Antibodies to PD-L1 and methods of use thereof

Abstract

The present invention is directed to human monoclonal antibodies that bind to the cell surface receptor PDL-1 (programmed death ligand 1), which can be used to treat cancer and chronic viral infections. TIFF2023504271000049.tif146170

Description

[Technical Field]

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

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

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction of either the patent document or the patent disclosure, as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

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

[0005] FIELD OF THE INVENTION The present invention is directed to antibodies against PD-L1 (also known as programmed cell death 1 ligand 1, or B7H1) and methods of use thereof. [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 on pro-B cells and is thought to play a role in their differentiation. PD-1, a member of the CD28 family, is upregulated on activated T cells, B cells, and monocytes. PD-1 has two identified ligands in the B7 family: PD-L1 (programmed cell death-1 ligand 1, also known as cluster of differentiation 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 CD8+ T cell proliferation in lymph nodes. PD-L2 expression tends to be more restricted and is found primarily on activated antigen-presenting cells (APCs), whereas PD-L1 expression is widespread, including on hematopoietic cells (including activated T cells, B cells, monocytes, dendritic cells, and macrophages) and peripheral non-lymphoid tissues (including cardiac, skeletal, muscle, placental, lung, kidney, and liver tissues). The widespread expression of PD-L1 indicates its important role in regulating PD-1 / PD-L1-mediated peripheral immune tolerance. Summary of the Invention

[0007] One aspect of the present invention is directed to an isolated monoclonal antibody that binds to human programmed death ligand 1 (PD-L1). In some embodiments, the antibody may be an antigen-binding fragment thereof 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)-SS-(X3X4) (SEQ ID NO: 48), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 49), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 50), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 51), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 52), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 53), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 54), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 55), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 56), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 57), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 58), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 59), G 13 X 14 )-(X3X4) (SEQ ID NO: 205), G-(X1)-TF-(X 13 X 14 )-Y-(X4) (SEQ ID NO: 206), CDR1 comprising I-(X8X9X 10 X 11 )-G-(X 12)-A (SEQ ID NO: 51) or II-(X 15 )-IFG-(X 16 )-A (SEQ ID NO:207), and / or a CDR3 comprising 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), 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). In another embodiment, the light chain comprises a CDR2 comprising SGSIDSNY (SEQ ID NO:18), S-(X 17 X 18 )I-(X 19 )-SNY (SEQ ID NO: 208), or NIG-(X5)-K-(X 20 ) (SEQ ID NO: 48), CDR1 containing EDN (SEQ ID NO: 20), (X 21 )-DN (SEQ ID NO: 209), (X 22)-NN (SEQ ID NO:210), or DD-X6 (SEQ ID NO:49), and / or a CDR3 comprising 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 fully human or 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 x 10-9 M. In embodiments, the antibody or fragment may further comprise a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In embodiments, X1, X2, X3, or X4 is a non-polar amino acid residue. In other embodiments, X1, X2, X4, or X5 are non-polar amino acid residues. 3、 Or X4 is glycine (G), tyrosine (Y), phenylalanine (F), leucine (L), or alanine (A). In some embodiments, X1, X2, or X4 is a hydrophobic amino acid residue, e.g., X1, X2, or X4 is 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 are non-polar hydrophobic amino acid residues.10 , or X 11 is isoleucine (I), proline (P), alanine (A), or phenylalanine (F). 10 , or X 12 is a polar hydrophilic amino acid residue. 10 , or X 12 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). 11 is glycine (G), leucine (L), asparagine (N), or phenylalanine (F). 12 is isoleucine (I), arginine (R), threonine (T), or histidine (H). In some embodiments, X5 is a non-polar 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 is a non-polar 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 is a non-polar hydrophobic amino acid residue. 17 , or X 20 is glycine (G). 13 , X 14, X 15 , X 16 , X 17 , X 18 , X 19 , or X 21 is a polar hydrophilic amino acid residue. 14 , or X 21 is serine (S) or arginine (R). 13 is serine (S) or threonine (T). 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). 16 is isoleucine (I). In one embodiment, X 18 is serine (S) or asparagine (N). 19 is glycine (G) or alanine (A). 19 is aspartic acid (D). In one embodiment, X 21 is alanine (A). In one embodiment, X 21 is glutamic acid (E).

[0008] One aspect of the present invention is directed to an isolated antibody or fragment thereof that binds to human programmed death-ligand 1 (PD-L1) protein and comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 4, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 6, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 48, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50. In some embodiments, X5 in SEQ ID NO: 48 is a non-polar, 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 comprises the amino acid sequence of SEQ ID NO: 26, 33, 40, or 44. In embodiments, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 28, 35, or 45. In an embodiment, the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 30, or 37.In some embodiments, the antibody of (b) described herein comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a VL CDR3 comprising SEQ ID NO: 30, or comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 37, or comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 37, or comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 44, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 37.

[0009] One aspect of the present invention is directed to an isolated antibody or fragment thereof that binds to human PD-L1 protein and comprises a heavy chain variable region comprising 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 comprising 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 embodiment of the present invention relates to a heavy chain variable region that binds to human PD-L1 protein and comprises a 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, a 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 a 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 a VH-CDR3 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.

[0011] One embodiment of the present invention provides an isolated antibody or fragment thereof that binds to 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), respectively; 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), respectively; and (b) a light chain variable region having three CDRs comprising the amino acid sequence GYTLS (SEQ ID NO: 2), ... (c) a heavy chain variable region having three CDRs comprising 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 comprising the amino acid sequences NIGSKG (SEQ ID NO: 26), DDR (SEQ ID NO: 28), and QVWDSGSDHWV (SEQ ID NO: 30), respectively; (d) a light chain variable region having three CDRs comprising the amino acid sequences NIGDKG (SEQ ID NO: 33), DDS (SEQ ID NO: 35), and QVWDSSSDHWV (SEQ ID NO: 37), respectively; (e) a heavy chain variable region having three CDRs comprising 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 comprising the amino acid sequences NIGDKG (SEQ ID NO: 40), DDS (SEQ ID NO: 35), and QVWDSSSDHWV (SEQ ID NO: 37), respectively; (e) a heavy chain variable region having three CDRs each comprising 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 each comprising the amino acid sequences NIGGKG (SEQ ID NO: 44), DDY (SEQ ID NO: 45), and QVWDSSSDHWV (SEQ ID NO: 37), (f) a heavy chain variable region having three CDRs each comprising 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 each comprising the amino acid sequences NIGGKG (SEQ ID NO: 44), DDY (SEQ ID NO: 45), and QVWDSSSDHWV (SEQ ID NO: 37),and ARVHAALYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs each comprising 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 each comprising 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 each comprising the amino acid sequences NIESRS (SEQ ID NO: 108), DDT (SEQ ID NO: 118), and QVWDSSGDLWV (SEQ ID NO: 126), respectively; (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 ARVHAALYYGMDV (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 QVWDSSSDHWV (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 ARVHAALYYGMDV (SEQ ID NO: 14); (j) 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), and / or a light chain variable region having three CDRs containing the amino acid sequences NIGSKG (SEQ ID NO: 26), DDS (SEQ ID NO: 35), and QVWDSSSDHWV (SEQ ID NO: 37), respectively; (k) a light chain variable region having three CDRs containing the amino acid sequences DFAFSSAW (SEQ ID NO: 84), IKSKTDGETT (SEQ ID NO: 91), and TTGGLGLVYPYYNYIDV (SEQ ID NO: 99), respectively; (k) a heavy chain variable region having three CDRs each comprising 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 comprising 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 comprising 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 comprising the amino acid sequences SGSIASNY (SEQ ID NO: 111), ADN (SEQ ID NO: 120), and QSYDSSNHWV (SEQ ID NO: 129); (m) a light chain variable region having three CDRs each comprising the amino acid sequence GYTLSSHG (SEQ ID NO: 1 (n) a heavy chain variable region having three CDRs comprising the amino acid sequences GYTFTSYG (SEQ ID NO: 85), ISAYNGHA (SEQ ID NO: 94), and ARVHAALYYGMDV (SEQ ID NO: 14), and / or a light chain variable region having three CDRs comprising the amino acid sequences NIGSKS (SEQ ID NO: 109), DDS (SEQ ID NO: 35), and QVWDSSSDHWV (SEQ ID NO: 37), respectively; or (o) a light chain variable region having three CDRs comprising the amino acid sequences NIGSKG (SEQ ID NO: 26), DDS (SEQ ID NO: 35), and QVWDSRSDHWV (SEQ ID NO: 130), respectively; (o) a heavy chain variable region having three CDRs comprising the amino acid sequences GGTFSSYA (SEQ ID NO: 87), IIPIFGTA (SEQ ID NO: 95), and ARDGSGYDSAGMDD (SEQ ID NO: 102), respectively; and / or the amino acid sequences RSNIGSNY (SEQ ID NO: 112), SNN (SEQ ID NO: 121), and AVWDDSLSGVV (SEQ ID NO: 131), respectively. (p) a heavy chain variable region having three CDRs comprising the amino acid sequences GFTFSSYA (SEQ ID NO: 88), ISYDGSNK (SEQ ID NO: 96), and ARGFGGPDY (SEQ ID NO: 103), respectively; and / or a light chain variable region having three CDRs comprising the amino acid sequences SGINVGTYR (SEQ ID NO: 113), YKSDSDK (SEQ ID NO: 122), and MIWHSSAYV (SEQ ID NO: 132), respectively; (q) a light chain variable region having three CDRs comprising the amino acid sequences GYTFSSYG (SEQ ID NO: 89), ISAHNGHA (SEQ ID NO: 12), respectively;and ARVHGALYYGMDV (SEQ ID NO: 104), and / or a light chain variable region having three CDRs comprising 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 comprising 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 comprising the amino acid sequences NIGSKG (SEQ ID NO: 26), DDR (SEQ ID NO: 28), and QVWDSSSDHWV (SEQ ID NO: 37), respectively; (s) a heavy chain variable region having three CDRs comprising the amino acid sequences GGTFSSYA (SEQ ID NO: 87), IIPILGIA (SEQ ID NO: 97), and ASGSIVGAAYAFDI (SEQ ID NO: 105), respectively; and / or a light chain variable region having three CDRs comprising the amino acid sequences NIGGRV (SEQ ID NO: 115), DDT ( (t) a light chain variable region having three CDRs containing the amino acid sequences GFTFSSYS (SEQ ID NO: 90), IISDGSAT (SEQ ID NO: 98), and ARDRSEGGFDP (SEQ ID NO: 106), respectively; and / or a heavy chain variable region having three CDRs containing the amino acid sequences SLRSYY (SEQ ID NO: 116), GKN (SEQ ID NO: 124), and NSRDISDNQWQWI (SEQ ID NO: 134), respectively. The present invention relates to an isolated antibody or fragment thereof comprising: (u) a heavy chain variable region having three CDRs comprising the amino acid sequences GGTFSRYA (SEQ ID NO: 86), IIPIFGRA (SEQ ID NO: 93), and AEEGAFNSLAI (SEQ ID NO: 107), respectively; and / or a light chain variable region having three CDRs comprising the amino acid sequences SGSIASHF (SEQ ID NO: 117), GDD (SEQ ID NO: 125), and QSYDSSNHVV (SEQ ID NO: 135), respectively.

[0012] One aspect of the present invention is directed to 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 heavy chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:8, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:24.

[0013] One aspect of the present invention is directed to 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 heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 16, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 31.

[0014] One aspect of the present invention is directed to 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 heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 16, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 38.

[0015] One aspect of the present invention is directed to 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 heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 16, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 42.

[0016] One aspect of the present invention is directed to 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 heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 16, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 46.

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

[0018] One aspect of the invention provides 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: (a) V having SEQ ID NO: 8 H V having the amino acid sequence SEQ ID NO: 24 L amino acid sequence, (b) V having SEQ ID NO: 16 H V having the amino acid sequence SEQ ID NO: 31 L amino acid sequence, (c) V having SEQ ID NO: 16 H V having the amino acid sequence SEQ ID NO: 38 L amino acid sequence, (d) V having SEQ ID NO: 16 H V having the amino acid sequence SEQ ID NO: 42 L amino acid sequence, (e) V having SEQ ID NO: 16 H V having the amino acid sequence SEQ ID NO: 46 L amino acid sequence, (f) V having SEQ ID NO: 52 H V having the amino acid sequence SEQ ID NO: 53 L amino acid sequence, (g) V having SEQ ID NO: 54 H V having the amino acid sequence SEQ ID NO: 55 L amino acid sequence, (h) V having SEQ ID NO: 56 H V having the amino acid sequence SEQ ID NO: 57 L amino acid sequence, (i) V having SEQ ID NO: 16 H V having the amino acid sequence SEQ ID NO: 59 L amino acid sequence, (j) V having SEQ ID NO: 60 H V having the amino acid sequence SEQ ID NO: 61 L amino acid sequence, (k) V having SEQ ID NO: 62 H V having the amino acid sequence SEQ ID NO: 63 L amino acid sequence, (l) V having SEQ ID NO: 64 H V having the amino acid sequence SEQ ID NO: 65 L amino acid sequence, (m) V having SEQ ID NO: 66 H V having the amino acid sequence SEQ ID NO: 67 L amino acid sequence, (n) V having SEQ ID NO: 68 H V having the amino acid sequence SEQ ID NO: 69 L amino acid sequence, (o) V having SEQ ID NO: 70 H V having the amino acid sequence SEQ ID NO: 71 L amino acid sequence, (p) V having SEQ ID NO: 72 H V having the amino acid sequence SEQ ID NO: 73 L amino acid sequence, (q) V having SEQ ID NO: 74 H V having the amino acid sequence SEQ ID NO: 75 L amino acid sequence, (r) V having SEQ ID NO: 76 H V having the amino acid sequence SEQ ID NO: 77 L amino acid sequence, (s) V having SEQ ID NO: 78 H V having the amino acid sequence SEQ ID NO: 79 L amino acid sequence, (t) V having SEQ ID NO: 80 HV having the amino acid sequence SEQ ID NO: 81 L an amino acid sequence, or (u) V having SEQ ID NO: 82 H V having the amino acid sequence SEQ ID NO: 83 L Amino acid sequence The present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising:

[0019] Aspects of the present invention are directed to isolated bispecific antibodies comprising a fragment of an antibody described herein and a second antigen-binding fragment having specificity for a molecule on an immune cell. In some embodiments, the molecule on an immune cell 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. 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 nucleic acids encoding the antibodies described herein.

[0021] Aspects of the invention are directed to vectors comprising nucleic acids encoding the antibodies described herein.

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

[0023] Aspects of the invention are directed to nucleic acids encoding the bispecific antibodies described herein.

[0024] Aspects of the invention are directed to vectors comprising nucleic acids encoding the bispecific antibodies described herein.

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

[0026] Aspects of the present invention are directed to pharmaceutical compositions comprising an antibody described herein or a fragment described herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition further comprises at least one additional therapeutic agent. In other embodiments, the therapeutic agent is a toxin, a radiolabel, an siRNA, a small molecule, or a cytokine.

[0027] Aspects of the present invention are directed to pharmaceutical compositions comprising a bispecific antibody described herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition further comprises at least one additional therapeutic agent. In other embodiments, the therapeutic agent is a toxin, a radiolabel, an siRNA, a small molecule, or a cytokine.

[0028] Aspects of the present invention are directed to isolated cells comprising one or more polynucleotides encoding an antibody or fragment thereof described herein.

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

[0030] An embodiment of the present invention is directed to a kit comprising at least one antibody described herein; a syringe, needle, or applicator for administering the at least one antibody to a subject; and instructions for use.

[0031] One aspect of the present invention is directed to a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an intracellular signaling domain, a transmembrane domain, and an extracellular domain, wherein the extracellular domain is an isolated monoclonal antibody or antigen-binding fragment thereof that binds to 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. In some embodiments, the heavy chain is G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 47), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 48), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 49), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 50), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 51), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 52), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 53), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 54), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 55), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 56), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 57), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 58), G-(X1)-T-(X2) 13 X 14 )-(X3X4) (SEQ ID NO: 205), G-(X1)-TF-(X 13 X 14 )-Y-(X4) (SEQ ID NO: 206), CDR1 comprising I-(X8X9X 10 X 11 )-G-(X 12 )-A (SEQ ID NO: 51) or II-(X 15 )-IFG-(X 16 )-A (SEQ ID NO:207), and / or a CDR3 comprising 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), 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). In another embodiment, the light chain comprises a CDR2 comprising SGSIDSNY (SEQ ID NO:18), S-(X 17 X 18 )I-(X 19 )-SNY (SEQ ID NO: 208), or NIG-(X5)-K-(X 20 ) (SEQ ID NO: 48), CDR1 containing EDN (SEQ ID NO: 20), (X 21 )-DN (SEQ ID NO: 209), (X 22)-NN (SEQ ID NO: 210), or DD-X6 (SEQ ID NO: 49), and / or a CDR3 comprising 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 CAR antibody is fully human or humanized. In other embodiments, the CAR antibody is monospecific, bispecific, or multispecific. In further embodiments, the CAR antibody is a single-chain antibody. In embodiments, X1, X2, X3, or X4 is a non-polar amino acid residue. 3、 Or X4 is glycine (G), tyrosine (Y), phenylalanine (F), leucine (L), or alanine (A). In some embodiments, X1, X2, or X4 is a hydrophobic amino acid residue, e.g., X1, X2, or X4 is 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 are non-polar hydrophobic amino acid residues. 10 , or X 11 is isoleucine (I), proline (P), alanine (A), or phenylalanine (F). 10, or X 12 is a polar hydrophilic amino acid residue. 10 , or X 12 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). 11 is glycine (G), leucine (L), asparagine (N), or phenylalanine (F). 12 is isoleucine (I), arginine (R), threonine (T), or histidine (H). In some embodiments, X5 is a non-polar 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 non-polar 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 is a non-polar hydrophobic amino acid residue. 17 , or X 20 is glycine (G). 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , or X 21is a polar hydrophilic amino acid residue. 14 , or X 21 is serine (S) or arginine (R). 13 is serine (S) or threonine (T). 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). 16 is isoleucine (I). In one embodiment, X 18 is serine (S) or asparagine (N). 19 is glycine (G) or alanine (A). 19 is aspartic acid (D). In one embodiment, X 21 is alanine (A). In one embodiment, X 21 is glutamic acid (E). In some embodiments, the transmembrane domain further comprises a stalk region disposed between the extracellular domain and the transmembrane domain. In other embodiments, the transmembrane domain comprises CD28. In some embodiments, the CAR further comprises one or more additional costimulatory molecules disposed between the transmembrane domain and the intracellular signaling domain. In further embodiments, the costimulatory molecule is CD28, 4-1BB, ICOS, or OX40. In some embodiments, the intracellular signaling domain comprises a CD3 zeta chain. In other embodiments, the antibody of the CAR is a Fab or scFV.

