Human monoclonal anti-PD-L1 antibodies and methods of use

Abstract

The present invention includes human monoclonal antibodies that bind to PD-L1 (also known as programmed death ligand 1 or B7H1). Binding of the antibodies of the present invention to PD-L1 inhibits binding to its receptor PD1 (programmed death 1) and ligand-mediated activity and can be used to treat cancer and chronic viral infections.

Description

[0001] This application is a continuation of a divisional application of an invention application with an application date of October 4, 2013, Chinese national application number of 201380063513.0, and invention name of “Human monoclonal anti-PD-L1 antibodies and methods of use” (Chinese national application number of 201711156800.2).

[0002] Related Applications

[0003] This application claims priority to and the benefit of U.S. Provisional Application No. 61 / 709,731, filed October 4, 2012, and U.S. Provisional Application No. 61 / 779,969, filed March 13, 2013, the contents of each of which are incorporated herein by reference in their entirety. Field of the Invention

[0004] The present invention generally relates to anti-PD-L1 (also known as programmed cell death 1 ligand 1 or B7H1) antibodies and methods of using the same. Background of the Invention

[0005] The immune system must achieve a balance between effective response to remove pathogens and maintain tolerance to prevent autoimmune diseases. T cells are critical for maintaining this balance, and their proper regulation is mainly coordinated 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) provides a key positive signal that not only initiates, improves and maintains T cell responses, but also promotes the key negative signals of limiting, terminating and / or weakening T cell responses when appropriate. A member of the CD28 family, called PD-1 (also known as programmed cell death-1), is upregulated on activated T cells, B cells and monocytes. PD-1 has two identified ligands in the B7 family, PD-L1 (also known as BH71 or programmed cell death-1 ligand 1) and PD-L2. While the expression of PD-L2 tends to be more restricted, being found primarily on activated antigen presenting cells (APCs), the expression of PD-L1 is more ubiquitous, including cells of the hematopoietic lineage (which includes activated T cells, B cells, monocytes, dendritic cells, and macrophages) and peripheral non-lymphoid tissues (which include heart, bone, muscle, placenta, lung, kidney, and liver tissues). The ubiquitous expression of PD-L1 suggests its important role in regulating PD-1 / PD-L1-mediated peripheral tolerance.

[0006] The combination between PD-L1 and PD-1 has a far-reaching effect on the regulation of T cell responses. Specifically, PD-L1 / PD-1 interactions inhibit the production of T cell proliferation and effector cytokines (which mediate T cell activity and immune responses, such as IL-2 and IFN-γ). This negative regulatory function is important for preventing T cell-mediated autoimmunity and immunopathology. However, it has also been shown that the PD-1 / PD-L1 axis plays a role in T cell exhaustion, whereby the negative regulatory function inhibits the response of T cells to the host's harm. Long-term or chronic antigen stimulation of T cells can induce negative immune feedback mechanisms, which inhibit antigen-specific responses and lead to immune escape of pathogens. T cell exhaustion can also lead to progressive physical deletion of antigen-specific T cells themselves. T-cell expression of PD-1 is upregulated during chronic antigen stimulation, and its binding to PD-L1 leads to blockade of effector function in both CD4+ (T helper cells) and CD8+ (cytotoxic T lymphocytes or CTLs) T cells, thus implicating PD-1 / PD-L1 interactions in the induction of T-cell exhaustion.

