoclonal antibody or its antigen-binding fragment that binds to the human programmed death ligand 1 (PD-L1) protein, and the monoclonal antibody or its fragment comprises a heavy chain, a light chain, or a combination thereof. The aforementioned heavy chain, G-(X １ )-T-(X ２ )-SS-(X ３ X ４ ) (Sequence No. 47), G-(X １ )-T-(X ２ ) - (X １３ X １４ ) - (X ３ X ４ ) (Sequence No. 205), or G-(X １ )-TF-(X １３ X １４ ) - Y - (X ４ CDR1 containing (Sequence ID 206), I - (X ８ X ９ X １０ X １１ )-G-(X １２ )-A (Sequence No. 51), or II-(X １５ )-IFG-(X １６ )-A (Sequence ID: 207) including CDR2, and / or CDR3 containing ARGRQMFGAGIDF (SEQ ID NO: 6), ARVHAALYYGMDV (SEQ ID NO: 14), TTGGLGLVYPYYNYIDV (SEQ ID NO: 99), AKVHPVFSYALDV (SEQ ID NO: 100), AEEGAFNSLAI (SEQ ID NO: 101), ARDGSGYDSAGMDD (SEQ ID NO: 102), ARGFGGGPY (SEQ ID NO: 103), ARVHGALYYYGMDV (SEQ ID NO: 104), ASGSIVGAAYAFDI (SEQ ID NO: 105), ARDRSEGGFDP (SEQ ID NO: 106), or AEEGAFNSLAI (SEQ ID NO: 107). Includes, The aforementioned light chain, S-(X １７ X １８ ) I - (X １９ )-SNY (Sequence No. 208) or NIG-(X ５ )-K-(X ２０ CDR1 containing (Sequence ID 48), (X ２１ )-DN (Sequence ID 209), (X ２２ )-NN (Sequence ID 210), or DD-X ６ CDR2 containing (Sequence ID 49), and / or QSYDSNNRHVI (Sequence ID 22), QVWDS-(X ７ ) - CDR3 containing SDHWV (SEQ ID NO: 50), QVWDSSGDLWV (SEQ ID NO: 126), AAWDDSLNGLV (SEQ ID NO: 127), QSYDGITVI (SEQ ID NO: 128), QSYDSSNHWV (SEQ ID NO: 129), AVWDDSLSGVV (SEQ ID NO: 131), MIWHSSAYV (SEQ ID NO: 132), NSRDISDNQWQWI (SEQ ID NO: 134), or QSYDSSNHVV (SEQ ID NO: 135) including, The aforementioned chimeric antigen receptor (CAR).

96. The CAR according to claim 95, wherein the transmembrane domain further comprises a stalk region located between the extracellular domain and the transmembrane domain.

97. The CAR according to claim 95, wherein the transmembrane domain includes CD28.

98. The CAR according to claim 95, further comprising one or more additional costimulatory molecules positioned between the transmembrane domain and the intracellular signaling domain.

99. The CAR according to claim 98, wherein the co-stimulatory molecule is CD28, 4-1BB, ICOS, or OX40.

100. The CAR according to claim 95, wherein the intracellular signaling domain includes a CD3 zeta chain.

101. The CAR according to claim 95, wherein the antibody is Fab or scFV.

102. A nucleic acid encoding a CAR as described in any one of the prior claims.

103. The nucleic acid according to claim 102, further comprising a nucleic acid encoding a polypeptide located after the intracellular signaling domain.