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

[0033] Aspects of the present invention are directed 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 disposed after the intracellular signaling domain, wherein the polypeptide comprises an isolated monoclonal antibody or antigen-binding fragment thereof that binds to 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. In some embodiments, the heavy chain is G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 47), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 48), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 49), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 50), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 51), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 52), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 53), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 54), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 55), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 56), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 57), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 13 X 14 )-(X3X4) (SEQ ID NO: 205), G-(X1)-TF-(X 13 X 14 )-Y-(X4) (SEQ ID NO: 206), CDR1 comprising I-(X8X9X 10 X 11 )-G-(X 12 )-A (SEQ ID NO: 51) or II-(X 15 )-IFG-(X 16 )-A (SEQ ID NO:207), and / or a CDR3 comprising 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), 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). In another embodiment, the light chain comprises a CDR2 comprising SGSIDSNY (SEQ ID NO:18), S-(X 17 X 18 )I-(X 19 )-SNY (SEQ ID NO: 208), or NIG-(X5)-K-(X 20 ) (SEQ ID NO: 48), CDR1 containing EDN (SEQ ID NO: 20), (X 21 )-DN (SEQ ID NO: 209), (X 22)-NN (SEQ ID NO: 210), or DD-X6 (SEQ ID NO: 49), and / or a CDR3 comprising 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 CAR antibody is fully human or humanized. In other embodiments, the CAR antibody is monospecific, bispecific, or multispecific. In further embodiments, the CAR antibody is a single-chain antibody. In embodiments, X1, X2, X3, or X4 is a non-polar amino acid residue. 3、 Or X4 is glycine (G), tyrosine (Y), phenylalanine (F), leucine (L), or alanine (A). In some embodiments, X1, X2, or X4 is a hydrophobic amino acid residue, e.g., X1, X2, or X4 is 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 are non-polar hydrophobic amino acid residues. 10 , or X 11 is isoleucine (I), proline (P), alanine (A), or phenylalanine (F). 10 , or X12 is a polar hydrophilic amino acid residue. 10 , or X 12 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). 11 is glycine (G), leucine (L), asparagine (N), or phenylalanine (F). 12 is isoleucine (I), arginine (R), threonine (T), or histidine (H). In some embodiments, X5 is a non-polar 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 non-polar 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 is a non-polar hydrophobic amino acid residue. 17 , or X 20 is glycine (G). 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , or X 21is a polar hydrophilic amino acid residue. 14 , or X 21 is serine (S) or arginine (R). 13 is serine (S) or threonine (T). 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). 16 is isoleucine (I). In one embodiment, X 18 is serine (S) or asparagine (N). 19 is glycine (G) or alanine (A). 19 is aspartic acid (D). In one embodiment, X 21 is alanine (A). In one embodiment, X 21 is glutamic acid (E). In some embodiments, the transmembrane domain further comprises a stalk region disposed between the extracellular domain and the transmembrane domain. In other embodiments, the transmembrane domain comprises CD28. In some embodiments, the CAR further comprises one or more additional costimulatory molecules disposed between the transmembrane domain and the intracellular signaling domain. In further embodiments, the costimulatory molecule is CD28, 4-1BB, ICOS, or OX40. In some embodiments, the intracellular signaling domain comprises a CD3 zeta chain. In other embodiments, the antibody of the CAR is a Fab or scFV.

[0034] Aspects of the invention are directed to vectors comprising a nucleic acid encoding a CAR as described herein.

[0035] Aspects of the invention are directed to cells hosting a vector comprising a nucleic acid encoding a CAR as described herein.

[0036] Aspects of the present invention are directed to genetically engineered cells that express the CARS described herein. In one embodiment, the cells express and carry on the cell surface membrane a chimeric antigen receptor described herein. In some embodiments, the cells are T cells or NK cells. In further embodiments, the T cells are CD4 + or CD8 + In another embodiment, the genetically engineered cells are CD4 + and CD8 cells + Contains a mixed population of

[0037] An aspect of the present invention is directed to genetically engineered cells that express and retain on their cell surface membrane a chimeric antigen receptor, which is further engineered to express and secrete a polypeptide, wherein the polypeptide is an isolated monoclonal antibody or antigen-binding fragment thereof that binds to 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. In some embodiments, the heavy chain is G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 47), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 48), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 49), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 50), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 51), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 52), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 53), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 54), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 55), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 56), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 57), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 58), G-(X1)-T-(X2)-SS-(X3 13 X 14 )-(X3X4) (SEQ ID NO: 205), G-(X1)-TF-(X 13 X 14 )-Y-(X4) (SEQ ID NO: 206), CDR1 comprising I-(X8X9X 10 X 11 )-G-(X 12 )-A (SEQ ID NO: 51) or II-(X 15 )-IFG-(X 16)-A (SEQ ID NO:207), and / or a CDR3 comprising 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), 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). In another embodiment, the light chain comprises a CDR2 comprising SGSIDSNY (SEQ ID NO:18), S-(X 17 X 18 )I-(X 19 )-SNY (SEQ ID NO: 208), or NIG-(X5)-K-(X 20 ) (SEQ ID NO: 48), CDR1 containing EDN (SEQ ID NO: 20), (X 21 )-DN (SEQ ID NO: 209), (X 22 )-NN (SEQ ID NO: 210), or DD-X6 (SEQ ID NO: 49), and / or a CDR3 comprising 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 CAR antibody is fully human or humanized. In other embodiments, the CAR antibody is monospecific, bispecific, or multispecific. In further embodiments, the CAR antibody is a single-chain antibody. In embodiments, X1, X2, X3, or X4 is a non-polar amino acid residue. 3、Or X4 is glycine (G), tyrosine (Y), phenylalanine (F), leucine (L), or alanine (A). In some embodiments, X1, X2, or X4 is a hydrophobic amino acid residue, e.g., X1, X2, or X4 is 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 are non-polar hydrophobic amino acid residues. 10 , or X 11 is isoleucine (I), proline (P), alanine (A), or phenylalanine (F). 10 , or X 12 is a polar hydrophilic amino acid residue. 10 , or X 12 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). 11 is glycine (G), leucine (L), asparagine (N), or phenylalanine (F). 12is isoleucine (I), arginine (R), threonine (T), or histidine (H). In some embodiments, X5 is a non-polar 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 non-polar 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 is a non-polar hydrophobic amino acid residue. 17 , or X 20 is glycine (G). 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , or X 21 is a polar hydrophilic amino acid residue. 14 , or X 21 is serine (S) or arginine (R). 13 is serine (S) or threonine (T). 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). 16 is isoleucine (I). In one embodiment, X 18 is serine (S) or asparagine (N). 19is glycine (G) or alanine (A). 19 is aspartic acid (D). In one embodiment, X 21 is alanine (A). In one embodiment, X 21 is glutamic acid (E).