[0007] Recently, it has been shown that some chronic viral infections and cancers develop immune evasion strategies that specifically exploit the PD-1 / PD-L1 axis by causing PD-1 / PD-L1-mediated T cell exhaustion. A variety of human tumor cells and tumor-associated antigen presenting cells express high levels of PD-L1, suggesting that the tumors induce T cell exhaustion to escape anti-tumor immune responses. During chronic HIV infection, HIV-specific CD8+T cells are functionally impaired, manifested by reduced ability to produce cytokines and effector molecules and reduced proliferation. Studies have shown that PD-1 is highly expressed on HIV-specific CD8+T cells in HIV-infected individuals, suggesting that blocking the PD-1 / PD-L1 pathway may have therapeutic potential for the treatment of HIV-infected and AIDS patients. Taken together, agents that block the PD-1 / PD-L1 pathway will provide new treatments for a variety of cancers, HIV infection, and / or other diseases and conditions associated with T-cell exhaustion. Therefore, there is an urgent need for agents that can block or prevent the PD-1 / PD-L1 interaction. Brief description of the invention

[0008] The present invention is based on the discovery of a monoclonal antibody that binds to PD-L1. The monoclonal antibody is a fully human antibody. The antibody binds to PD-L1. The antibody is referred to herein as huPD-L1 antibody.

[0009] PD-L1 is also known as programmed cell death 1 ligand 1, programmed death ligand 1, PDCD1 ligand 1, PDCD1L1, PDL1, B7 homolog 1, B7H1, B7-H, CD274, and CD274 antigen.