104. The nucleic acid according to claim 103, wherein the polypeptide is an antibody or a cytokine.

105. The nucleic acid according to claim 104, wherein the antibody is scFV.

106. A nucleic acid encoding a CAR, wherein the CAR comprises an intracellular signaling domain, a transmembrane domain, and an extracellular domain, and further comprises a nucleic acid encoding a polypeptide positioned after the intracellular signaling domain, wherein the polypeptide comprises an isolated monoclonal antibody or an antigen-binding fragment thereof that binds to a human programmed death ligand 1 (PD-L1) protein, and the monoclonal antibody or fragment thereof comprises a heavy chain, a light chain, or a combination thereof. The aforementioned heavy chain, G-(X １ )-T-(X ２ )-SS-(X ３ X ４ ) (Sequence No. 47), G-(X １ )-T-(X ２ ) - (X １３ X １４ ) - (X ３ X ４ ) (Sequence No. 205), or G-(X １ )-TF-(X １３ X １４ ) - Y - (X ４ CDR1 containing (Sequence ID 206), I - (X ８ X ９ X １０ X １１ )-G-(X １２ )-A (Sequence No. 51), or II-(X １５ )-IFG-(X １６ )-A (Sequence ID: 207) including CDR2, and / or CDR3 containing ARGRQMFGAGIDF (SEQ ID NO: 6), ARVHAALYYGMDV (SEQ ID NO: 14), TTGGLGLVYPYYNYIDV (SEQ ID NO: 99), AKVHPVFSYALDV (SEQ ID NO: 100), AEEGAFNSLAI (SEQ ID NO: 101), ARDGSGYDSAGMDD (SEQ ID NO: 102), ARGFGGGPY (SEQ ID NO: 103), ARVHGALYYYGMDV (SEQ ID NO: 104), ASGSIVGAAYAFDI (SEQ ID NO: 105), ARDRSEGGFDP (SEQ ID NO: 106), or AEEGAFNSLAI (SEQ ID NO: 107). Includes, The aforementioned light chain, S-(X １７ X １８ ) I - (X １９ )-SNY (Sequence No. 208) or NIG-(X ５ )-K-(X ２０ CDR1 containing (Sequence ID 48), (X ２１ )-DN (Sequence ID 209), (X ２２ )-NN (Sequence ID 210), or DD-X ６ CDR2 containing (Sequence ID 49), and / or QSYDSNNRHVI (Sequence ID 22), QVWDS-(X ７ ) - CDR3 containing SDHWV (SEQ ID NO: 50), QVWDSSGDLWV (SEQ ID NO: 126), AAWDDSLNGLV (SEQ ID NO: 127), QSYDGITVI (SEQ ID NO: 128), QSYDSSNHWV (SEQ ID NO: 129), AVWDDSLSGVV (SEQ ID NO: 131), MIWHSSAYV (SEQ ID NO: 132), NSRDISDNQWQWI (SEQ ID NO: 134), or QSYDSSNHVV (SEQ ID NO: 135) including, The nucleic acid that codes for the aforementioned CAR.

107. A vector comprising the nucleic acid according to any one of claims 102 to 106.

108. A cell comprising the vector according to claim 107.

109. A genetically modified cell that expresses and retains a chimeric antigen receptor according to any one of claims 95 to 101 on its cell surface membrane.

110. A genetically modified cell according to claim 108 or 109, which is a T cell or an NK cell.

111. The aforementioned T cells, CD4 ＋ or CD8 ＋ The genetically modified cell according to claim 110.

112. CD4 ＋ and CD8 ＋ The genetically modified cells according to claim 111, comprising a mixed population of cells.

113. Genetically engineered cells expressing and retaining a chimeric antigen receptor on the cell surface membrane, further engineered to express and secrete a polypeptide, wherein the polypeptide is an isolated monoclonal antibody or its antigen-binding fragment that binds to the human programmed death ligand 1 (PD-L1) protein, and the monoclonal antibody or fragment comprises a heavy chain, a light chain, or a combination thereof. The aforementioned heavy chain, G-(X １ )-T-(X ２ )-SS-(X ３ X ４ ) (Sequence No. 47), G-(X １ )-T-(X ２ ) - (X １３ X １４ ) - (X ３ X ４ ) (Sequence No. 205), or G-(X １ )-TF-(X １３ X １４ ) - Y - (X ４ CDR1 containing (Sequence ID 206), I - (X ８ X ９ X １０ X １１ )-G-(X １２ )-A (Sequence No. 51), or II-(X １５ )-IFG-(X １６ )-A (Sequence ID: 207) including CDR2, and / or CDR3 containing ARGRQMFGAGIDF (SEQ ID NO: 6), ARVHAALYYGMDV (SEQ ID NO: 14), TTGGLGLVYPYYNYIDV (SEQ ID NO: 99), AKVHPVFSYALDV (SEQ ID NO: 100), AEEGAFNSLAI (SEQ ID NO: 101), ARDGSGYDSAGMDD (SEQ ID NO: 102), ARGFGGGPY (SEQ ID NO: 103), ARVHGALYYYGMDV (SEQ ID NO: 104), ASGSIVGAAYAFDI (SEQ ID NO: 105), ARDRSEGGFDP (SEQ ID NO: 106), or AEEGAFNSLAI (SEQ ID NO: 107). Includes, The aforementioned light chain, S-(X １７ X １８ ) I - (X １９ )-SNY (Sequence No. 208) or NIG-(X ５ )-K-(X ２０ CDR1 containing (Sequence ID 48), (X ２１ )-DN (Sequence ID 209), (X ２２ )-NN (Sequence ID 210), or DD-X ６ CDR2 containing (Sequence ID 49), and / or QSYDSNNRHVI (Sequence ID 22), QVWDS-(X ７ ) - CDR3 containing SDHWV (SEQ ID NO: 50), QVWDSSGDLWV (SEQ ID NO: 126), AAWDDSLNGLV (SEQ ID NO: 127), QSYDGITVI (SEQ ID NO: 128), QSYDSSNHWV (SEQ ID NO: 129), AVWDDSLSGVV (SEQ ID NO: 131), MIWHSSAYV (SEQ ID NO: 132), NSRDISDNQWQWI (SEQ ID NO: 134), or QSYDSSNHVV (SEQ ID NO: 135) including, The aforementioned genetically modified cells.

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