[0038] One aspect of the present invention is directed to a method for treating cancer in a subject. In some embodiments, the method comprises administering to a subject in need thereof 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] [The present invention 1001] 1. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Programmed Death Ligand 1 (PD-L1) protein, comprising a heavy chain, a light chain, or a combination thereof, the 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), I-(X 8 X 9 X 10 X 11 )-G-(X 12 )-A (SEQ ID NO: 51), or II-(X 15 )-IFG-(X 16 )-A (SEQ ID NO: 207), and / or CDR3 comprising 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), 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). Including, the light chain S-(X 17 X 18 )I-(X 19 )-SNY (SEQ ID NO: 208) or NIG-(X 5 )-K-(X 20 ) (SEQ ID NO: 48), (X 21 )-DN (SEQ ID NO: 209), (X 22 )-NN (SEQ ID NO: 210), or DD-X 6 (SEQ ID NO: 49), and / or QSYDSNNRHVI (SEQ ID NO: 22), QVWDS-(X 7 )-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 antigen-binding fragment thereof, comprising: [The present invention 1002] 1001. An antibody of the invention which is fully human or humanized. [The present invention 1003] The antibody of the present invention 1001, which is monospecific, bispecific, or multispecific. [The present invention 1004] The antibody of the present invention is a single-chain antibody. [The present invention 1005] At least 3.3 x 10 -9 1001. An antibody of the present invention having a binding affinity of M. [The present invention 1006] The antibody or fragment of the present invention 1001 further comprising a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. [The present invention 1007] X 1 、X 2 、X 3、 or X 4 is a nonpolar amino acid residue. [The present invention 1008] X 1 、X 2 、X 3、 or X 4 is glycine (G), tyrosine (Y), phenylalanine (F), leucine (L), or alanine (A). [The present invention 1009] X 1 、X 2 , or X 4 is a hydrophobic amino acid residue. [The present invention 1010] X 1 、X 2、 or X 4 is glycine (G), leucine (L), or alanine (A). [The present invention 1011] X 3 is a hydrophilic polar amino acid residue. [The present invention 1012] X 3 is histidine (H). [The present invention 1013] X 1 is phenylalanine (F), glycine (G), or tyrosine (Y). [The present invention 1014] X 2 is phenylalanine (F) or leucine (L). [The present invention 1015] X 3 is histidine (H) or tyrosine (Y). [The present invention 1016] X 4 is serine (S), glycine (G), or alanine (A). [The present invention 1017] X 8 、X 9 、X 10 , or X 11 is a non-polar hydrophobic amino acid residue. [The present invention 1018] X 8 、X 9、 X 10 , or X 11 is isoleucine (I), proline (P), alanine (A), or phenylalanine (F). [The present invention 1019] X 8 、X 10、 or X 12 is a polar hydrophilic amino acid residue. [The present invention 1020] X 8 、X 10 , or X 12 is histidine (H), serine (S), asparagine (N), or threonine (T). [The present invention 1021] X 8 is alanine (A), isoleucine (I), or serine (S). [The present invention 1022] X 9 is proline (P), tyrosine (Y), serine (S), or alanine (A). [The present invention 1023] X 10 is tyrosine (Y), aspartic acid (D), isoleucine (I), or histidine (H). [The present invention 1024] X 11 is glycine (G), leucine (L), asparagine (N), or phenylalanine (F). [The present invention 1025] X 12 is isoleucine (I), arginine (R), threonine (T), or histidine (H). [The present invention 1026] X 5 is a non-polar hydrophobic amino acid residue. [The present invention 1027] X 5 is glycine (G). [The present invention 1028] X 5 is a polar hydrophilic amino acid residue. [The present invention 1029] X 5 is serine (S), asparagine (N), or aspartic acid (D). [The present invention 1030] X 6 is a nonpolar amino acid residue. [The present invention 1031] X 6 is tyrosine (Y). [The present invention 1032] X 6 is a polar hydrophilic amino acid residue. [The present invention 1033] X 6 is serine (S), threonine (T), or arginine (R). [The present invention 1034] X 7 、X 15 、X 16 、X 17 、X 19 、X 20 , or X 21 is a non-polar hydrophobic amino acid residue. [This invention 1035] X 7 、X 17 , or X 20 is glycine (G). [The present invention 1036] 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. [This invention 1037] X 7 、X 14 , or X 21 is serine (S) or arginine (R). [The present invention 1038] X 13 is serine (S) or threonine (T). [This invention 1039] X 15 is proline (P). [The present invention 1040] X 15 、X 17 , or X 20 is serine (S). [The present invention 1041] X 16 is threonine (T) or arginine (R). [The present invention 1042] X 16 is isoleucine (I). [This invention 1043] X 18 is serine (S) or asparagine (N). [This invention 1044] X 19 is glycine (G) or alanine (A). [This invention 1045] X 19 is aspartic acid (D). [The present invention 1046] X 21 is alanine (A). [This invention 1047] X 21 is glutamic acid (E). [This invention 1048] binds to the human programmed death-ligand 1 (PD-L1) protein, and (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 4, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 6, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 48, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50 An isolated antibody or fragment thereof comprising: [This invention 1049] X in SEQ ID NO: 48 5 is a non-polar hydrophobic amino acid residue. [The present invention 1050] X 5 is glycine (G). [This invention 1051] X 5 is a polar hydrophilic amino acid residue. [This invention 1052] X 5 is serine (S), asparagine (N), or aspartic acid (D). [This invention 1053] X in SEQ ID NO: 49 6 is a nonpolar amino acid residue. [This invention 1054] X 6 is tyrosine (Y). [This invention 1055] X 6 is a polar hydrophilic amino acid residue. [This invention 1056] X 6 is serine (S), threonine (T), or arginine (R). [This invention 1057] X in SEQ ID NO:50 7 is a non-polar hydrophobic amino acid residue. [This invention 1058] X 7 is glycine (G). [This invention 1059] X 7 is a polar hydrophilic amino acid residue. [The present invention 1060] X 7 is serine (S) or arginine (R). [This invention 1061] The antibody of the present invention 1048, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 26, 33, 40, or 44. [This invention 1062] The antibody of the present invention 1048, wherein the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 28, 35, or 45. [The present invention 1063] The antibody of the present invention 1048, wherein the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 30 or 37. [This invention 1064] An antibody of the present invention 1048, wherein the antibody (b) comprises a VL CDR1 having the amino acid sequence of SEQ ID NO: 26, a VL CDR2 having the amino acid sequence of SEQ ID NO: 28, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 30, or a VL CDR1 having the amino acid sequence of SEQ ID NO: 33, a VL CDR2 having the amino acid sequence of SEQ ID NO: 35, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 37, or a VL CDR1 having the amino acid sequence of SEQ ID NO: 40, a VL CDR2 having the amino acid sequence of SEQ ID NO: 35, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 37, or a VL CDR1 having the amino acid sequence of SEQ ID NO: 44, a VL CDR2 having the amino acid sequence of SEQ ID NO: 45, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 37. [This invention 1065] binds to the human PD-L1 protein, and a heavy chain variable region comprising 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 comprising 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; An isolated antibody or fragment thereof comprising: [The present invention 1066] 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; a 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 a heavy chain variable region comprising 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; a 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. a light chain variable region comprising An isolated antibody or fragment thereof comprising: [This invention 1067] 1. An isolated antibody or fragment thereof that binds to human PD-L1 protein, wherein the antibody: (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), respectively, 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), respectively; (b) a heavy chain variable region having three CDRs comprising 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 comprising the amino acid sequences NIGSKG (SEQ ID NO: 26), DDR (SEQ ID NO: 28), and QVWDSGSDHWV (SEQ ID NO: 30), respectively; (c) a heavy chain variable region having three CDRs comprising 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 comprising 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 comprising 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 comprising the amino acid sequences NIGNKG (SEQ ID NO: 40), DDS (SEQ ID NO: 35), and QVWDSSSDHWV (SEQ ID NO: 37), respectively; (e) a heavy chain variable region having three CDRs comprising 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 comprising the amino acid sequences NIGGKG (SEQ ID NO: 44), DDY (SEQ ID NO: 45), and QVWDSSSDHWV (SEQ ID NO: 37), respectively; (f) a heavy chain variable region having three CDRs comprising 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 comprising 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 comprising 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 comprising the amino acid sequences NIGSKG (SEQ ID NO: 26), DDS (SEQ ID NO: 35), and QVWDSSSDHWV (SEQ ID NO: 37), respectively; (h) a heavy chain variable region having three CDRs comprising 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 comprising the amino acid sequences NIGSKS (SEQ ID NO: 109), DDS (SEQ ID NO: 35), and QVWDSSSDHWV (SEQ ID NO: 37); (i) a heavy chain variable region having three CDRs comprising 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 comprising the amino acid sequences NIGSKG (SEQ ID NO: 26), DDS (SEQ ID NO: 35), and QVWDSSSDHWV (SEQ ID NO: 37), respectively; (j) a heavy chain variable region having three CDRs comprising the amino acid sequences DFAFSSAW (SEQ ID NO: 84), IKSKTDGETT (SEQ ID NO: 91), and TTGGLGLVYPYYNYIDV (SEQ ID NO: 99), respectively, and / or a light chain variable region having three CDRs comprising 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 comprising the amino acid sequences GYTFTSYG (SEQ ID NO: 85), TSPHNGLT (SEQ ID NO: 92), and AKVHPVFSYALDV (SEQ ID NO: 100), respectively, and / or a light chain variable region having three CDRs comprising the amino acid sequences SGSIASNY (SEQ ID NO: 111), EDN (SEQ ID NO: 20), and QSYDGITVI (SEQ ID NO: 128), respectively; (l) a heavy chain variable region having three CDRs comprising the amino acid sequences GGTFSRYA (SEQ ID NO: 86), IIPIFGRA (SEQ ID NO: 93), and AEEGAFNSLAI (SEQ ID NO: 101), respectively, and / or a light chain variable region having three CDRs comprising the amino acid sequences SGSIASNY (SEQ ID NO: 111), ADN (SEQ ID NO: 120), and QSYDSSNHWV (SEQ ID NO: 129), respectively; (m) a heavy chain variable region having three CDRs comprising 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 comprising 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 comprising the amino acid sequences GYTFTSYG (SEQ ID NO: 85), ISAYNGHA (SEQ ID NO: 94), and ARVHAALYYGMDV (SEQ ID NO: 14), respectively, and / or a light chain variable region having three CDRs comprising the amino acid sequences NIGSKG (SEQ ID NO: 26), DDS (SEQ ID NO: 35), and QVWDSRSDHWV (SEQ ID NO: 130), respectively; (o) a heavy chain variable region having three CDRs comprising the amino acid sequences GGTFSSYA (SEQ ID NO: 87), IIPIFGTA (SEQ ID NO: 95), and ARDGSGYDSAGMDD (SEQ ID NO: 102), respectively, and / or a light chain variable region having three CDRs comprising the amino acid sequences RSNIGSNY (SEQ ID NO: 112), SNN (SEQ ID NO: 121), and AVWDDSLSGVV (SEQ ID NO: 131), respectively; (p) a heavy chain variable region having three CDRs comprising the amino acid sequences GFTFSSYA (SEQ ID NO: 88), ISYDGSNK (SEQ ID NO: 96), and ARGFGGPDY (SEQ ID NO: 103), respectively, and / or a light chain variable region having three CDRs comprising the amino acid sequences SGINVGTYR (SEQ ID NO: 113), YKSDSDK (SEQ ID NO: 122), and MIWHSSAYV (SEQ ID NO: 132), respectively; (q) a heavy chain variable region having three CDRs comprising the amino acid sequences GYTFSSYG (SEQ ID NO: 89), ISAHNGHA (SEQ ID NO: 12), and ARVHGALYYGMDV (SEQ ID NO: 104), respectively, and / or a light chain variable region having three CDRs comprising 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 comprising 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 comprising the amino acid sequences NIGSKG (SEQ ID NO: 26), DDR (SEQ ID NO: 28), and QVWDSSSDHWV (SEQ ID NO: 37), respectively; (s) a heavy chain variable region having three CDRs comprising the amino acid sequences GGTFSSYA (SEQ ID NO: 87), IIPILGIA (SEQ ID NO: 97), and ASGSIVGAAYAFDI (SEQ ID NO: 105), respectively, and / or a light chain variable region having three CDRs comprising the amino acid sequences NIGGRV (SEQ ID NO: 115), DDT (SEQ ID NO: 123), and QVWDSRSDHPV (SEQ ID NO: 133), respectively; (t) a heavy chain variable region having three CDRs comprising the amino acid sequences GFTFSSYS (SEQ ID NO: 90), IISDGSAT (SEQ ID NO: 98), and ARDRSEGGFDP (SEQ ID NO: 106), respectively, and / or a light chain variable region having three CDRs comprising the amino acid sequences SLRSYY (SEQ ID NO: 116), GKN (SEQ ID NO: 124), and NSRDISDNQWQWI (SEQ ID NO: 134), respectively; or (u) a heavy chain variable region having three CDRs comprising the amino acid sequences GGTFSRYA (SEQ ID NO: 86), IIPIFGRA (SEQ ID NO: 93), and AEEGAFNSLAI (SEQ ID NO: 107), respectively, and / or a light chain variable region having three CDRs comprising the amino acid sequences SGSIASHF (SEQ ID NO: 117), GDD (SEQ ID NO: 125), and QSYDSSNHVV (SEQ ID NO: 135), respectively. The isolated antibody or fragment thereof comprising: [The present invention 1068] 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 heavy chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:8, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:24. [The present invention 1069] 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 heavy chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO: 16, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO: 31. [The present invention 1070] 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 heavy chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO: 16, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO: 38. [This invention 1071] 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 heavy chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO: 16, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO: 42. [This invention 1072] 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 heavy chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO: 16, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO: 46. [This invention 1073] 1. 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, (a) the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 52 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 53; (b) the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 54 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 55; (c) the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 56 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 57; (d) the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 16 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 59; (e) the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 60 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 61; (f) the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 62 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 63; (g) the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 64 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 65; (h) the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 66 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 67; (i) the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 68 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 69; (j) the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 70 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 71; (k) the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 72 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 73; (l) the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 74 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 75; (m) the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 76 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 77; (n) the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 78 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 79; (o) the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 80 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 81; or (p) the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 82 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 83; The isolated monoclonal antibody or antigen-binding fragment thereof. [This invention 1074] 1. 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: (a) V having SEQ ID NO: 8 H V having the amino acid sequence SEQ ID NO: 24 L amino acid sequence, (b) V having SEQ ID NO: 16 H V having the amino acid sequence SEQ ID NO: 31 L amino acid sequence, (c) V having SEQ ID NO: 16 H V having the amino acid sequence SEQ ID NO: 38 L amino acid sequence, (d) V having SEQ ID NO: 16 H V having the amino acid sequence SEQ ID NO: 42 L amino acid sequence, (e) V having SEQ ID NO: 16 H V having the amino acid sequence SEQ ID NO: 46 L amino acid sequence, (f) V having SEQ ID NO: 52 H V having the amino acid sequence SEQ ID NO: 53 L amino acid sequence, (g) V having SEQ ID NO: 54 H V having the amino acid sequence SEQ ID NO: 55 L amino acid sequence, (h) V having SEQ ID NO: 56 H V having the amino acid sequence SEQ ID NO: 57 L amino acid sequence, (i) V having SEQ ID NO: 16 H V having the amino acid sequence SEQ ID NO: 59 L amino acid sequence, (j) V having SEQ ID NO: 60 H V having the amino acid sequence SEQ ID NO: 61 L amino acid sequence, (k) V having SEQ ID NO: 62 H V having the amino acid sequence SEQ ID NO: 63 L amino acid sequence, (l) V having SEQ ID NO: 64 H V having the amino acid sequence SEQ ID NO: 65 L amino acid sequence, (m) V having SEQ ID NO: 66 H V having the amino acid sequence SEQ ID NO: 67 L amino acid sequence, (n) V having SEQ ID NO: 68 H V having the amino acid sequence SEQ ID NO: 69 L amino acid sequence, (o) V having SEQ ID NO: 70 H V having the amino acid sequence SEQ ID NO: 71 L amino acid sequence, (p) V having SEQ ID NO: 72 H V having the amino acid sequence SEQ ID NO: 73 L amino acid sequence, (q) V having SEQ ID NO: 74 H V having the amino acid sequence SEQ ID NO: 75 L amino acid sequence, (r) V having SEQ ID NO: 76 H V having the amino acid sequence SEQ ID NO: 77 L amino acid sequence, (s) V having SEQ ID NO: 78 H V having the amino acid sequence SEQ ID NO: 79 L amino acid sequence, (t) V having SEQ ID NO: 80 H V having the amino acid sequence SEQ ID NO: 81 L an amino acid sequence, or (u) V having SEQ ID NO: 82 H V having the amino acid sequence SEQ ID NO: 83 L Amino acid sequence The isolated monoclonal antibody or antigen-binding fragment thereof, comprising: [This invention 1075] 1. An isolated bispecific antibody comprising a fragment of 1001, 1048, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, or 1074 and a second antigen-binding fragment having specificity for a molecule on an immune cell. [This invention 1076] 1075. A bispecific antibody of the invention, wherein said 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. [This invention 1077] The bispecific antibody of the present invention 1075, wherein each of said fragment and said second fragment is independently selected from a Fab fragment, a single chain variable fragment (scFv), or a single domain antibody. [This invention 1078] A bispecific antibody of the present invention 1075, further comprising an Fc fragment. [This invention 1079] A nucleic acid encoding any one of the antibodies 1001 to 1074 of the present invention. [The present invention 1080] A nucleic acid encoding any one of the bispecific antibodies of the present invention 1075 to 1078. [This invention 1081] A pharmaceutical composition comprising any one of the antibodies of the present invention 1001 to 1074 or a fragment thereof, and a pharmaceutically acceptable carrier or excipient. [This invention 1082] The pharmaceutical composition of invention 1081 further comprising at least one additional therapeutic agent. [This invention 1083] 1082. The pharmaceutical composition of claim 1082, wherein said therapeutic agent is a toxin, a radiolabel, an siRNA, a small molecule, or a cytokine. [This invention 1084] A pharmaceutical composition comprising any of the bispecific antibodies of the present invention 1075 to 1078 and a pharmaceutically acceptable carrier or excipient. [This invention 1085] The pharmaceutical composition of the present invention 1084 further comprising at least one additional therapeutic agent. [This invention 1086] 1085. The pharmaceutical composition of claim 1085, wherein said therapeutic agent is a toxin, a radiolabel, an siRNA, a small molecule, or a cytokine. [This invention 1087] An isolated cell comprising one or more polynucleotides encoding any one of the antibodies or fragments thereof of the present invention 1001 to 1074. [This invention 1088] An isolated cell comprising one or more polynucleotides encoding the bispecific antibody or a fragment thereof of any one of 1075 to 1078 of the present invention. [This invention 1089] A vector comprising the nucleic acid of the present invention 1079 or 1080. [The present invention 1090] A cell comprising a vector of the present invention. [This invention 1091] A kit comprising at least one antibody composition of the invention 1081 or 1084; a syringe, needle, or applicator for administering said at least one antibody to a subject; and instructions for use. [This invention 1092] A method for treating cancer in a subject, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of a composition comprising an antibody of any of 1001 to 1074 of the present invention, a bispecific antibody of any of 1075 to 1078 of the present invention, or a pharmaceutical composition of 1081 or 1084 of the present invention, or a composition of a CAR of any of 1095 to 1101 of the present invention. [This invention 1093] The method of claim 1092, further comprising administering to said subject a chemotherapeutic agent. [This invention 1094] The method of claim 1092, wherein the cancer is a checkpoint inhibition cancer. [This invention 1095] 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 antigen-binding fragment thereof that binds to 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 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), I-(X 8 X 9 X 10 X 11 )-G-(X 12 )-A (SEQ ID NO: 51), or II-(X 15 )-IFG-(X 16 )-A (SEQ ID NO: 207), and / or CDR3 comprising 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), 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). Including, the light chain S-(X 17 X 18 )I-(X 19 )-SNY (SEQ ID NO: 208) or NIG-(X 5 )-K-(X 20 ) (SEQ ID NO: 48), (X 21 )-DN (SEQ ID NO: 209), (X 22 )-NN (SEQ ID NO: 210), or DD-X 6 (SEQ ID NO: 49), and / or QSYDSNNRHVI (SEQ ID NO: 22), QVWDS-(X 7 )-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 chimeric antigen receptor (CAR). [This invention 1096] The CAR of the present invention 1095, wherein the transmembrane domain further comprises a stalk region disposed between the extracellular domain and the transmembrane domain. [This invention 1097] The CAR of the present invention, wherein the transmembrane domain comprises CD28. [This invention 1098] The CAR of the present invention 1095, further comprising one or more additional costimulatory molecules disposed between the transmembrane domain and the intracellular signaling domain. [This invention 1099] The CAR of the present invention, wherein the costimulatory molecule is CD28, 4-1BB, ICOS, or OX40. [The present invention 1100] The CAR of the present invention, wherein the intracellular signaling domain comprises a CD3 zeta chain. [The present invention 1101] The CAR of the present invention, wherein the antibody is a Fab or scFV. [The present invention 1102] A nucleic acid encoding any of the CARs of the present invention. [The present invention 1103] The nucleic acid of claim 1102, further comprising a nucleic acid encoding a polypeptide disposed after said intracellular signaling domain. [The present invention 1104] The nucleic acid of the present invention 1103, wherein the polypeptide is an antibody or a cytokine. [This invention 1105] 1104. The nucleic acid of the present invention, wherein the antibody is an scFV. [The present invention 1106] a nucleic acid encoding a CAR, the CAR comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain, further comprising a nucleic acid encoding a polypeptide disposed after the intracellular signaling domain, the polypeptide comprising an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) protein, the monoclonal antibody or fragment thereof comprising a heavy chain, a light chain, or a combination thereof; the 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), I-(X 8 X 9 X 10 X 11 )-G-(X 12 )-A (SEQ ID NO: 51), or II-(X15 )-IFG-(X 16 )-A (SEQ ID NO: 207), and / or CDR3 comprising 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), 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). Including, the light chain S-(X 17 X 18 )I-(X 19 )-SNY (SEQ ID NO: 208) or NIG-(X 5 )-K-(X 20 ) (SEQ ID NO: 48), (X 21 )-DN (SEQ ID NO: 209), (X 22 )-NN (SEQ ID NO: 210), or DD-X 6 (SEQ ID NO: 49), and / or QSYDSNNRHVI (SEQ ID NO: 22), QVWDS-(X 7 )-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, A nucleic acid encoding the CAR. [This invention 1107] A vector comprising any one of the nucleic acids of 1102 to 1106 of the present invention. [This invention 1108] A cell comprising the vector of the present invention 1107. [This invention 1109] A genetically engineered cell that expresses any one of the chimeric antigen receptors of the present inventions 1095 to 1101 and retains it on the cell surface membrane. [The present invention 1110] The genetically engineered cell of invention 1108 or 1109, which is a T cell or an NK cell. [The present invention 1111] The T cells are CD4 + or CD8 + The genetically engineered cell of the present invention 1110. [The present invention 1112] CD4 + and CD8 cells + The genetically engineered cells of the present invention, comprising a mixed population of: [The present invention 1113] A genetically engineered cell that expresses and retains on its cell surface membrane a chimeric antigen receptor, which is further engineered to express and secrete a polypeptide, wherein the polypeptide is an isolated monoclonal antibody or antigen-binding fragment thereof that binds to 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 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), I-(X 8 X 9 X 10 X 11 )-G-(X 12 )-A (SEQ ID NO: 51), or II-(X 15 )-IFG-(X 16 )-A (SEQ ID NO: 207), and / or CDR3 comprising 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), 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). Including, the light chain S-(X 17 X 18 )I-(X 19 )-SNY (SEQ ID NO: 208) or NIG-(X 5 )-K-(X 20 ) (SEQ ID NO: 48), (X 21 )-DN (SEQ ID NO: 209), (X 22 )-NN (SEQ ID NO: 210), or DD-X 6 (SEQ ID NO: 49), and / or QSYDSNNRHVI (SEQ ID NO: 22), QVWDS-(X 7 )-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 genetically engineered cells. Other objects and advantages of the present invention will become readily apparent from the description that follows. [Brief explanation of the drawings]

[0040] [Figure 1] 1 shows a schematic diagram of the bispecific GITR-PDL1 light chain fusion. [Figure 2-1] Figure 2 shows FACS plots and binding curves of PD-L1 antibodies. [Figure 2-2] See description of 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 description of Figure 3-1. [Figure 4] Schematic showing kinetic measurements of aPDL1 antibodies (top image). Based on a previous series of competitive matrices, a representative clone was used in the final matrix (bottom image). [Figure 5] 1 is a graph showing 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. ELISA plate development shows that some clones are comparable to ATEZ. [Figure 7] 1 is a bar graph showing the results of MLR using αPD-L1 antibody. [Figure 8] Figure 1 is a bar graph showing the results of an MLR using αPD-L1 antibody (150 nM). [Figure 9] Figure 1 is a bar graph showing the results of an MLR using αPD-L1 antibody (150 nM). [Figure 10-1] FIG. 10 is a schematic representation of the variable region heavy chain germline alignment (amino acid sequence). [Figure 10-2] See description of Figure 10-1. DETAILED DESCRIPTION OF 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 present invention can be embodied in various forms. Therefore, the specific details disclosed herein should not be construed as limiting, but as a basis for the claims and as a representative basis for teaching one skilled in the art how to use the present invention in any suitable manner.

[0042] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The use of the words "a" or "an" when used in conjunction with the word "comprising" in the claims and / or specification can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0043] Whenever any of the phrases "for example," "such as," "including," etc. are used herein, unless expressly stated otherwise, it is understood that the phrase "without limitation" is also involved. Similarly, "one example," "exemplary," etc. are understood to be non-limiting.

[0044] The term "substantially" permits deviations from the descriptor that do not adversely affect the intended purpose. It is understood that a descriptor is modified by the term "substantially" even if the word "substantially" is not explicitly recited.

[0045] Terms such as "comprising," "including," "having," and "involving" (and similarly, "comprises," "includes," "has," and "involves") are used interchangeably and have the same meaning. Specifically, each term is defined consistent with the general U.S. patent law definition of "comprising" and, therefore, is construed as an open term meaning "at least the following," and not excluding additional features, limitations, aspects, etc. Thus, for example, "a process comprising steps a, b, and c" means that the process includes 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 interpretation is meaningless in the context.

[0046] As used herein, the term "about" is used herein to mean approximately, roughly, around, or within a range. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 20 percent above or below (high or low).

[0047] PD-L1 Programmed T-cell death 1 (PD-1) is a transmembrane protein present on the surface of T cells that, when bound to programmed T-cell death-ligand 1 (PD-L1) on tumor cells, leads to the suppression of T-cell activity and a reduction in T-cell-mediated cytotoxicity. PD-1 and PD-L1 are thus immune downregulators or immune checkpoint "off switches."