[0010] The present invention provides an isolated humanized monoclonal antibody having: a heavy chain having three CDRs (which respectively comprise the amino acid sequences SYGIS (SEQ ID NO: 58), WISAYNGNTNYAQKLQG (SEQ ID NO: 71) and ALPSGTILVGGWFDP (SEQ ID NO: 86)) and a light chain having three CDRs (which respectively comprise the amino acid sequences TRSSGNIASNYVQ (SEQ ID NO: 101), EDNQRPS (SEQ ID NO: 115) and QSYDSSNLWV (SEQ ID NO: 127)); a heavy chain having three CDRs (which respectively comprise the amino acid sequences SYALS (SEQ ID NO: 59), AISGGGGSTYYADSVKD (SEQ ID NO: 72) and DVFPETFSMNYGMDV (SEQ ID NO: 87)) and a light chain having three CDRs (which respectively comprise the amino acid sequences QGDSLRSYYAS (SEQ ID NO: 102), GKNNRPS (SEQ ID NO: 114) and QSYDSSNLWV (SEQ ID NO: 127)). NO: 116) and NSRDSSGNHYV (SEQ ID NO: 128)); a heavy chain having three CDRs comprising the amino acid sequences DYAMH (SEQ ID NO: 60), LISGDGGSTYYADSVKD (SEQ ID NO: 73) and VLLPCSSTSCYGSVGAFDI (SEQ ID NO: 88), respectively, and a light chain having three CDRs comprising the amino acid sequences GGSDIGRKSVH (SEQ ID NO: 103), SDRDRPS (SEQ ID NO: 117) and QVWDNNSDHYV (SEQ ID NO: 129), respectively; a heavy chain having three CDRs comprising the amino acid sequences NYDMS (SEQ ID NO: 61), RVNWNGGSTTYADAVKD (SEQ ID NO: 74) and EFVGAYDL (SEQ ID NO: 89) and a heavy chain having three CDRs comprising the amino acid sequences TGTSSDVGGYNYVS (SEQ ID NO: 104), DVSNRPS (SEQ ID NO: 116) and NSRDSSGNHYV (SEQ ID NO: 128) respectively. ID NO: 118) and SSYTSSTLP (SEQ ID NO: 130));A heavy chain having three CDRs comprising the amino acid sequences of GLYIH (SEQ ID NO:62), WIIPIFGTANYAQKFED (SEQ ID NO:75), and GLRWGIWGWFDP (SEQ ID NO:90), respectively, and a light chain having three CDRs comprising the amino acid sequences of RASQSIGNSLA (SEQ ID NO:105), GASSRAT (SEQ ID NO:119), and QQHTIPTFS (SEQ ID NO:131), respectively; a heavy chain having three CDRs comprising the amino acid sequences of DNAIS (SEQ ID NO:63), WIIPIFGKPNYAQKFED (SEQ ID NO:76), and TMVRGFLGVMDV (SEQ ID NO:91), respectively, and a light chain having three CDRs comprising the amino acid sequences of RASQGIGSYLA (SEQ ID NO:106), AASTLQS (SEQ ID NO:120), and QQLNNYPIT (SEQ ID NO:131), respectively. NO: 132)); a light chain having three CDRs comprising the amino acid sequence SYAMS (SEQ ID NO: 64), AISGSGGSTYYADSVKD (SEQ ID NO: 77), and DQFVTIFGVPRYGMDV (SEQ ID NO: 92), respectively, and a light chain having three CDRs comprising the amino acid sequence SGDKLGNKYAY (SEQ ID NO: 107), QDIKRPS (SEQ ID NO: 121), and QTWDNSVV (SEQ ID NO: 133); a heavy chain having three CDRs comprising the amino acid sequence SYAIS (SEQ ID NO: 57), WIIPIFGTANYAQKFED (SEQ ID NO: 78), and GRQMFGAGIDF (SEQ ID NO: 93), respectively, and a heavy chain having three CDRs comprising the amino acid sequence TRSSGSIDSNYVQ (SEQ ID NO: 108), EDNQRPS (SEQ ID NO: 115), and QSYDSNNRHVI (SEQ ID NO: 137) respectively. A light chain having three CDRs comprising the amino acid sequences TYALN (SEQ ID NO: 65), RIVPLIGLVNYAHNFED (SEQ ID NO: 79), and EVYGGNSDY (SEQ ID NO: 94), respectively, and a light chain having three CDRs comprising the amino acid sequences TRSSGNIGTNYVQ (SEQ ID NO: 109), EDYRRPS (SEQ ID NO: 122), and QSYHSSGWE (SEQ ID NO: 135), respectively;A heavy chain having three CDRs comprising the amino acid sequences SHGIT (SEQ ID NO: 66), WISAHNGHASNAQKVED (SEQ ID NO: 80), and VHAALYYGMDV (SEQ ID NO: 95), respectively, and a light chain having three CDRs comprising the amino acid sequences GGNNIGSKGVH (SEQ ID NO: 110), DDSDRPS (SEQ ID NO: 123), and QVWDSSSDHWV (SEQ ID NO: 136), respectively; a heavy chain having three CDRs comprising the amino acid sequences RHGMH (SEQ ID NO: 67), VISHDGSVKYYADSMKD (SEQ ID NO: 81), and GLSYQVSGWFDP (SEQ ID NO: 96), respectively, and a light chain having three CDRs comprising the amino acid sequences TRSSGSIASNYVQ (SEQ ID NO: 111), EDNQRPS (SEQ ID NO: 115), and QSYDSTTPSV (SEQ ID NO: 137), respectively. a light chain having three CDRs comprising the amino acid sequences SYGIS (SEQ ID NO: 58), WTSPHLTAFAQILED (SEQ ID NO: 82), and VHPVFSYALDV (SEQ ID NO: 97), respectively, and a light chain having three CDRs comprising the amino acid sequences TRSSGSIASNYVQ (SEQ ID NO: 112), EDNQRPS (SEQ ID NO: 115), and QSYDGITVI (SEQ ID NO: 138), respectively; a heavy chain having three CDRs comprising the amino acid sequences TYAFS (SEQ ID NO: 68), RIIPILGIANYAQKFED (SEQ ID NO: 83), and DGYGSDPVL (SEQ ID NO: 98), respectively, and a heavy chain having three CDRs comprising the amino acid sequences TRSSGSIASHYVQ (SEQ ID NO: 113), EDNKRPS (SEQ ID NO: 124), and QSYDSSNRWV (SEQ ID NO: 139), respectively. NO: 139)) of the light chain; or a heavy chain having three CDRs (which respectively comprise the amino acid sequences NYGIS (SEQ ID NO: 69), WISAYNGNTNYAQKVED (SEQ ID NO: 84) and GDFRKPFDY (SEQ ID NO: 99)) and a light chain having three CDRs (which respectively comprise the amino acid sequences TLRSGLNVGSYRIY (SEQ ID NO: 114), YKSDSNKQQAS (SEQ ID NO: 125) and MIWYSSAVV (SEQ ID NO: 140));wherein the antibody binds to human PD-L1.;

[0011] In one aspect, the antibody is monovalent or divalent. In another aspect, the antibody is a single chain antibody.

[0012] The present invention provides a single-chain antibody, which comprises: a V comprising SEQ ID NO: 1 H Nucleotide sequence and V containing SEQ ID NO:3 L Nucleotide sequence; V containing SEQ ID NO:5 H Nucleotide sequence and V containing SEQ ID NO:7 L Nucleotide sequence; V containing SEQ ID NO:9 H Nucleotide sequence and V containing SEQ ID NO:11 L Nucleotide sequence; V containing SEQ ID NO: 13 H Nucleotide sequence and V containing SEQ ID NO:15 L Nucleotide sequence; V containing SEQ ID NO: 17 H Nucleotide sequence and V containing SEQ ID NO: 19 L Nucleotide sequence; V containing SEQ ID NO: 21 H Nucleotide sequence and V containing SEQ ID NO:23 L Nucleotide sequence; V containing SEQ ID NO: 25 H Nucleotide sequence and V containing SEQ ID NO:27 L Nucleotide sequence; V containing SEQ ID NO: 29 H Nucleotide sequence and V containing SEQ ID NO:31 L Nucleotide sequence; V containing SEQ ID NO:33 H Nucleotide sequence and V containing SEQ ID NO:35 L Nucleotide sequence; V containing SEQ ID NO: 37 H Nucleotide sequence and V containing SEQ ID NO:39 L Nucleotide sequence; V containing SEQ ID NO:41 H Nucleotide sequence and V containing SEQ ID NO:43 L Nucleotide sequence; V containing SEQ ID NO:45 H Nucleotide sequence and V containing SEQ ID NO:47 L Nucleotide sequence; V containing SEQ ID NO:49 H Nucleotide sequence and V containing SEQ ID NO:51 LNucleotide sequence; or V containing SEQ ID NO:53 H Nucleotide sequence and V containing SEQ ID NO:55 L Nucleotide sequence.

[0013] In another aspect, the present invention provides a single-chain antibody comprising: a V sequence comprising SEQ ID NO: 2 H Amino acid sequence and V containing SEQ ID NO:4 L Amino acid sequence; V containing SEQ ID NO:6 H Amino acid sequence and V containing SEQ ID NO:8 L Amino acid sequence; V containing SEQ ID NO: 10 H Amino acid sequence and V containing SEQ ID NO:12 L Amino acid sequence; V containing SEQ ID NO: 14 H Amino acid sequence and V containing SEQ ID NO:16 L Amino acid sequence; V containing SEQ ID NO: 18 H Amino acid sequence and V containing SEQ ID NO:20 L Amino acid sequence; V containing SEQ ID NO: 22 H Amino acid sequence and V containing SEQ ID NO:24 L Amino acid sequence; V containing SEQ ID NO: 26 H Amino acid sequence and V containing SEQ ID NO:28 L Amino acid sequence; V containing SEQ ID NO: 30 H Amino acid sequence and V containing SEQ ID NO:32 L Amino acid sequence; V containing SEQ ID NO:34 H Amino acid sequence and V containing SEQ ID NO:36 L Amino acid sequence; V containing SEQ ID NO: 38 H Amino acid sequence and V containing SEQ ID NO:40 L Amino acid sequence; V containing SEQ ID NO:42 H Amino acid sequence and V containing SEQ ID NO:44 L Amino acid sequence; V containing SEQ ID NO:46 H Amino acid sequence and V containing SEQ ID NO:48 L Amino acid sequence; V containing SEQ ID NO: 50 H Amino acid sequence and V containing SEQ ID NO:52 LAmino acid sequence; or V comprising SEQ ID NO: 54 H Amino acid sequence and V containing SEQ ID NO:56 L Amino acid sequence.