[0048] The immune system must maintain a balance between effective responses to eliminate pathogens and maintaining tolerance to prevent autoimmune disease. T cells are central to maintaining this balance, and their proper regulation is primarily mediated by molecules of the B7-CD28 family. The interaction between B7 family members, which function as ligands, and CD28 family members, which function as receptors, not only provides important positive signals that initiate, enhance, and sustain T cell responses, but also contributes important negative signals that limit, terminate, and / or dampen T cell responses as needed. PD-1 is a member of the CD28 family.

[0049] Binding between PD-L1 and PD-1 has a profound effect on regulating T cell responses. Specifically, PD-L1 / PD-1 interaction inhibits T cell proliferation and the production of effector cytokines, such as IL-2 and IFN-γ, that mediate T cell activity and immune responses. This negative regulatory function is important 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 exhaustion, thereby inhibiting T cell responses and adversely affecting the host. Long-term or chronic antigen stimulation of T cells can induce a negative immunological feedback mechanism, inhibiting antigen-specific responses and potentially leading to immune evasion by pathogens. T cell exhaustion can also lead to the physical loss of antigen-specific T cells themselves. PD-1 expression on T cells is upregulated during chronic antigen stimulation, and its binding to PD-L1 blocks effector function in both CD4+ (T helper cells) and CD8+ (cytotoxic T lymphocytes or CTL) T cells, thus implicating PD-1 / PD-L1 interactions in the induction of T cell exhaustion.

[0050] Recent studies have demonstrated that some chronic viral infections and cancers have developed immune evasion tactics that specifically exploit the PD-1 / PD-L1 axis by inducing PD-1 / PD-L1-mediated T cell depletion. 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 antitumor immune responses. For example, during chronic HIV infection, HIV-specific CD8+ T cells are functionally impaired, including a reduced ability to produce cytokines and effector molecules and a reduced proliferation capacity. Studies have shown that PD-1 is highly expressed on HIV-specific CD8+ T cells in HIV-infected individuals, suggesting the therapeutic potential of blocking the PD-1 / PD-L1 pathway in the treatment of HIV infection and AIDS patients. Collectively, 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 PD-1 / PD-L1 interaction.

[0051] Overexpression of PD-L1 has been detected in various cancers. For example, overexpression of PD-L1 in breast cancer is associated with a high-risk prognosis. PD-L1 is upregulated in renal cell carcinoma, and increased expression of PD-1 is also observed on tumor-infiltrating leukocytes. Anti-PD-L1 and anti-PD-1 antibodies have shown some clinical efficacy in Phase I trials of renal cell carcinoma. Therapeutic agents capable of binding to PD-1 or PD-L1 may be useful for specifically targeting tumor cells. Agents capable of blocking the PD-1 / PD-L1 interaction may be further useful in treating cancers that have evaded antitumor T cell activity by inducing T cell exhaustion. The use of such agents alone or in combination with other anticancer therapeutics can effectively target tumor cells that overexpress PD-L1, enhancing antitumor T cell activity and thereby enhancing the immune response against targeted tumor cells.

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

[0053] An embodiment of the present invention provides an isolated monoclonal antibody specific for PDL-1. When used herein with reference to a cell, nucleic acid, such as DNA or RNA, the term "isolated" refers to a molecule separated from other DNA or RNA, respectively, present in the natural source of the macromolecule. The term "isolated" can also refer to a nucleic acid or peptide that is substantially free of 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, an "isolated nucleic acid" can include nucleic acid fragments that are not naturally occurring as fragments and would not be found in their natural state. "Isolated" can also refer to a cell or polypeptide that has been isolated from other cellular proteins or tissues. An isolated polypeptide can encompass both purified and recombinant polypeptides. The isolated antibodies were identified through the use of a 27 billion human single-chain antibody (scFv) phage display library using PDL-1 as the library selection target. These antibodies represent a new 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," with reference to a polypeptide (such as an antibody) or polynucleotide, may refer to a form of the polypeptide or polynucleotide that is not naturally occurring, a non-limiting example of which may be made by combining polynucleotides or polypeptides that are not normally found together.

[0055] The nucleic acid and amino acid sequences of the monoclonal PD-L1 antibodies are provided below; the amino acid sequences of the heavy and light chain complementarity determining regions (CDRs) of the PD-L1 antibodies are provided below. It is attached with TIFF0007795459000001.tif5155.

[0056] Table 1: Amino acid sequence of Ab 40mut variable region TIFF0007795459000002.tif50143

[0057] Table 2B: Ab 50-6B6.1 mut variable region amino acid sequence TIFF0007795459000003.tif56143

[0058] Table 3B: Ab 50-6B6.2 variable region amino acid sequence TIFF0007795459000004.tif56143

[0059] Table 4B. Ab 50-6B6.2 variable region amino acid sequence TIFF0007795459000005.tif56143

[0060] Table 5B: Ab 50-5B9 variable region amino acid sequences TIFF0007795459000006.tif56143

[0061] Table 6: Ab 14C61 variable region amino acid sequence TIFF0007795459000007.tif56143

[0062] Table 7. Ab 1A2 variable region amino acid sequence TIFF0007795459000008.tif56143

[0063] Table 8: Ab 1A3 variable region amino acid sequence TIFF0007795459000009.tif56143

[0064] Table 9: Ab 1A6 variable region amino acid sequence TIFF0007795459000010.tif56143

[0065] Table 10: Ab 1B4 variable region amino acid sequences TIFF0007795459000011.tif55143

[0066] Table 11. Ab 1C1 variable region amino acid sequences TIFF0007795459000012.tif56143

[0067] Table 12: Ab 1C4 variable region amino acid sequences TIFF0007795459000013.tif56143

[0068] Table 13: Ab 1C6 variable region amino acid sequences TIFF0007795459000014.tif56143

[0069] Table 14: Ab 1D1 variable region amino acid sequences TIFF0007795459000015.tif56143

[0070] Table 15. Ab 1D2 variable region amino acid sequences TIFF0007795459000016.tif56143

[0071] Table 16. Ab 1D4 variable region amino acid sequences TIFF0007795459000017.tif61143

[0072] Table 17. Ab 1E1 variable region amino acid sequences TIFF0007795459000018.tif56143

[0073] Table 18. Ab 1F1 variable region amino acid sequences TIFF0007795459000019.tif56143

[0074] Table 19. Ab 1G1 variable region amino acid sequences TIFF0007795459000020.tif56143

[0075] Table 20. Ab 1H2 variable region amino acid sequences TIFF0007795459000021.tif56143

[0076] Table 21. Ab 1H5 variable region amino acid sequences TIFF0007795459000022.tif56143

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

[0078] Table 6A. Heavy chain (V) of PDL-1 antibody H ) complementarity-determining regions (CDRs) TIFF0007795459000023.tif218134

[0079] Table 6B. Light chain (V) of PDL-1 antibody L ) complementarity-determining regions (CDRs) TIFF0007795459000024.tif218132

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

[0081] Table 7A. Heavy chain (V) of PDL-1 antibody H ) framework region (FR) TIFF0007795459000025.tif84156TIFF0007795459000026.tif232156TIFF0007795459000027.tif155156

[0082] Table 7B. Heavy chain (V) of PDL-1 antibody L ) framework region (FR) TIFF0007795459000028.tif32153TIFF0007795459000029.tif221153TIFF0007795459000030.tif209153

[0083] The PD-L1 antibodies described herein bind to PD-L1. In one embodiment, the PD-L1 antibodies have 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 antibodies described herein. For example, "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which may be aligned for comparison purposes. If a position in the compared sequences is 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 more amino acid sequence identity when compared to a particular region or the full length of any one of the anti-PD-L1 antibodies described herein. For example, an antibody may have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more nucleic acid sequence identity when compared to a particular region or the full length of any one of the anti-PD-L1 antibodies described herein. Sequence identity or similarity for the nucleic acids and proteins of the present invention can be determined by methods known in the art, such as sequence comparison and / or alignment using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. For example, sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection can be used to determine the percent sequence identity or similarity for the nucleic acids and proteins of the present invention.

[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 chain of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids can refer to a "polypeptide" herein, and the term "polypeptide" can be used in place of or interchangeably with any of these terms. "Polypeptide" can also refer to post-expression modified products of a polypeptide, such as, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology and need not necessarily be translated from a specific nucleic acid sequence. It can be generated 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 a nucleic acid, peptide, polypeptide, or protein sequence that alter, 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 alteration results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing 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 replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art as follows: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, the amino acid chain can be replaced with a structurally similar chain that differs in the order and / or composition of the side chain family members.

[0086] antibody As used herein, "antibody" or "antigen-binding polypeptide" can refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody, any antigen-binding fragment, or a single chain thereof. For example, an "antibody" can include any protein- or peptide-containing molecule containing at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Non-limiting examples include the complementarity-determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, 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" can refer to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts" means that the antibody reacts with one or more antigenic determinants of a desired antigen and not with other polypeptides.

[0087] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to an antibody fragment of F (ab’)2 , F (ab)2 , F ab ', F ab"Antibody fragment" refers to a portion of an antibody, such as a Fv, scFv, or the like. Regardless of structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" can encompass aptamers (such as spiegelmers), 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 to form a complex. 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, dAbs (domain antibodies), minibodies, disulfide-linked Fv (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies.

[0088] A "single-chain variable fragment" or "scFv" is a fragment of an immunoglobulin heavy chain (V H ) and light chain (V L (scFv) refers to a fusion protein of the variable regions of a single-chain Fv ("scFv") polypeptide molecule. A single-chain Fv ("scFv") polypeptide molecule is a covalently linked VH:VL heterodimer that can be expressed from a gene fusion comprising VH- and VL-encoding genes linked by a peptide-encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883.) In some embodiments, the regions are 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, where the V H N-terminus and V LThe C-terminus of the scFv molecule can be linked to the C-terminus of the antibody V region, 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. Many methods have been described for identifying chemical structures for converting the naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules that will fold into a three-dimensional structure substantially similar to that of an antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513, 5,892,019, 5,132,405, and 4,946,778, each of which is incorporated by reference in its entirety.

[0089] Very large naive human scFv libraries have been and can be generated to provide a large source of rearranged antibody genes against a large number of 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 in the nature of the heavy chains present in the molecule. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses within each (e.g., γ1-γ4). Certain classes also have subclasses, such as IgG1, IgG2, IgG3, and IgG4, as well as others. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, and IgG5, are well characterized and are known to confer functional specificity. For IgG, a typical 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 joined by disulfide bonds in a "Y" configuration, with the light chains surrounding the heavy chains, starting at the mouth of the "Y" and continuing through the variable region. The immunoglobulin or antibody molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

[0091] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can combine with either kappa or lambda light chains. Generally, light and heavy chains are covalently linked to each other, and when immunoglobulins are produced by either 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 the heavy chains, the amino acid sequence extends from the N-terminus at the forked end of the Y-shape to the C-terminus at the bottom of each chain.

[0092] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used in a functional sense. The variable domains (VL and VH) of both the light and heavy chains determine antigen recognition and specificity. Conversely, the constant domains (CL, and CH1, CH2, or CH3) of the light and heavy chains confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, and complement fixation. The term "antigen-binding site" or "binding portion" can refer to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues in the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent sections within the V regions of the heavy and light chains, called "hypervariable regions," are interspersed with more conserved adjacent sections known as "framework regions" or "FRs." Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions of immunoglobulins. In an antibody molecule, 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 to be bound, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions," or "CDRs." The VH and VL regions, including the CDRs and framework regions (FRs), of the PD-1 antibody are shown in Tables 1A-15B.

[0093] The six CDRs present in each antigen-binding domain are short, noncontiguous sequences of amino acids that are specifically arranged to form the antigen-binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids of the antigen-binding domain, the FR regions, show little inter-molecular variability. The framework regions primarily adopt a beta-sheet conformation, and the CDRs form loops that connect them and, in some cases, form part of the beta-sheet structure. The framework regions function to form a scaffold that orients the CDRs through inter-chain non-covalent interactions. The antigen-binding domain formed by the arranged CDRs provides a surface complementary to the epitope on the antigen in an immune response, promoting non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and framework regions, respectively, can be readily identified for heavy or light chain variable regions by one skilled in the art, as they have been previously identified (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., USDapartment of Health and Human Services, (1983), and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0094] When there are more than one definition for a term used and / or accepted in the art, the definition of the term used herein is intended to encompass all such meanings unless specifically and explicitly stated to the contrary. A specific example is the use of the term "complementarity-determining region" ("CDR") to describe the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. This particular region is described by Kabat et al., U.S. Department 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 entireties. The Kabat and Chothia definitions of CDRs include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, the application of either definition to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein. For comparison, the appropriate amino acid residues encompassing the CDRs defined by each of the above cited references are set forth in the following table. The exact residue numbers which comprise a particular CDR will vary depending on the sequence and size of the CDR, and one of skill in the art can routinely determine which residues make up a particular CDR given the variable region amino acid sequence of an antibody. TIFF0007795459000031.tif53128

[0095] Kabat et al. defined a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence, without relying on other experimental data from the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0096] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine ​​residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins 15 residues after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins approximately 33 amino acid residues after the end of CDR-H2, includes 3-25 amino acids, and ends with the sequence WGXG (where X is any amino acid). CDR-L1 begins approximately 24 (i.e., following the cysteine ​​residue), includes approximately 10-17 residues, and ends at the next tryptophan residue. CDR-L2 begins approximately 16 residues after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins approximately 33 residues after the end of CDR-L2 (i.e., following the cysteine ​​residue), includes 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" can include any protein determinant capable of specific binding to an immunoglobulin, scFv, or T-cell receptor. The variable region enables an antibody to selectively recognize and specifically bind to an epitope on an antigen. For example, the VL and VH domains of an antibody, or a subset of complementarity-determining regions (CDRs), combine to form the variable regions that define the three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end 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 typically have specific three-dimensional structural and charge characteristics. For example, antibodies can be raised against N- or C-terminal peptides of a polypeptide. More specifically, the antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) on each of the VH and VL chains. In one embodiment, the antibody comprises a PD-L1 comprising the amino acid sequence of SEQ ID NO: 204 (Genbank Accession No. NP_054862; 290 amino acid residues in length): TIFF0007795459000032.tif18140 can be the target.

[0098] As used herein, the terms "immunological binding" and "immunological binding properties" can refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity, of an immunological binding interaction is determined by the dissociation constant (K d ) and smaller K d represents a 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, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Therefore, both "on rate constants" (K on) and "off rate constant" (K off ) can be determined by calculation of the concentration and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). K off / K on The ratio of α to β cancels all parameters unrelated to affinity, and the dissociation constant K D (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473.) The antibodies of the present invention have an equilibrium binding constant (K) as measured by kinetic assays such as radioligand binding assays or similar assays known to those skilled in the art, such as BIAcore or Octet (BLI). D ) is ≦1 μM, ≦10 μM, ≦10 nM, ≦10 pM, or ≦100 pM to about 1 pM. For example, in some embodiments, D is between approximately 1E-12M and 1E-11M. D In some embodiments, K D is approximately between 1E-11M and 1E-10M. D In some embodiments, K D is between approximately 1E-10M and 1E-9M. D In some embodiments, K D is approximately between 1E-9M and 1E-8M. D In some embodiments, K D is between approximately 1E-8M and 1E-7M. D In some embodiments, K D is between approximately 1E-7M and 1E-6M. D For example, in some embodiments, K D is about 1E-12M, and in other embodiments, K D is about 1E-11M. In some embodiments, K D is about 1E-10 M, and in other embodiments, K D is about 1E-9M. In some embodiments, K D is about 1E-8M, and in other embodiments, KD is about 1E-7M. In some embodiments, K D is about 1E-6M, and in other embodiments, K D is about 1E-5M. In some embodiments, for example, K D is about 3E-11M, and in other embodiments, K D is about 3E-12M. In some embodiments, K D is approximately 6E-11M. "Specifically binds" or "having specificity for" 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 readily than it binds to a random, unrelated epitope.

[0099] For example, PD-L1 antibodies can be monovalent or bivalent and include single or two chains. Functionally, the binding affinity of PD-L1 antibodies can be greater than or equal to 10 -5 M~10 -12 For example, the binding affinity of PD-L1 antibodies is in the range of 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's M.

[0100] The PD-L1 proteins of the invention, or derivatives, fragments, analogs, homologs or orthologs thereof, can be used as immunogens in the generation of antibodies that immunologically specifically bind to these protein components, for example, amino acid residues comprising SEQ ID NO: 204. The PD-L1 proteins coupled to proteoliposomes, or derivatives, fragments, analogs, homologs or orthologs thereof, can be used as immunogens in the generation of antibodies that immunologically specifically bind to these protein components.

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

[0102] Another method for determining whether a human monoclonal antibody has the specificity of a human monoclonal antibody of the invention is to preincubate the human monoclonal antibody of the invention with the PD-L1 protein with which it is normally reactive, 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 a functionally equivalent, epitope specificity as the monoclonal antibody of the invention. Screening of the human monoclonal antibodies of the invention can also be carried out using PD-L1 to determine whether the monoclonal antibody to be tested can neutralize PD-L1.

[0103] Various procedures known in the art can be used for the production of polyclonal or monoclonal antibodies directed against the proteins of the invention or against their derivatives, fragments, analogs, homologs, or orthologs (see, e.g., 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 immune serum. Subsequently, or alternatively, the specific antigen that is the target of the desired immunoglobulin, or an epitope thereof, can be immobilized on a column, and immune-specific antibodies can be purified by immunoaffinity chromatography. Immunoglobulin purification 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 term "monoclonal antibody" or "mAb" or "Mab" or "monoclonal antibody composition" can refer to a population of antibody molecules containing only one molecular species of antibody molecule, consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity-determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. MAbs contain an antigen-binding site capable of immunoreacting with an epitope of an antigen characterized by a unique binding affinity for it.