[0014] In some aspects, the antibody has -5 M-10 -12 The binding affinity is in the range of M.

[0015] In another aspect, the antibody is a bispecific antibody that also binds to a tumor-associated antigen, a cytokine, or a cell surface receptor. For example, the tumor-associated antigen is CAIX. For example, the cytokine is IL-10. For example, the cell surface receptor is CCR4, IL21R, BTLA, HVEM, or TIM3.

[0016] The present invention provides antibodies linked to therapeutic agents. For example, the therapeutic agent is a toxin, a radiolabel, a siRNA, a small molecule, or a cytokine.

[0017] The present invention provides cells that produce any of the above antibodies.

[0018] The present invention also provides a method for selectively killing tumor cells, comprising contacting the cells with any of the above antibodies. In one aspect, selective killing is produced by antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP). In another aspect, the tumor cells express PD-L1.

[0019] The present invention also provides a method for preventing or reversing T cell exhaustion, which comprises administering a composition containing any of the above antibodies to a subject in need thereof.

[0020] The present invention also provides a method for improving the immune response to an antigen, comprising administering a composition containing any of the above antibodies to a subject in need thereof. In one aspect, the antigen is a viral antigen, a bacterial antigen, or a tumor-associated antigen. In another aspect, the viral antigen is HIV. In another aspect, the tumor-associated antigen is CAIX. In another aspect, the antibody is administered before or after exposure to the antigen. In another aspect, the administration of the antibody causes an increase in antigen-specific T cell activity. In another aspect, the T-cell is an effector T cell.

[0021] The present invention also provides a method for treating or alleviating the symptoms of cancer, comprising administering a composition containing any of the above antibodies to a subject in need thereof. For example, the cancer is renal cell carcinoma or breast cancer. For example, the cancer is a cancer in which PD-L1 is overexpressed. In another example, the cancer is a cancer that induces T cell exhaustion.

[0022] The present invention also provides a method for treating or alleviating the symptoms of chronic viral infection, which comprises administering a composition containing any of the above antibodies to a subject in need thereof. For example, the chronic viral infection is HIV infection. For example, the chronic viral infection is a viral infection that induces T cell exhaustion.

[0023] The present invention provides nucleic acid sequences comprising the nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53 or 55.

[0024] In another aspect, the invention provides a nucleic acid sequence encoding a polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 or 56.

[0025] In another aspect, the invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 or 56.

[0026] In another aspect, the invention provides a vector comprising a nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or 55. The invention provides a vector comprising a nucleic acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56. The invention further provides a cell comprising any one of the above vectors.

[0027] Routes of administration in any of the methods of the present disclosure include, but are not limited to, parenteral (eg, intravenous), intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal administration.

[0028] The subject in any method of the present disclosure is, for example, a mammal. The mammal is, for example, a human.