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

[0107] The immunizing agent can include a protein antigen, a fragment thereof, or a fusion protein thereof. For example, if cells of human origin are desired, peripheral blood lymphocytes can be used, or if a non-human mammalian source is desired, spleen cells or lymph node cells can be used. The lymphocytes are then fused with an immortalized cell line using a suitable fusing 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 can be transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. For example, rat or mouse myeloma cell lines can be used. The hybridoma cells can be cultured in a suitable medium containing one or more substances that inhibit the growth or survival of unfused, immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent growth of HGPRT-deficient cells.

[0108] Useful immortalized cell lines are those that fuse efficiently, maintain stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Immortalized cell lines are, for example, 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 heteromyeloma 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] The culture medium in which hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies against the antigen.For example, the binding specificity of the monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).Such techniques and assays are known in the art.The binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson and Pollard, Anal.Biochem.,107:220(1980).In addition, in the therapeutic use of monoclonal antibodies, it is important to identify antibodies that have high specificity and high binding affinity for target antigens.

[0110] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by 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's medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0111] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite 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 (incorporated herein by reference in its entirety). DNA encoding the monoclonal antibodies of the present invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). The hybridoma cells of the present invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into 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 protein, to obtain the synthesis of the monoclonal antibody in the recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (see U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)), or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or for the variable domains of one antigen-binding site of an antibody of the invention to create a chimeric bivalent antibody.

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

[0114] A "humanized antibody" can be an antibody derived from a non-human species (such as a mouse) whose light and heavy chain protein sequences have been modified to enhance similarity to antibody variants produced in humans. Humanized antibodies are antibody molecules derived from a non-human species antibody that binds to a desired antigen, having one or more complementarity-determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions 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, such as by modeling the interactions between CDRs and framework residues to identify framework residues important for antigen binding and comparing sequences to identify unusual framework residues at specific positions. (See, e.g., Queen et al., US Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), both of which are incorporated herein by reference in their entireties.) For example, the non-human portions of the antibody (such as the CDRs of the light and / or heavy chains) can bind to the target antigen.

[0115] Antibodies can be humanized using various techniques known in the art, such as, for example, CDR grafting (EP 239,400, PCT Publication WO 91 / 09967, U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106, EP 519,596, 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. Pat. No. 5,565,332, incorporated by reference in its entirety). "Humanization" (also called reshaping or CDR grafting) is an established technique well known to those skilled in the art for reducing the immunogenicity of monoclonal antibodies (mAbs) derived from heterologous sources (usually rodents) and improving their activation by the human immune system (see, e.g., 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 grafting. See also Safdari et al. (2013) Biotechnol Genet Eng Rev.; 29:175-86. Furthermore, humanized antibodies can be produced in transgenic plants, providing an inexpensive alternative to existing mammalian production systems. For example, the transgenic plant can be a tobacco plant, i.e., Nicotiania benthamiana, and Nicotiana tabaccum. The antibody is purified from the leaves of the plant.Stable transformation of plants can be achieved using Agrobacterium tumefaciens or particle bombardment. For example, a nucleic acid expression vector containing at least the heavy and light chain sequences can be expressed via transformation in bacterial culture, i.e., A. tumefaciens strain BLA4404. Infiltration of plants can be achieved by injection. Soluble leaf extracts can be prepared by crushing leaf tissue in a mortar and centrifugation. Isolation and purification of antibodies can be easily carried out by many methods known to those skilled in the art. Other methods for 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. Thus, the present invention further provides any cell or plant containing a vector encoding or producing an antibody of the present invention.

[0116] Human monoclonal antibodies, including 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), EBV hybridoma technology 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 are available and can be produced by using human hybridomas (see Cote, et al, 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (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 made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. After challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, as well as 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 65-93 (1995).

[0118] Human antibodies can also be produced using transgenic non-human animals that are modified to produce fully human antibodies in response to antigen challenge rather than the animal's endogenous antibodies. (See PCT Publication No. WO 94 / 02602 and U.S. Patent No. 6,673,986.) The endogenous genes encoding heavy and light immunoglobulin chains in the non-human host are disabled, and active loci encoding human heavy and light immunoglobulin chains are inserted into the host's genome. Human genes are incorporated, for example, using yeast artificial chromosomes containing the necessary human DNA segments. Animals that provide all the desired modifications are then obtained as progeny by breeding intermediate transgenic animals containing less than the full complement of modifications. A non-limiting example of such a non-human animal is a mouse, referred to as the Xenomouse™, as disclosed in PCT Publication Nos. WO 96 / 33735 and WO 96 / 34096. The animal produces B cells that secrete fully human immunoglobulins. Antibodies can be obtained directly from animals after immunization with an immunogen of interest, e.g., as polyclonal antibody preparations, or alternatively, from immortalized B cells derived from animals, such as hybridomas, that produce monoclonal antibodies. In addition, genes encoding immunoglobulins with human variable regions can be recovered and expressed to obtain antibodies directly or further modified to obtain antibody analogs, such as, for example, single-chain Fv (scFv) molecules.

[0119] Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology 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, e.g., PCT Publication Nos. WO 98 / 24893, WO 96 / 34096, 96 / 33735, U.S. Patent Nos. 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 by reference in their entireties. Additionally, companies such as Creative BioLabs (Shirley, NY) offer services to provide human antibodies directed against a selected antigen using technology similar to that 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 rearrangement of the locus and the formation of transcripts of the rearranged immunoglobulin heavy chain locus, the deletion being carried out by a targeting vector containing a gene encoding a selectable marker, and producing a transgenic mouse from the embryonic stem cells, the somatic and germ cells of which contain the gene encoding the 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 the heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding the light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses the antibody containing the heavy and light chains.

[0122] In a further improvement of this procedure, methods for identifying clinically relevant epitopes on immunogens and corresponding methods for selecting antibodies that immunospecifically bind with high affinity to the relevant epitopes are disclosed in PCT Publication No. WO 99 / 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 can include liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, etc. Vectors can include chemical conjugates such as those described in WO93 / 64701, which have 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 are fusion proteins containing a targeting moiety (e.g., an antibody specific to a target cell) and a nucleic acid binding moiety (e.g., protamine), plasmids, phages, viral vectors, etc. Vectors can be chromosomal, non-chromosomal, or synthetic. Retroviral vectors can also be used, including Moloney murine leukemia virus.

[0125] DNA viral vectors may also be used, including pox vectors such as orthopox or avipox vectors, and herpes virus vectors such as herpes simplex virus type I (HSV) vectors (Geller, AI 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, AI et al, Proc Natl. Acad. Sci.: USA 90:7603 (1993); Geller, AI, et al, Proc Natl. Acad. Sci USA 87:1149 (1990)), Adenovirus Vectors (see 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 neural cells. Adenovirus vectors result in shorter-term expression (approximately 2 months) than adeno-associated virus (approximately 4 months), which in turn results in shorter expression than HSV vectors. The specific vector selected 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 viral vectors.

[0127] Vectors can be used to target essentially any desired target cell. For example, stereotactic injection can be used to guide vectors (e.g., adenovirus, HSV) to the desired location. In addition, particles can be delivered by intracerebroventricular (icv) injection using a minipump infusion system such as the SynchroMed Infusion System. A method based on bulk flow, called convection, has also proven effective in delivering large molecules to extended areas 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 administration routes.

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

[0129] Techniques can be adapted to produce single chain antibodies specific to antigenic proteins of the invention (see, e.g., U.S. Pat. No. 4,946,778). In addition, methods can be used to ab Monoclonal Fs with desired specificity for the protein, or derivatives, fragments, analogs, or homologs thereof, are suitable for constructing expression libraries (see, e.g., Huse, et al., 1989 Science 246:1275-1281). abAntibody fragments containing the idiotype to a protein antigen can be produced by techniques known in the art, including, but not limited to, (i) F produced by pepsin digestion of antibody molecules. (ab’)2 Fragment, (ii)F (ab’)2 F generated by reducing the disulfide bridges of the fragment ab (iii) F fragments produced by treating antibody molecules with papain and a reducing agent. ab fragments, and (iv) F v Contains fragments.

[0130] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies, for example, allow immune system cells to target unwanted cells (see U.S. Pat. No. 4,676,980) and allow the treatment of HIV infection (see PCT Publication Nos. WO91 / 00360 and WO92 / 20373). It is contemplated that antibodies can be prepared in vitro using known methods in the field of protein synthetic chemistry, such as using cross-linking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of reagents suitable for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate, and those disclosed, for example, in U.S. Pat. No. 4,676,980.

[0131] The antibodies of the present invention can be modified with respect to effector function, for example, to enhance the effectiveness of the antibody in treating cancer. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, antibodies can be engineered with dual Fc regions, thereby enhancing complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)).

[0132] In certain embodiments, antibodies of the present invention can include Fc variants containing amino acid substitutions that alter the antigen-independent effector function of the antibody, particularly its circulating half-life. Such antibodies exhibit either increased or decreased binding to FcRn and therefore have increased or decreased serum half-lives, respectively, when compared with antibodies lacking these substitutions. Fc variants with improved affinity for FcRn are expected to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where a long half-life of the administered antibody is desired, for example, to treat chronic diseases or disorders. In contrast, Fc variants with reduced FcRn binding affinity are expected to have shorter half-lives, and such molecules are also useful, for example, for administration to mammals where a shortened circulation time would be advantageous, for example, for in vivo diagnostic imaging, or in situations where the starting antibody has toxic side effects if present in the circulation for an extended period of time. Fc variants with reduced FcRn-binding affinity are also less likely to cross the placenta and are therefore useful in treating diseases or disorders in pregnant women. Additionally, other applications in which reduced FcRn-binding affinity may be desirable include applications in which localization to the brain, kidney, and / or liver is desirable. In one embodiment, an Fc variant-containing antibody may exhibit reduced transport from the vasculature across the epithelium of renal glomeruli. 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 altered FcRn binding comprises an Fc domain with one or more amino acid substitutions within the "FcRn-binding loop" of the Fc domain. The FcRn-binding loop is composed of amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions that alter FcRn-binding activity are disclosed in PCT Publication No. WO 05 / 047327, incorporated herein by reference. In certain exemplary embodiments, an antibody of the invention, or a fragment thereof, comprises an Fc domain with 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 the mAb to alter the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the mAb. For example, the mutation is a LALA mutation in the CH2 domain. In one embodiment, an antibody (e.g., a human mAb or a bispecific Ab) contains a mutation on one scFv unit of the heterodimeric mAb that reduces ADCC activity. In another embodiment, the mAb contains mutations on both chains of the heterodimeric mAb that completely eliminate ADCC activity. For example, the mutation introduced into one or both scFv units of the mAb is a 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 minimal 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 an IgG1 or IgG4 heavy chain constant region, that is altered to reduce or eliminate glycosylation. For example, antibodies of the present invention can also include Fc variants containing amino acid substitutions that alter the glycosylation of the antibody. For example, Fc variants can have reduced glycosylation (e.g., N-linked or O-linked glycosylation). In some embodiments, the Fc variant has reduced glycosylation of the N-linked glycan normally found at amino acid position 297 (EU numbering). In another embodiment, the antibody has an amino acid substitution near or within a glycosylation motif, such as an N-linked glycosylation motif comprising the amino acid sequence NXT or NXS. In a specific embodiment, the antibody comprises an Fc variant with an amino acid substitution at amino acid position 228 or 299 (EU numbering). In a more specific embodiment, the antibody comprises an IgG1 or IgG4 constant region comprising S228P and T299A mutations (EU numbering).

[0135] Exemplary amino acid substitutions that reduce or alter glycosylation are disclosed in PCT Publication No. WO 05 / 018572, which is incorporated herein by reference. In some embodiments, antibodies of the invention, or fragments thereof, are modified to eliminate glycosylation. Such antibodies, or fragments thereof, may be referred to as "agly" antibodies, or fragments thereof (e.g., "agly" antibodies). Without being bound by theory, "agly" antibodies, or fragments thereof, may have an improved safety and stability profile in vivo. An exemplary agly antibody, or fragment thereof, comprises a deglycosylated Fc region of an IgG4 antibody that lacks Fc effector function, thereby eliminating the potential for Fc-mediated toxicity to normal living tissues and cells that express PD-L1. In yet other embodiments, antibodies of the invention, or fragments thereof, comprise an altered glycan. For example, the antibody may have a reduced number of fucose residues on the N-glycan at Asn297 of the Fc region, i.e., be defucosylated. In another embodiment, the antibody can have an altered number of sialic acid residues on the N-glycan at Asn297 of the Fc region.

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

[0137] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, soapwort inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes. A variety of 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 an example.

[0138] Conjugates of antibodies and cytotoxic agents are prepared using various bifunctional protein binding agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as triene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin 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 conjugating radionucleotides to antibodies (see PCT Publication No. WO 94 / 11026 and U.S. Patent No. 5,736,137).

[0139] Those skilled in the art will recognize that a wide variety of possible moieties can be attached to a given antibody or other molecule of the invention (see, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0140] Conjugation can be achieved by any chemical reaction that will link two molecules, so long as the antibody and other moiety retain their respective activities. This conjugation can involve many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordinate bonding, and complex formation. In one embodiment, the conjugation is a covalent bond. Covalent bonding can be achieved by direct condensation of existing side chains or by incorporating an external crosslinking molecule. Many bivalent or multivalent linking agents are useful for linking protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative linking agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various classes of linking agents known in the art, but rather is illustrative of more common linking 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. (See, for example, 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. Pat. No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives linked to antibodies by oligopeptide linkers.Non-limiting examples of useful linkers that can be used with the antibodies of the invention include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride), (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G), (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat. No. 21651G), (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propionamido]hexanoate (Pierce Chem. Co., Cat. No. 2165-G), and (v) sulfo-NHS (-hydroxysulfo-succinimide) conjugated to EDC (Pierce Chem. Co., Cat. No. 2165-G). Chem. Co., catalog number 24510).

[0141] The linkers described herein contain components with different attributes, thereby resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, the linker SMPT contains a sterically hindered disulfide bond, allowing for the formation of 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 carbodiimide bonds. Carbodiimide bonds (such as EDC) when used in combination with sulfo-NHS form esters that are more resistant to hydrolysis than the carbodiimide bond reaction alone.

[0142] The antibody disclosed herein can also be formulated as immunoliposome.The liposome containing antibody is prepared by the method known in the art, such as 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. Patent No. 4,485,045 and U.S. Patent No. 4,544,545.Liposome with extended circulation time is disclosed in U.S. Patent No. 5,013,556.

[0143] Non-limiting examples of useful liposomes can be generated by reverse-phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to generate liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to 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 that can recognize two or more different antigens. For example, a bispecific antibody (bsAb) is an antibody that contains two variable domains or scFv units, such that the resulting antibody recognizes two different antigens. For example, a trispecific antibody (tsAb) is an antibody that contains two variable domains or scFv units, such that the resulting antibody recognizes three different antigens. The present invention provides multispecific antibodies, such as bispecific antibodies that recognize PD-L1 and a second antigen. For example, PD-L1 is both an immune checkpoint molecule and a tumor antigen. As a tumor antigen targeting molecule, an antibody or antigen-binding fragment specific for PD-L1 can be combined with a second antigen-binding fragment specific for an immune cell to generate a bispecific antibody. In some embodiments, the immune cell is 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 (KIR), 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 invention comprise the heavy and light chain combination of a PD-L1 antibody, or an scFv, disclosed herein.

[0145] For example, the nucleic acid and amino acid sequences of bispecific PD-L1 antibodies (such as GITR-PD-1L-fusions) are provided below, along with exemplary constant regions that are useful in combination with the VH and VL sequences provided herein.

[0146] (Table 11A) Ab#E1-3H7 variable region nucleic acid sequence TIFF0007795459000033.tif82143

[0147] Table 11B: Ab#E1-3H7 variable region amino acid sequence TIFF0007795459000034.tif39143

[0148] Table 12A. Ab#E1-3H7 constant region nucleic acid sequence - wild-type IgG1 monomer TIFF0007795459000035.tif169143

[0149] Table 12B: Ab#E1-3H7 constant region amino acid sequence - wild-type IgG1 monomer TIFF0007795459000036.tif17170TIFF0007795459000037.tif83143

[0150] Table 13A: Ab#E1-3H7 constant region nucleic acid sequence - IgG1 LALA-aPDL-1 40mut TIFF0007795459000038.tif164143

[0151] Table 13B: Ab#E1-3H7 constant region amino acid sequence - IgG1 LALA-aPDL-1 40mut TIFF0007795459000039.tif94143

[0152] Table 14A: Ab#E1-3H7 constant region nucleic acid sequence - IgG1 LALA-aPD-L1 50-6B6.1 mut TIFF0007795459000040.tif175143

[0153] Table 14B: Ab#E1-3H7 constant region amino acid sequence - IgG1 LALA-aPD-L1 50-6B6.1 mut TIFF0007795459000041.tif93143

[0154] Table 15A: Ab#E1-3H7 constant region nucleic acid sequence - IgG1 LALA-aPD-L1 50-6B6.2 TIFF0007795459000042.tif175143

[0155] Table 15B: Ab#E1-3H7 constant region amino acid sequence - IgG1 LALA-aPD-L1 50-6B6.2 TIFF0007795459000043.tif94143

[0156] Table 16A: Ab#E1-3H7 constant region nucleic acid sequence - IgG1 LALA-aPD-L1 50-7B3 TIFF0007795459000044.tif180143

[0157] Table 16B: Ab#E1-3H7 constant region amino acid sequence - IgG1 LALA-aPD-L1 50-7B3 TIFF0007795459000045.tif99143

[0158] (Table 17A) Ab#E1-3H7 constant region nucleic acid sequence - IgG1 LALA-aPD-L1 50-5B9 TIFF0007795459000046.tif175143

[0159] Table 17B: Ab#E1-3H7 constant region amino acid sequence - IgG1 LALA-aPD-L1 50-5B9 TIFF0007795459000047.tif94143

[0160] Multispecific antibodies (e.g., bispecific and trispecific antibodies) of the present invention can be constructed using methods known in the art. In some embodiments, bispecific antibodies are single polypeptides in which two scFv fragments are joined by a long linker polypeptide of sufficient length to allow intramolecular association between the two scFv units to form the antibody. In other embodiments, bispecific antibodies are two or more polypeptides linked by covalent or non-covalent bonds. In some embodiments, the amino acid linker (GGGGSGGGGS; "(G4S)2") that can be used with the scFv fusion constructs described herein can be generated with a longer G4S linker to improve flexibility. For example, the linker can be "(G4S)3" (e.g., GGGGSGGGGSGGGGGS), "(G4S)4" (e.g., GGGGSGGGGSGGGGSGGGGS), "(G4S)5" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGGS), "(G4S)6" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS), "(G4S)7" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGGS), etc. For example, the use of a (G4S)5 linker can 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 where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Non-limiting examples of linkers known to those of skill in the art that can be used are described in U.S. Pat. No. 9,708,412, U.S. Patent Application Publication Nos. 2018 / 0134789 and 2020 / 0148771, and PCT Publication No. WO2019 / 051122, the entire contents of each of which are incorporated by reference.

[0161] In another embodiment, multispecific antibodies (e.g., bispecific and trispecific antibodies) can be constructed using the "knobs-into-holes" 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 heavy chain pairing while preserving the heavy-light chain pairing. Two heavy-light chain heterodimers that recognize two different antigens are mixed to promote heteroligation pairing mediated through engineered "knobs-into-holes" in the CH3 domains.

[0162] In another embodiment, multispecific antibodies (e.g., bispecific and trispecific antibodies) can be constructed by exchanging heavy-light chain dimers from two or more different antibodies to generate a hybrid antibody, 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 generate a hybrid antibody, 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 similar 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 domains of each heavy chain with disulfide bridges. The presence of a specific amino acid in the CH3 domain (R409) has been shown to promote dimer exchange and the assembly of IgG4 molecules. Heavy chain pairing is also further stabilized by inter-heavy chain disulfide bridges in the antibody hinge region. 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, which contains the sequence Cys-Pro-Pro-Cys). This difference in the sequence of serine at position 229 is associated with the tendency of IgG4 to form intrachain disulfides in the hinge region (Van der Neut Kolfschoten, M. et al., 2007, Science 317:1554-1557 and Labrijn, A. F. et al., 2011, Journal of Immunol 187:3238-3246).

[0163] Thus, bispecific antibodies of the present invention can be generated through the introduction of the R409 residue in the CH3 domain and a Cys-Pro-Ser-Cys sequence in the hinge region of an antibody that recognizes PD-L1 or a second antigen, such that the heavy-light chain dimers are swapped to produce an antibody molecule having one heavy-light chain dimer that recognizes PD-L1 and a second heavy-light chain dimer that recognizes a second antigen, where the second antigen is any antigen disclosed herein. Known IgG4 molecules can also be engineered such that the heavy and light chains recognize PD-L1 or a second antigen, as disclosed herein. The use of this method to construct bispecific antibodies of the present invention can be advantageous due to the unique characteristics of IgG4 molecules, whose Fc region differs from other IgG subtypes in that it interacts poorly 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 must bind to a target and functionally alter target-associated signaling pathways, but does not induce effector activity.

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

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

[0166] Use of antibodies against PD-L1 Antibodies of the invention that specifically bind to the PD-L1 protein or a fragment thereof can be administered to treat a PD-L1-associated disease or disorder. A "PD-L1-associated disease or disorder" includes disease states and / or symptoms associated with disease states in which elevated levels of PD-L1 and / or activation of cell signaling pathways involving PD-L1 are observed. Exemplary PD-L1-associated diseases or disorders include, but are not limited to, cancer and autoimmune diseases.

[0167] The antibodies of the present invention, including 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, improve vaccine efficacy, or enhance natural immune responses. Antibody preparations, e.g., those with high specificity and high affinity for their target antigen, are administered to a subject and generally will produce an effect resulting from binding to the target. Administration of the antibody can neutralize, inhibit, or interfere with the activity of the PD-L1 protein.

[0168] Antibodies of the present invention that specifically bind to the PD-L1 protein or a fragment thereof can be administered for the treatment of cancer in the form of pharmaceutical compositions. Principles and considerations involved in the preparation of therapeutic pharmaceutical compositions containing antibodies, as well as guidance in the selection of ingredients, 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 regimen for a particular patient depends on various factors, such as the specific antibody, its variant or derivative used, the patient's age, weight, general health, sex, and diet, as well as the administration time, excretion frequency, concomitant use of drugs, and the severity of the specific disease being treated.The judgment of such factors by medical professionals is within the skill of those skilled in the art.The amount will also depend on the individual patient being treated, the administration route, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect.The amount used can be determined by pharmaceutical and pharmacokinetic principles well known in the art.

[0170] A therapeutically effective amount of an antibody of the present invention can be the amount necessary to achieve a therapeutic goal. As described above, the therapeutic goal can be the binding interaction between the antibody and its target antigen, interfering with target function in certain cases. The amount required to be administered further depends on the binding affinity of the antibody for its specific antigen and also on the rate at which the administered antibody is depleted from the free volume of the other subject to which it is administered. The dosage of an antigen-binding polypeptide described herein administered to a subject (e.g., a patient) is typically 0.1 mg / kg to 100 mg / kg of patient body weight, 0.1 mg / kg to 20 mg / kg of patient body weight, or 1 mg / kg to 10 mg / kg of 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 foreign polypeptides. Therefore, lower dosages and less frequent administration of human antibodies are often possible. Furthermore, the dosage and frequency of administration of the antibodies of the present disclosure can be reduced by enhancing antibody uptake and tissue (e.g., brain) penetration through modifications such as lipidation. A typical range for therapeutically effective administration of an antibody or antibody fragment of the invention can be, by way of non-limiting example, about 0.1 mg / kg body weight to about 50 mg / kg body weight, and a typical administration frequency can range, for example, from twice daily to once weekly.

[0171] When an antibody fragment is used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the variable region sequence of the antibody. 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 can also contain two or more active compounds necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can contain an agent that enhances its function, such as, for example, a cytotoxic agent, a cytokine (e.g., IL-15), a chemotherapeutic agent, or a growth inhibitor. Such molecules are suitably 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 by interfacial polymerization, e.g., 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] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0174] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable the release of molecules for more than 100 days, while certain hydrogels release proteins for shorter periods of time.

[0175] The antibodies of the present invention can be used as agents for detecting the presence of PD-L1 (or a protein fragment thereof) in a sample. For example, the antibody can comprise a detectable label. The antibody can be polyclonal or monoclonal. The intact antibody, or a fragment thereof (e.g., F ab , scFv, or F (ab)2) can be used. With respect to probes or antibodies, the term "labeled" can encompass direct labeling of the probe or antibody by conjugating (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 detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling 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 a subject. Thus, the use of the term "biological sample" includes 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 analyte mRNA, protein, or genomic DNA in biological samples in vitro and in vivo. For example, in vitro techniques for detecting analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations.

[0176] Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, J.R.Crowther (Ed.) Human Press, Totowa, NJ, 1995, "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996, and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Additionally, in vivo techniques for detecting an analyte protein include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

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

[0178] Antibodies specific for the PD-L1 protein of the invention can be used to isolate PD-L1 polypeptides by standard techniques, such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies to the PD-L1 protein (or fragments thereof) can be used diagnostically to monitor protein levels in tissues as part of a clinical testing procedure, for example to determine the effectiveness of a given therapeutic regimen.

[0179] Detection can be facilitated by conjugating (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. Examples of luminescent materials include luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include 125 I, 131 I, 35 S, 32 P or 3 H is one example.

[0180] The antibodies or agents of the present invention (also referred to herein as "active compounds"), as well as their derivatives, fragments, analogs, and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody or agent and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles, such as liposomes and fixed oils, can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0181] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of administration routes 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: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate, or phosphate, and an agent for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0182] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous 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 to the extent that easy syringability exists. It can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of 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, thimerosal, etc. In many cases, it will be preferable to include isotonic agents in the composition, such as sugars, polyalcohols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0183] Sterile injectable solution can be prepared by incorporating the required amount of active compound into the appropriate solvent with one or combination of the ingredients listed above as needed, and then sterilize by filtration.For example, dispersion is prepared by incorporating active compound into the sterile vehicle that contains the basic dispersion solvent and the other ingredients listed above.For the preparation of sterile injectable solution, the method of preparation is vacuum drying and freeze-drying, which produces powder of active compound and any additional desired ingredients from the solution that has been previously sterile filtered.

[0184] Oral compositions include inert diluents or edible carriers. They can be enclosed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using fluid carriers for use as mouthwashes, in which the compound in the fluid carrier is orally applied, swirled in the mouth, and expectorated or swallowed. Pharmaceutically compatible binders and / or adjuvants can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, tragacanth gum, or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, primogel, or corn starch; a lubricant such as magnesium stearate or sterols; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavor.

[0185] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0186] Systemic administration can also be by transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and for example, for transmucosal administration, include detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be achieved through the use of nasal sprays or suppositories.For transdermal administration, active compounds are formulated into ointments, salves, gels, or creams generally known in the art.

[0187] The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0188] In one embodiment, the active compound is prepared with a carrier that will protect the compound against rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells 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. Pat. No. 4,522,811.

[0189] For the sake of ease of administration and uniformity of dosage, oral or parenteral compositions can be formulated in dosage unit form.Dosage unit form as used herein refers to a physically separate unit that is suitable as a single dose for the subject to be treated, and each unit contains a predetermined amount of active compound that is calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification of dosage unit form of the present invention is determined by and directly depends on the specific characteristics of active compound and the specific therapeutic effect that should be achieved, and the inherent limitation of the technology that formulates this active compound for individual treatment.

[0190] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0191] Treatment method As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow (alleviate) an undesirable physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in extent of disease, stable (i.e., not worsening) state of disease, slowing or delaying of disease progression, improvement or palliation of disease state, remission (partial or total), whether detectable or not. "Treatment" refers to prolonging survival as compared to expected survival in the absence of treatment. Those in need of treatment include those already suffering from the condition or disorder, as well as those susceptible to the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0192] The present invention provides both prophylactic and therapeutic methods for treating subjects at risk of (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 aberrant expression of PD-L1. For example, the method is used to treat, prevent, or alleviate the symptoms of cancer. In one embodiment, the method is used to treat, prevent, or alleviate the symptoms of solid tumors. Non-limiting examples of other tumors that can be treated by embodiments herein include lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, or gastric cancer. Additionally, the method of the present invention can be used to treat blood 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 aspect, the invention provides a method for preventing, treating, or alleviating symptomatic cancer or cell proliferative disease or disorder in a patient by administering to the patient a monoclonal antibody, scFv antibody, or bispecific antibody of the invention. For example, an anti-PD-L1 antibody can be administered in a therapeutically effective amount.

[0194] Subjects at risk for cancer or cell proliferation-related diseases or disorders may include patients with a family history of cancer or subjects who have been exposed to substances known or suspected to cause cancer. Administration of a prophylactic agent can occur prior to the onset of cancer, such that the disease is prevented or, alternatively, its progression is delayed.

[0195] In another embodiment, tumor cell growth is inhibited by contacting the cell with an anti-PD-L1 antibody of the invention. The cell can be any cell that expresses 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 treatment of subjects at risk of a disease, disorder, or condition associated with T cell depletion, or at risk of developing T cell depletion. The present invention also provides therapeutic methods for both prophylactic and therapeutic treatment of subjects at risk of a disease, disorder, or condition associated with T cell depletion, or at risk of developing T cell depletion. 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] The present invention also encompasses methods for increasing or enhancing an immune response to an antigen. The immune response is increased or enhanced by administering a monoclonal antibody, scFv antibody, or bispecific antibody of the present invention to a subject. The immune response is enhanced, for example, by enhancing antigen-specific T effector function. The antigen is a viral (e.g., HIV), bacterial, parasitic, or tumor antigen. The immune response is a natural immune response. A natural immune response refers to an immune response resulting from an infectious disease. The infectious disease is a chronic infectious disease. 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 transcriptional profile. Alternatively, the immune response is a response induced by vaccination.

[0198] Thus, in another aspect, the present invention provides a method for increasing vaccine efficacy by administering to a subject a monoclonal antibody or scFv antibody of the present invention and a vaccine, wherein the antibody and vaccine are administered sequentially or simultaneously. The vaccine may be a tumor 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. Chemotherapeutic agents that can be administered with the compositions described herein include antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, and sulfuric acid). vincristine); hormones (e.g., medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives (e.g., mephalen, colambucil, mechlorethamine (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 invention described herein can be administered in combination with cytokines, including but 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 can be administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

[0202] In some embodiments, the compositions described herein can be administered in combination with other immunotherapeutic agents, including, but not limited to, simtuzumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, sitatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, and durizumab. Gotumab, ducizitumab, detumomab, decetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, fizitumumab, framvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imagatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab , iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minletumomab, mitumomab, moxetumomab, narunatumumab, naptumomab, necitumumab, nimotuzumab, nofetumomab, ocaratuzumab, ofatumumab, olaratuzumab, onartuzumab, oportuzumab, oregovomab, panitumumab, palsatuzumab, patritumab, pemtumomab, These include pertuzumab, pintumomab, pritumumab, racotumomab, radletuzumab, rilotumumab, rituximab, lobatumumab, satumomab, sibrotuzumab, siltuximab, solitomab, tacatuzumab, taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab, borsetuzumab, votumumab, zalutumumab, 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 on the PD-L1 protein, or alternatively, two different epitopes on 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 another embodiment, cancer can be treated by administering a bispecific antibody that binds to PD-L1 and also to a protein other than PD-L1. For example, the protein other than PD-L1 includes, but is not limited to, GITR. For example, the protein other than PD-L1 can be a tumor-associated antigen, or the protein other than PD-L1 can be a cytokine.

[0204] In some embodiments, the invention provides for the administration of anti-PD-L1 antibodies, alone or in combination with an additional antibody that recognizes another protein other than PD-L1, together with cells capable of achieving or enhancing an immune response. For example, these cells can 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] Additionally, the present invention provides for the administration of antibodies that bind to PD-L1 protein and other therapeutic agents, including anti-neoplastic agents, such as small molecules, growth factors, cytokines, or biomolecules, such as peptides, peptidomimetics, peptoids, polynucleotides, lipid-derived mediators, small biogenic amines, hormones, neuropeptides, and proteases. Small molecules include, but are not limited to, inorganic molecules 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 Also provided herein are cell therapies such as chimeric antigen receptor (CAR) T-cell therapy. CAR T-cell therapy redirects a patient's T cells to kill tumor cells through exogenous expression of a CAR. CARs can be transmembrane fusion proteins linking the antigen-recognition domain of an antibody to the intracellular signaling domains of a T-cell receptor and co-receptor. Suitable cells can be used that are contacted with the anti-PD-L1 antibodies of the present invention (or engineered to express the anti-PD-L1 antibodies as described herein). Solid tumors present unique challenges for CAR-T therapy. Unlike hematological cancers, tumor-associated target proteins are overexpressed between tumors and healthy tissues, resulting in on-target / off-tumor T-cell killing of healthy tissues. Furthermore, immunosuppression in the tumor microenvironment (TME) limits the activation of CAR-T cells toward tumor killing. Following such contact or engineering, the cells can then be introduced into a cancer patient in need of treatment. The cancer patient may have any of the types of cancer disclosed herein. The cell (e.g., T cell) can be, but is not limited to, for example, 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 its entirety.

[0207] In one embodiment, the PD-L1 antibodies discussed herein can be used in the construction of multispecific antibodies or as a payload for CAR-T cells. For example, in one embodiment, the anti-PD-L1 antibodies discussed herein can be used for targeting CARS (i.e., as a targeting moiety). In another embodiment, the anti-PD-L1 antibodies discussed herein can be used as a targeting moiety, and a different PD-L1 antibody targeting a different epitope can be used as the payload. In another embodiment, the payload can be an immunomodulatory antibody payload. In some embodiments, the PD-L1 antibodies described herein can be used as a targeting moiety in a CAR (e.g., PDL-1 + They can be used as tumor cell killers, or as secreted checkpoint blockade antibodies to reverse T cell exhaustion.

[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 antigen-binding fragment thereof that binds to the human programmed death ligand 1 (PD-L1) protein. For example, the monoclonal antibody or fragment thereof may comprise a heavy chain, a light chain, or a combination thereof, wherein the heavy chain is G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 47), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 48), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 49), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 50), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 51), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 52), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 53), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 54), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 55), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 56), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 57), G-(X1)-T-(X2)-SS-(X3X4) (SEQ ID NO: 58), G-(X1)-T-(X2)-SS-( 13 X 14 )-(X3X4) (SEQ ID NO: 205), G-(X1)-TF-(X 13 X 14 )-Y-(X4) (SEQ ID NO: 206), CDR1 comprising I-(X8X9X 10 X 11 )-G-(X 12 )-A (SEQ ID NO: 51), or II-(X 15 )-IFG-(X 16)-A (SEQ ID NO:207), and / or a CDR3 comprising 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); 17 X 18 )I-(X 19 )-SNY (SEQ ID NO: 208) or NIG-(X5)-K-(X 20 ) (SEQ ID NO: 48), CDR1 comprising (X 21 )-DN (SEQ ID NO: 209), (X 22 )-NN (SEQ ID NO:210), or DD-X6 (SEQ ID NO:49), and / or CDR3 comprising 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).