[0029] Other features and advantages of the invention will be apparent from the following detailed description and claims, and are encompassed therein. Sequence Listing <110> Dana-Farber Cancer Institute, Inc. Marasco, Wayne A. Sui, Jianhua <120> Human monoclonal anti-PD-L1 antibodies and methods of use <130> 20363-068001WO <140> PCT / US13 / 63509 <141> 2013-10-04 <150> US 61 / 709,731 <151> 2012-10-04 <150> US 61 / 779,969 <151> 2013-03-13 <160> 140 <170> PatentIn version 3.5 <210> 1 <211> 372 <212> DNA <213> Artificial sequence <220> <223> Variable heavy chain of Ab-14 <400> 1 caggtgcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta cacctttacc agctatggta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcagcgctt acaatggtaa cacaaactat 180 gcacagaagc tccagggcag agtcaccatg accacagaca catccacgag cacagcctac 240 atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagagctcta 300 cctagtggga ctatactggt cggaggttgg ttcgacccct ggggccaggg aaccctggtc 360 accgtctcct ca 372 <210> 2 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-14 <400> 2 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Leu Pro Ser Gly Thr Ile Leu Val Gly Gly Trp Phe Asp 100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 3 <211> 333 <212> DNA <213> Artificial sequence <220> <223> Variable light chain of Ab-14 <400> 3 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60 tcctgcaccc gcagcagtgg caacattgcc agcaattatg tgcagtggta ccaacagcgc 120 ccgggcagtg cccccaccac tgtgatctat gaggataacc aaagaccctc tggggtccct 180 gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240 ctgaagactg aggacgaggc tgactactac tgtcagtctt atgatagcag caatctttgg 300 gtgttcggcg gagggaccaa gctgaccgtc cta 333 <210> 4 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Variable light chain of Ab-14 <400> 4 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Asn Ile Ala Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 5 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-16 <400> 5 gaggtgcagc tggtgcagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgccc tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggtg gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagacgtg 300 tttccagaga ctttttcgat gaactacggt atggacgtct ggggccaagg aaccctggtc 360 accgtctcct ca 372 <210> 6 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-16 <400> 6 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Val Phe Pro Glu Thr Phe Ser Met Asn Tyr Gly Met Asp 100 105 110 Click Download to save Val Trp Gly Gln Gly Thr Leu mp3 youtube com 115 120 <210> 7 <211> 324 <212> DNA <213> The snowstorm <220> <223> Ab-16 snowflakes <400> 7 tcttctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc acatgccaag gagacagcct cagaagctat tatgcaagct ggtaccagca gaagccagga caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcgga gatgaggctg actattactg taactcccgg gacagcagtg gtaaccatta tgtcttcgga actgggacca aggtcaccgt ccta <210> 8 <211> 108 <212> PRT <213> The snowstorm <220> <223> Ab-16 snowflakes <400> 8 Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His 85 90 95 Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 <210> 9 <211> 384 <212> DNA <213> Synthetic sequence <220> <223> Variable heavy chain of Ab-22 <400> 9 caggtgcagc tggtgcagtc tgggggaggc gtggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttgat gattatgcca tgcactgggt ccgtcaagct 120 ccagggaagg gtctggagtg ggtctctctt attagtgggg atggtggtag cacatactat 180 gcagactctg tgaagggccg attcaccatc tccagagaca acagcaaaaa ctccctgtat 240 ctgcaaatga acagtctgag aactgaggac accgccttgt attactgtgc aaaagtgctc 300 ctcccctgta gtagtaccag ctgctatgga agcgtcggtg cttttgatat ctggggccaa 360 gggaccacgg tcaccgtctc ctca 384 <210> 10 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-22 <400> 10 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Val Leu Leu Pro Cys Ser Ser Thr Ser Cys Tyr Gly Ser Val 100 105 110 Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 11 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Variable light chain of Ab-22 <400> 11 taggacgatg agctcggtcc cagctccgaa gacataatga tcactattat tatcccacac 60 ctgacagtaa taatcggcct catcaccggc ttcgaccctg ctgatggtca gggtggccgt 120 gttcccagag ttggagccag agaatcgctc agagatccct gagggccggt ccctatcaga 180 gtagatgacc aacgcagggg cctggcctgg cttctgctgg taccagtgca cactcttcct 240 tccaatgtcg cttcccccac aggtaatcct ggccgtcttt cctggggcca ctgacactga 300 gggtgcctga gtcagcacag gcag 324 <210> 12 