[0209] A CAR according to the present invention may comprise at least one transmembrane polypeptide comprising at least one extracellular ligand-binding domain and 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 oligo- or polypeptide capable of binding to a ligand. For example, the domain may be capable of interacting with a cell surface molecule. For example, the 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 can comprise an antigen-binding domain derived from an antibody directed against a target antigen. For example, the target can be PD-L1. Thus, the CAR can be specific for PD-L1. In one embodiment, the extracellular ligand-binding domain is a single-chain antibody fragment (scFv) comprising the light chain (VL) and heavy chain (VH) variable fragments of a target antigen-specific monoclonal antibody joined by a flexible linker. For example, the scFv antibody is specific for PD-L1. However, it is understood that binding domains other than scFvs can also be used for predefined targeting of lymphocytes, such as, for example, but not limited to, camelid single-domain antibody fragments or receptor ligands, antibody binding domains, antibody hypervariable loops, or CDRs.

[0212] In embodiments, the transmembrane domain comprises a stalk region between the extracellular ligand-binding domain and the transmembrane domain. The term "stalk region" can refer to 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 comprise up to 300 amino acids, such as 10 to 100 amino acids. In embodiments, the stalk region comprises 25 to 50 amino acids. The stalk region can 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 all or part of an antibody constant region. Alternatively, the stalk region can be a synthetic sequence corresponding to a naturally occurring stalk sequence or can be a completely synthetic stalk sequence. In a preferred embodiment, the stalk region is a portion of the human CD8 alpha chain.

[0213] In embodiments, the transmembrane domain may comprise CD28.

[0214] The signal transduction domain or intracellular signal transduction domain of the CAR of the present invention is involved in intracellular signal transduction after the extracellular ligand binding domain binds to a target, resulting in the activation of immune cells and immune responses. In other words, the signal transduction domain is involved in activating at least one of the normal effector functions of the immune cells expressing the CAR. For example, the effector function of T cells can be cytolytic activity or helper activity, including cytokine secretion. Therefore, the term "signal transduction domain" can refer to the portion of a protein that converts effector signal function signals and instructs cells to perform specific functions.

[0215] The signal transduction domain can comprise two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation and those that act antigen-independently to provide secondary or costimulatory signals. Primary cytoplasmic signaling sequences can comprise signaling motifs known as ITAMs (immunoreceptor tyrosine-based activation motifs). ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that function as binding sites for the syk / zap70 class of tyrosine kinases. Examples of ITAMs used in the present invention can include, but are not limited to, those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the signaling domain of the CAR can comprise the CD3 zeta signaling domain or the cytoplasmic domain of the Fc epsilon RI beta or gamma chain. In another preferred embodiment, signaling is provided by CD3 zeta, with costimulation provided by CD28 and a tumor necrosis factor receptor (TNFr), such as, for example, 4-1BB or OX40.

[0216] In embodiments, the intracellular signaling domain of the CAR of the present invention comprises a costimulatory signal molecule. In some embodiments, the intracellular signaling domain contains two, three, four, or more costimulatory molecules in tandem. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response.

[0217] A "costimulatory ligand" can refer to a molecule on an antigen-presenting cell that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal that mediates T cell responses, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by, for example, binding of a peptide-loaded MHC molecule to the TCR / CD3 complex. Costimulatory ligands include CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, Toll ligand receptor, and in particular a ligand that specifically binds B7-H3. Costimulatory ligands can include, but are not limited to, agonists or antibodies that bind to costimulatory molecules present on T cells, such as, but 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 cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, such as, 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 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 ligands that specifically bind to CD83.

[0219] In embodiments, the selection of CD28 as the costimulatory domain of the CAR may be based on the fact that CD28 CARs direct active proliferative responses and enhance effector function, whereas 4-1BB-based CARs induce more progressive T cell accumulation, which may offset 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-associated factor 2 (TRAF2)-binding motif in the cytoplasmic tail of a costimulatory TNFR family member. The cytoplasmic tail of a costimulatory 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] The distinctive 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 a predetermined target cell. The different transmembrane polypeptides of the CAR of the present invention, including extracellular ligand binding domains and / or signaling domains, interact together to participate in signal transduction after binding to the target ligand and induce an immune response. The transmembrane domain can be derived from either natural or synthetic origin. The transmembrane domain can be derived from any membrane-bound or transmembrane protein.

[0222] As used herein, the term "portion" can refer to any subset of a molecule, i.e., a shorter peptide. Alternatively, functional variants of a polypeptide's amino acid sequence can be prepared by mutation of the DNA encoding the polypeptide. Such variants or functional variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct has the desired activity, in particular, specific anti-target cellular immune activity. The functionality of the CAR of the present invention in host cells can be detected by an assay suitable for demonstrating the signal transduction ability of the CAR upon binding of a specific target. Such assays are available to those skilled in the art. For example, the assay allows for the detection of signal transduction pathways triggered upon target binding, such as assays involving measurement of increased calcium ion release, intracellular tyrosine phosphorylation, inositol phosphate turnover, or the resulting production of interleukin (IL) 2, interferon gamma, GM-CSF, IL-3, and IL-4.

[0223] CAR-expressing cells Embodiments of the present invention include cells expressing a CAR (i.e., a CART). The cells can be of any type, including immune cells capable of expressing a CAR for cancer therapy, or cells such as bacterial cells harboring an expression vector encoding a CAR. As used herein, the terms "cell," "cell line," and "cell culture" can be used interchangeably. All of these terms include their progeny, that is, any subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, a "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 exogenous nucleic acid is transferred or introduced into a host cell. A transformed cell includes the primary subject cell and its progeny. As used herein, the terms "engineered" and "recombinant" or host cell are intended to refer to a cell into which an exogenous nucleic acid sequence, such as a vector, has been introduced. Thus, recombinant cells are distinguishable from naturally occurring cells that do not contain a recombinantly introduced nucleic acid. In an embodiment of the invention, the host cell is a T cell, including a cytotoxic T cell (also known as a TC, cytotoxic T lymphocyte, CTL, T killer cell, cytolytic T cell, CD8+ T cell, or killer T cell); NK cells and NKT cells are also included in the invention.

[0224] Some vectors may use control sequences that allow them to be replicated and / or expressed in both prokaryotic and eukaryotic cells. Those skilled in the art will further understand the conditions for incubating all of the above host cells to maintain them and allow vector replication. Also understood and known are the techniques and conditions that allow large-scale production of vectors and the production of nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

[0225] The cells can be autologous, syngeneic, allogeneic, and in some cases, xenogeneic.

[0226] In many situations, it may be desirable to be able to kill modified CTLs when treatment termination is desired, when cells become tumorigenic, in studies where the absence of cells after their presence is of interest, or in other events. To this end, one can provide for the expression of a specific gene product, such as an inducible suicide gene, that can kill modified cells under controlled conditions.

[0227] Armed CART The present invention further includes CARTs engineered to secrete one or more polypeptides. Armed CARTs have the advantage of simultaneously secreting polypeptides at target sites, such as tumor sites. The polypeptides can be, for example, antibodies or cytokines. For example, the antibodies are specific for PD-L1, such as the antibodies and fragments described herein. In other embodiments, the secreted antibodies can be specific for CAIX, GITR, PD-L2, PD-1, or CCR4 (see, for example, the sequences set forth in PCT Publication No. WO2016 / 1009085, the entire application of which is incorporated by reference).

[0228] An armed CART can be constructed by including a nucleic acid encoding a secreted polypeptide of interest after an intracellular signaling domain. In embodiments, an internal ribosome entry site (IRES) is located between the intracellular signaling domain and the polypeptide of interest. Those skilled in the art will appreciate that multiple IRES sequences can be used in tandem to express more than one polypeptide.

[0229] In embodiments, CART cells can be maintained using cytokines such as, for example, 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 enrichment of tumor-specific CD8 T cells and has increased antitumor efficacy in mouse melanoma models compared with IL-2 or IL-15.

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

[0232] Transduction of constructs into CTLs The expression vector encoding the CAR can be introduced as one or more DNA molecules or constructs, optionally with at least one marker present that allows for selection of host cells containing the construct.

[0233] Constructs can be prepared using conventional methods, and the genes and regulatory regions can be isolated, ligated, cloned into a suitable cloning host, and analyzed by restriction or sequencing or other convenient means. In particular, PCR can be used to isolate individual fragments containing all or part of the functional unit, and one or more mutations can be introduced using primer repair, ligation, in vitro mutagenesis, or other suitable methods. Once a construct is complete and has been demonstrated to have the correct sequence, it can then be introduced into CTLs by any convenient means. Constructs may be packaged and integrated into non-replicating, defective viral genomes, such as adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), or retroviral or lentiviral vectors, for cell infection and transduction. Constructs may optionally include viral sequences for transfection. Alternatively, constructs may be introduced by fusion, electroporation, biolistic methods, transfection, lipofection, or other methods. Host cells can be grown and expanded in culture before introducing the construct, and then the construct can be introduced and treated appropriately to integrate the construct. The cells are then expanded and screened for the marker present in the construct. Various markers that have been successfully used include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc.

[0234] In some cases, if it is desired that the construct be integrated into a specific locus, it may have a target site for homologous recombination. For example, a ) can knock out an endogenous gene and replace it with a 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 the 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 construct can be introduced as a single DNA molecule that encodes at least CAR and optionally another gene, or as different DNA molecules that have one or more genes.Other genes include, for example, genes that encode therapeutic molecules or suicide genes.The constructs can be introduced simultaneously or sequentially, and each has the same or different markers.

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

[0237] Methods of using cells expressing CAR The cells described herein can be used to treat cancer or other cell proliferation-related diseases or disorders, including, but not limited to, diseases or disorders associated with aberrant expression of PD-L1. In another embodiment, the isolated cells according to the present invention can be used in the manufacture of a medicament for treating cancer or other cell proliferation-related diseases or disorders, including, but not limited to, diseases or disorders associated with aberrant expression of PD-L1.

[0238] Embodiments described herein rely on methods for treating a patient in need thereof, the method comprising at least one of the steps of: (a) providing chimeric antigen receptor cells according to the invention; and (b) administering the cells to the patient.

[0239] The treatment may be ameliorative, curative, or preventative. It may be part of an autoimmune therapy or part of an allogeneic immunotherapy treatment. Autologous means that the cells, cell lines, or cell populations used to treat a patient are derived from the patient or from a human leukocyte antigen (HLA)-matched donor. Allogeneic means that the cells or cell populations used to treat a patient are derived from a donor, rather than from the patient.

[0240] The present invention is particularly suitable for allogeneic immunotherapy insofar as it allows the transformation of T cells, typically obtained from a donor, into non-allo-reactive cells. This can be performed under standard protocols and replicated as many times as necessary. The resulting modified T cells can be pooled and administered to one or several patients, making them available as an "off-the-shelf" therapeutic product.

[0241] Cancers that can be treated using the antibodies or CAR compositions described herein include tumors that are not vascularized or have not yet been substantially vascularized, as well as vascularized tumors. Cancers can include non-solid tumors (e.g., hematological tumors, such as leukemia and lymphoma) or solid tumors. Cancer types 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 leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included. For example, cancers in which checkpoint inhibition is the standard treatment for multiple malignancies (referred to herein as "checkpoint inhibition 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 microsatellite instability-high (MSI-H) or DNA mismatch repair deficient (dMMR) adult and pediatric solid tumors (doi:10.1016 / j.csbj.2019.03.006). The treatments described herein may also include other cancers that are under investigation for checkpoint inhibitor therapy. Without wishing to be bound by theory, RCC and B-CLL mouse models can be used for treatment with CAR T factories, which are models relevant to human disease.

[0242] For example, the treatment can 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 radiation therapy.

[0243] According to an embodiment of the present invention, the treatment can be administered to a patient undergoing immunosuppressive therapy. Indeed, the present invention may rely on cells or cell populations that have been made resistant to at least one immunosuppressant due to inactivation of a gene encoding a receptor for such an immunosuppressant. In this aspect, the immunosuppressive therapy should aid in the selection and expansion of T cells according to the present invention in the patient.

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

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

[0246] Depending on the nature of the cells, cells can be introduced into a host organism, e.g., a mammal, in a variety of ways. In certain embodiments, the cells can be introduced into the site of a tumor, while in alternative embodiments, the cells home to the cancer or are modified to home to the cancer. The number of cells used depends on various circumstances, the purpose of the introduction, the lifespan of the cells, the protocol used, e.g., the number of administrations, the ability of the cells to proliferate, the stability of the recombinant construct, etc. The cells may be applied as a dispersion, typically injected at or near the site of interest. The cells may be in a physiologically acceptable medium.

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

[0248] The cells can be administered as desired. A variety of protocols can be used depending on the desired response, the method of administration, the longevity of the cells, and the number of cells present. The number of administrations will depend, at least in part, on the factors described above.

[0249] Administration of the cells or cell populations of the present invention can be by any convenient method, including aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, intravenously, or intralymphatically, or intraperitoneally. In one embodiment, the cell compositions of the present invention are preferably administered by intravenous injection.

[0250] The administration of cells or cell populations was 10 per kg of body weight. 4 ~10 9 cells, e.g., 105 ~10 6 The effective amount of cells may consist of administering 100 or more cells / kg body weight, including all integer values ​​within these ranges. The cells or cell populations can be administered in one or more doses. In another embodiment, the effective amount of cells is administered as a single dose. In another embodiment, the effective amount of cells is administered as 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 clinical condition of the patient. The 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 amounts of a given cell type for a particular disease or condition is within the skill of the art. An effective amount refers to an amount that provides a therapeutic or prophylactic benefit. The dosage administered will depend on the age, health, and weight of the recipient, the type of concurrent treatment, if any, the frequency of treatment, and the nature of the desired effect.

[0251] It will be appreciated that the system is influenced by many variables that may change over time and circumstances, such as cellular response to ligand, expression efficiency and, if appropriate, secretion levels, activity of the expressed product, the specific needs of the patient, the rate of cell loss or loss of cellular activity as a result of expression activity of individual cells, etc. Thus, even if there are pluripotent cells that can be administered to an entire population for each individual patient, it is expected that each patient will be monitored for the appropriate dosage for that individual, 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. Recombinant 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 where it can be replicated. The nucleic acid sequence can be "exogenous," meaning that the sequence is foreign to the cell into which the vector is introduced, or that the sequence is homologous to a sequence in the cell, but is located in a location within the host cell nucleic acid where the sequence is not normally found. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), artificial chromosomes (YACs, etc.). Those skilled in the art will be well equipped to construct vectors using standard recombinant techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both of which are incorporated herein by reference).

[0254] The term "expression vector" can refer to any type of genetic construct that contains a nucleic acid encoding an RNA that can be transcribed. 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 "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors can contain nucleic acid sequences that perform other functions as well, as described below.

[0255] A "promoter" can refer to a regulatory sequence, which is a region of a nucleic acid sequence that controls the initiation and rate of transcription. It can include genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind to initiate specific transcription of a nucleic acid sequence. The phrases "operably positioned," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional location and / or orientation with respect to a nucleic acid sequence to control transcription initiation and / or expression of that sequence.

[0256] A promoter may contain sequences that function to position the start site for RNA synthesis. The most well-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoters of the mammalian terminal deoxynucleotidyl transferase gene and the SV40 late genes, separate elements surrounding the start site themselves help to anchor the start site. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, but some promoters have been shown to contain functional elements downstream of the start site as well. To place a coding sequence "under the control" of a promoter, the 5' end of the transcription start site of the transcriptional reading frame is positioned "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates DNA transcription, promoting expression of the encoded RNA.

[0257] The spacing between promoter elements is often flexible, allowing elements to be inverted or moved relative to one another while still maintaining promoter function. In the tk promoter, the spacing between promoter elements can be increased by up to 50 bp before activity begins to decline. Depending on the promoter, individual elements may function cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

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

[0259] Naturally, it will be important to use a promoter and / or enhancer that effectively directs expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology generally know 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 constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high-level expression of the introduced DNA segment, which is advantageous for large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

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

[0261] The identity of tissue-specific promoters or elements, as well as assays to characterize their activity, are well known to those of skill in the art.

[0262] Specific initiation signals may also be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would be able to readily determine this and provide the necessary signals.

[0263] In certain embodiments of the present invention, the use of internal ribosome entry site (IRES) elements is used to create multigenic, or polycistronic, messages, which may be used in the present invention.

[0264] Vectors can contain a multiple cloning site (MCS), a nucleic acid region containing multiple restriction enzyme sites, any of which can be used to digest the vector in conjunction with standard recombinant techniques. "Restriction enzyme digestion" refers to the catalytic cleavage of a nucleic acid molecule by an enzyme that functions only at specific locations in the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is widely understood by those skilled in the art. Restriction enzymes that cut within the MCS are often used to linearize or fragment a vector so that an exogenous sequence can be ligated into the vector. "Ligation" refers to the process of forming phosphodiester bonds 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 recombinant technology.

[0265] Splice sites, termination signals, origins of replication, and selectable markers may also be used.

[0266] In certain embodiments, plasmid vectors can be used to transform host cells. Plasmid vectors containing replicon and control sequences derived from species compatible with the host cell can be used in connection with these hosts. The vector usually carries a replication site as well as marking sequences capable of providing 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 E. coli species. pBR322 contains genes for ampicillin and tetracycline resistance, allowing easy identification of transformed cells. pBR plasmids, or other microbial plasmids or phages, must also contain, or be modified to contain, promoters that can be used by the microorganism for expression of its own proteins, for example.

[0267] Furthermore, phage vectors containing replicon and control sequences compatible with host microorganisms can be used as transforming vectors in connection with these hosts. For example, phage lambda GEM TM.11 can be used to generate recombinant phage vectors that can be used to transform host cells such as E. coli LE392.

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

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

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

[0271] Methods for nucleic acid delivery include the use of adenoviral expression vectors. Adenoviral vectors are known to have a low ability to integrate into genomic DNA, but this feature is offset by the high gene transfer efficiency achieved by these vectors. The term "adenoviral expression vector" refers to a construct containing sufficient adenoviral sequences (a) to support packaging of the construct and (b) to ultimately express a tissue- or cell-specific construct cloned therein. Due to the genetic makeup or knowledge that adenovirus is a 36 kb, linear, double-stranded DNA virus, large segments of adenoviral DNA can be replaced with foreign sequences up to 7 kb in size (Grunhaus and Horwitz, 1992).

[0272] Adenovirus-assisted transfection can be used to introduce nucleic acids into cells. Increased transfection efficiency has been reported in cell lines using adenovirus coupled systems (Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno-associated virus (AAV) is an attractive vector system for use in the cells of the present invention because of its high integration frequency and ability to infect non-dividing cells, and is therefore useful for gene delivery to mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a wide infectious host range (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Details regarding the generation and use of rAAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368, each of which is incorporated herein by reference.

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

[0274] To construct a retroviral vector, a nucleic acid (e.g., encoding a desired sequence) is inserted into the viral genome in place of a specific viral sequence, producing a replication-deficient virus. To produce virions, a packaging cell line containing the gag, pol, and env genes but without the long-term repeat (LTR) and packaging components is constructed (Mann et al., 1983). When a recombinant plasmid containing a cDNA along with the retroviral long-term repeat (LTR) and packaging sequence is introduced into a specialized cell line (e.g., by calcium phosphate precipitation), the packaging sequence allows the RNA transcripts 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 a variety of cell types. However, integration and stable expression require host cell division (Paskind et al., 1975).

[0275] Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. Lentiviral vectors are well known in the art (see, e.g., Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; U.S. Patent Nos. 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency viruses (HIV-1, HIV-2) and simian immunodeficiency viruses (SIV). Lentiviral vectors are generated by multiple attenuation of HIV pathogenicity genes, e.g., deletion of genes env, vif, vpr, vpu, and nef, resulting in biologically safe vectors.

[0276] Recombinant lentiviral vectors can infect non-dividing cells and can be used for gene transfer and expression of nucleic acid sequences both in vivo and ex vivo. For example, a recombinant lentivirus capable of infecting non-dividing cells in which suitable host cells have been transfected with two or more vectors carrying packaging functions, i.e., gag, pol, and env, and rev and tat, is described in U.S. Patent No. 5,994,136, incorporated herein by reference. To target receptors on specific cell types, recombinant viruses can be targeted by binding of the envelope protein to antibodies or specific ligands. 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] Other viral vectors can be used as vaccine constructs in the present invention. 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 offer several attractive features in various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

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

[0279] Another approach to targeting recombinant retroviruses was designed, using biotinylated antibodies against retroviral envelope proteins and specific cellular receptors. The antibodies were conjugated via the biotin moiety 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 bearing 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, direct delivery of DNA by ex vivo transfection, injection, etc.By applying techniques known in the art, cells can be stably or transiently transformed.

[0281] Ex vivo transformation Methods for transfecting eukaryotic cells and tissues ex vivo that have been removed from a living organism are known to those skilled in the art. Thus, it is contemplated that the nucleic acids of the present invention can be used to remove and transfect cells or tissues ex vivo. In certain embodiments, the transplanted cells or tissues can be placed in an organism. In a preferred aspect, the nucleic acid is expressed in the transplanted cells.

[0282] Kits of the Invention Any of the compositions described herein can be included in a kit.

[0283] Some components of the kit may be packaged either in aqueous media or in lyophilized form. The container means of the kit generally includes at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed, preferably appropriately dispensed. When two or more components are present in the kit, the kit will usually also include a second, third, or other additional container into which the additional components may be individually placed. However, various combinations of components may be included in vials. The kits of the present invention also typically include a means for containing the components in close confinement for commercial sale. Such containers may include injection-molded or blow-molded plastic containers into which the desired vials are retained.

[0284] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly useful. In some cases, the container means itself may be a syringe, pipette, and / or other similar device from which the formulation may be applied to an 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 also be provided as dry powders. When reagents and / or components are provided as dry powders, the powder can be reconstituted by adding a suitable solvent. It is envisioned that the solvent may also be provided in another container means. The kit may also comprise a second container means for containing a sterile pharmaceutically acceptable buffer and / or other diluent.

[0286] In embodiments of the present invention, the cells used for cell therapy are provided in a kit, and in some cases, the cells are essentially the only 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 for amplifying the desired sequence, nucleotides, suitable buffers or buffer reagents, salts, etc., and in some cases, the reagents include a vector and / or DNA encoding a CAR described herein and / or regulatory elements therefor.

[0287] In certain embodiments, there are present in the kit one or more devices suitable for extracting one or more samples from an individual. The device may be a syringe, a scalpel, or the like.

[0288] In some instances of the present invention, in addition to the cell therapy embodiment, the kit also includes a second cancer therapy, such as, for example, chemotherapy, hormone therapy, and / or immunotherapy. The kit can be tailored to an individual's particular cancer and can include each second cancer therapy for the individual.

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

[0290] In some embodiments, for diagnostic purposes, the anti-PD-L1 antibodies of the invention are linked to a detectable moiety to provide a method for detecting cancer cells, for example, in a subject at risk for or afflicted with cancer.

[0291] The detectable moiety can be conjugated directly to the antibody or fragment, 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.A chimera or a fusion protein containing an antibody or antibody fragment bound 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) describes a method for creating a vector construct that can express a fusion protein gene of aequorin and an antibody in mammalian cells.

[0292] As used herein, the term "labeled" with respect to a probe or antibody can encompass both direct labeling of the probe or antibody by conjugating (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 detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling a DNA probe with biotin for detection 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, the detection methods of the present invention can be used to detect cells expressing PD-L1 in biological samples in vitro and in vivo. For example, in vitro techniques for detecting PD-L1 include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. Additionally, in vivo techniques for detecting PD-L1 include introducing a labeled anti-PD-L1 antibody into a subject. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0293] For "targeted" conjugates, i.e., conjugates containing a targeting moiety, which is 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 when equilibrium between the bound "localized" entity and the unbound "free" entity within the subject is essentially achieved. The rate at which such equilibrium is achieved depends on the route of administration. For example, a conjugate administered by intravenous injection can achieve localization within minutes of injection, whereas an orally administered conjugate may take several hours to achieve localization. Alternatively, localization can simply refer to the location of an entity within a subject or animal at a selected period of time after the entity is administered. As another example, 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 one skilled in the art. Furthermore, the state of localization as a function of time can be tracked by imaging a detectable moiety (e.g., a luminescent conjugate) according to the method of the present invention, such as with a photodetector device. The "photodetector device" used should be sensitive enough to allow imaging of weak light from within a mammal in a reasonable time and to use the signal from such a device to construct an image.

[0295] If it is possible to use extremely bright light-generating moieties and / or detect light-generating fusion proteins localized near the surface of the object or animal being imaged, "night vision" goggles or standard highly sensitive video cameras such as Silicon Intensified Tube (SIT) cameras (e.g., from Hammamatsu Photonic Systems, Bridgewater, NJ) can be used. More typically, however, more sensitive light detection methods are required.

[0296] At extremely low light levels, the photon flux per unit area becomes so low that the scene being imaged no longer appears continuous. Instead, it is represented by individual photons that differ from each other both temporally and spatially. When viewed on a monitor, such an image appears as sparkling 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 the signal at each image point is assigned an intensity value, in photon-counting imaging, the amplitude of the signal is immaterial. The objective is simply to detect the presence of a signal (photon) and count its occurrence relative to its location over time.

[0297] At least two types of photodetector devices, described below, can detect individual photons and generate a signal that can be analyzed by an image processor. Noise-reducing photodetector devices achieve sensitivity by reducing the background noise of the photon detector rather than amplifying the photon signal. Noise is primarily reduced by cooling the detector array. Devices include charge-coupled device (CCD) cameras called "back-thinned" cooled CCD cameras. In more sensitive instruments, cooling is achieved using, for example, liquid nitrogen, which brings the temperature of the CCD array to approximately -120°C. "Back-thinned" refers to an ultra-thin backplate that reduces the path length photons must travel before detection, thereby increasing quantum efficiency. A particularly sensitive back-thinned cryogenic CCD camera is the "TECH 512" Series 200 camera, available from Photometries, Ltd. (Tucson, Arizona).

[0298] "Photon amplification devices" amplify photons before they hit the detection screen. This class includes CCD cameras equipped with intensifier tubes, such as microchannel intensifier tubes. Microchannel intensifier tubes typically contain a metal array of channels perpendicular to and coextensive with the camera's detection screen. The microchannel array is positioned between the sample, subject, or animal being imaged and the camera. Most of the photons that enter the channels of the array 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] Even greater sensitivity can be achieved by arranging intensifying microchannel arrays in series, so that electrons generated in the first stage in turn result in an amplified signal of electrons in the second stage. However, the increased sensitivity is achieved at the expense of spatial resolution, which decreases with each additional stage of amplification. An exemplary microchannel intensifier-tube-based single-photon detection device is the C2400 series available from Hamamatsu.

[0300] Image processors process signals generated by photon-counting photodetector devices to construct images that can be displayed on a monitor or printed on a video printer, for example. Such image processors are typically sold as part of systems that include the highly sensitive photon-counting cameras described above, and are therefore available from the same sources. Image processors are usually connected to personal computers, such as IBM-compatible PCs or Apple Macintosh computers (Apple Computer, Cupertino, Calif.), which may or may not be included as part of a purchased imaging system. Once images are in the form of digital files, they can be manipulated and printed using a variety of image processing programs (e.g., Adobe Photoshop, Adobe Systems, Adobe Systems, Mt. View, Calif.).

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

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

[0303] Other embodiments While the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative, but not limiting, of the scope of the invention, which is 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 described in the claims. [Example]

[0305] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary modes of making and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, which are for illustrative purposes only, as alternative methods may be used to obtain similar results.

[0306] Example 1 - Antibody panning The PD-L1 antibodies of the present invention were discovered via PMPL panning. Briefly, to increase their affinity to 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 generate the #42-light chain shuffling (LCS) library and #52 LCS library. Each library was then panned against PD-L1-mouse Fc soluble antigen for four rounds at decreasing antigen concentrations (1 μg / ml PD-L1-mFc for the third round, 0.5 μg / ml for the fourth round, and 0.1 μg / ml for the fourth round; 1 ml was used to coat the tube). Single colonies were screened against soluble PD-L1-mFc by ELISA. Positive clones were enriched after the second round of panning, increasing affinity. If desired, phage eluted after the third round of panning were also cloned into a yeast display library and screened for high-affinity binders via flow cytometry. Anti-PD-L1 antibodies #42 and #50, previously discovered in Dr. Marasco's laboratory, have lower affinity for PD-L1 compared to benchmark commercially available antibodies. Knowing that the heavy chains of #42 and #50 primarily contribute to their PD-L1 binding specificity, a new single-chain Fv phage display library was generated using a light chain shuffling technique. In this technique, either the #42 or #50 heavy chain variable region was fused to random kappa and lambda light chain variable regions. The resulting #42 LCS and #50 LCS libraries, respectively, have a diversity of approximately 2 x 10E8. Panning the new #42 and #52 LCS libraries at reduced antigen concentrations uncovered 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 with naive phage libraries.

[0307] Example 2 - Dual Binding 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. 3-fold serial dilutions of the bispecific GITR-PDL1 Lc fusion were made in a separate 96-well plate at a starting concentration of 9 μg / ml. 50 μl of Ab dilutions were added to 100 μl of buffer containing the cells to give 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 (the extracellular domain of PD-L1) and incubated at 4°C for 30 minutes. The cells were washed twice with 250ul of MACS buffer and resuspended in 100ul of MACS buffer containing 2ug / ml of FITC donkey anti-rabbit IgG (minimum x reactivity) antibody (BioLegend catalog number 406403). The cells were incubated for 20 minutes at 4°C. The cells were washed twice with 250ul of MACS buffer and resuspended in 200ul of MACS buffer. The plate was then read on a Fortessa HTS FACS plate reader.

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

[0309] Example 3 - Mixed Lymphocyte Reaction (MLR) Protocol CD14+ monocytes were isolated using Miltenyi CD14+ microbeads. The cells were cultured in Miltenyi Mo-DC medium (pre-prepared medium containing GM-CSF and IL4). After 5 days of culture, 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 DCs. T cells were isolated on the day of the MLR experiment (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 cultures were incubated for 5 days.

[0310] Supernatants were saved for ELISA screening (eg, IFNγ).

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

[0312] Anti-PDL1 Abs in scFv-Fc format were tested against a commercial formulation of atezolizumab, anti-PDL1#42 scFv-Fc, and a nonspecific Ab control. As shown in Figures 6-9, the addition of specific anti-PDL1 Abs, such as atezolizumab and anti-PDL1#42, increases cytokine production compared to the nonspecific control.

[0313] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific substances and procedures specifically described herein which equivalents are considered to be within the scope of this invention and covered by the appended claims.

Claims

1. An isolated antibody or fragment thereof that binds to human PD-L1 protein, wherein the isolated antibody and fragment thereof are both: a heavy chain variable region comprising the amino acid sequence GYTLSSSHG (SEQ ID NO: 10) as heavy chain CDR1, the amino acid sequence ISAHNGHA (SEQ ID NO: 12) as heavy chain CDR2, and the amino acid sequence ARVHAALYYGMDV (SEQ ID NO: 14) as heavy chain CDR3; a light chain variable region comprising the amino acid sequence NIGSKG (SEQ ID NO: 26) as light chain CDR1, the amino acid sequence DDR (SEQ ID NO: 28) as light chain CDR2, and the amino acid sequence QVWDSGSDHWV (SEQ ID NO: 30) as light chain CDR3; The isolated antibody or fragment thereof,

2. 2. The isolated antibody or fragment thereof according to claim 1, wherein the isolated antibody and fragment thereof are each: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 31; The isolated antibody or fragment thereof,

3. 10. An isolated bispecific antibody comprising the fragment of claim 1 or 2 and a second antigen-binding fragment having specificity for a molecule on an immune cell.

4. 4. The isolated bispecific antibody of claim 3, (i) the molecule is GITR; (ii) each of the fragment and the second antigen-binding fragment is independently selected from a Fab fragment, or a single-chain variable fragment (scFv); or (iii) the bispecific antibody further comprises an Fc fragment. The isolated bispecific antibody.

5. 1. An isolated cell comprising one or more polynucleotides encoding (i) an isolated antibody or fragment thereof of claim 1 or 2, or (ii) an isolated bispecific antibody of claim 3 or 4.

6. A chimeric antigen receptor (CAR) comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain, an isolated monoclonal antibody or antigen-binding fragment thereof, the extracellular domain of which binds to human programmed death-ligand 1 (PD-L1) protein; The isolated monoclonal antibody and antigen-binding fragment thereof comprise the isolated antibody or fragment thereof of claim 1 or 2 or the isolated bispecific antibody of claim 3 or 4. The chimeric antigen receptor (CAR).

7. The CAR according to claim 6, (i) the transmembrane domain further comprises a stalk region disposed between the extracellular domain and the transmembrane domain; (ii) the transmembrane domain comprises the transmembrane domain of CD28; (iii) the CAR further comprises an intracellular signaling domain of one or more additional costimulatory molecules disposed between the transmembrane domain and the intracellular signaling domain, optionally wherein the costimulatory molecule is CD28, 4-1BB, ICOS, or OX40; (iv) the intracellular signaling domain comprises the intracellular signaling domain of the CD3 zeta chain; or (v) the antibody comprises a Fab fragment or an scFv; The CAR.

8. 8. A composition comprising the isolated antibody or fragment thereof of claim 1 or 2, the isolated bispecific antibody of claim 3 or 4, or the CAR of claim 6 or 7, for use in a method of treating cancer in a subject.

9. 9. The composition of claim 8, (i) the method further comprises administering to the subject a chemotherapeutic agent; or (ii) the cancer is a checkpoint inhibition cancer; The composition.

10. A genetically engineered cell that expresses the chimeric antigen receptor of claim 6 or 7 and retains it on its cell surface membrane.

11. 11. The genetically engineered cell of claim 10, the cell is a T cell or an NK cell; Optionally, the T cells are CD4 ＋ or CD8 ＋ and Optionally, the cells are CD4 ＋ and CD8 ＋ containing a mixed population of cells, The genetically engineered cells.

12. 1. A genetically engineered cell that expresses and retains on its cell surface membrane a chimeric antigen receptor, which is further engineered to express and secrete a polypeptide, wherein the polypeptide is an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) protein, and wherein the monoclonal antibody or fragment thereof comprises the isolated antibody or fragment thereof of claim 1 or 2, or the isolated bispecific antibody of claim 3 or 4.

13. 8. A nucleic acid encoding the isolated antibody of claim 1 or 2, the isolated bispecific antibody of claim 3 or 4, or the CAR of claim 6 or 7.

14. A vector comprising the nucleic acid of claim 13.

15. An isolated cell comprising the vector of claim 14.

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