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Variable light chain of Ab-22 <400> 12 Leu Pro Val Leu Thr Gln Ala Pro Ser Val Ser Val Ala Pro Gly Lys 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Ser Asp Ile Gly Arg Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Ala Leu Val Ile Tyr 35 40 45 Ser Asp Arg Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn Asn Ser Asp His 85 90 95 Tyr Val Phe Gly Ala Gly Thr Glu Leu Ile Val Leu 100 105 <210> 13 <211> 351 <212> DNA <213> Artificial sequence <220> <223> Variable heavy chain of Ab-30 <400> 13 caggtgcagc tggtgcagtc tgggggaagt gtggtacggc ctggggaatc cctcagactc 60 tcctgtgtag cctctggatt catctttgat aattatgaca tgagttgggt ccgccaagtt 120 ccagggaagg ggctggagtg ggtctctcgt gttaattgga atggtggtag cacaacttat 180 gcagacgctg tgaagggccg attcaccatc tccagagaca acaccaagaa ctccctgtat 240 ctacaaatga acaacctgag agccgaagac acggccgtgt attactgtgt gcgcgagttt 300 gtcggtgctt atgatctctg gggccagggg accacggtca ccgtctcctc a 351 <210> 14 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-30 <400> 14 Gln Val Gln Leu Val Gln Ser Gly Gly Ser Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Ile Phe Asp Asn Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Val Asn Trp Asn Gly Gly Ser Thr Thr Tyr Ala Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Glu Phe Val Gly Ala Tyr Asp Leu Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 15 <211> 327 <212> DNA <213> Artificial sequence <220> <223> Variable light chain of Ab-30 <400> 15 cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60 tcctgcactg gaaccagcag tgacgttggt ggttataact atgtctcctg gtaccaacaa 120 cacccaggca aagcccccaa actcatgatt tatgatgtca gtaatcggcc ctcaggggtt 180 tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240 caggctgagg acgaggctga ttattactgc agctcatata caagcagcac tctgccgttc 300 ggcggaggga ccaagctgac cgtccta 327 <210> 16 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Variable light chain of Ab-30 <400> 16 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Thr Leu Pro Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 17 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Variable heavy chain of Ab-31 <400> 17 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc caggggccac agtgaaggtc 60 tcctgcaagg tttttggaga caccttccgc ggcctctata tacactgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcccta tctttggtac agcaaactac 180 gcacagaagt tccagggcag agtcacgatt accacggacg aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagcggacta 300 cgttggggga tctggggctg gttcgacccc tggggccagg gcaccctggt caccgtctcc 360 tca 363 <210> 18 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Variable Heavy Chain of Ab-31 <400> 18 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Val Ser Cys Lys Val Phe Gly Asp Thr Phe Arg Gly Leu 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Gly Leu Arg Trp Gly Ile Trp Gly Trp Phe Asp Pro Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 19 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Variable light chain of Ab-31 <400> 19 gaaattgtgt tgacgcagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtattggc aacagcttag cctggtacca gcagaaacct 120 ggccaggctc ccaggctcct catgtatggt gcatccagca gggccactgg catcccagac 180 aggttcagtg gcagtggggc tgggacagac ttcactctca ccatcagcag cctagagcct 240 gaagattttg caacgtatta ctgtcagcag catactatcc caacattctc tttcggccct 300 gggaccaaag tggaagtcaa a 321 <210> 20 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Variable light chain of Ab-31 <400> 20 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Asn Ser 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Met 35 40 45 Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ala Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Thr Ile Pro Thr Phe 85 90 95 Ser Phe Gly Pro Gly Thr Lys Val Glu Val Lys 100 105 <210> 21 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Variable heavy chain of Ab-32 <400> 21 gaggtgcagc tggtgcagtc tggggctgag ctgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttttggagg caccttcagt gacaatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg gatggggggc atcattccta tctttggaaa accaaactac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cactgcctac 240 atggtcctga gcagcctgag atctgaggac acggccgtat attactgtgc gagaactatg 300 gttcggggct ttcttggggt tatggacgtc tggggccaag ggaccacggt caccgtctcc 360 tca 363 <210> 22 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-32 <400> 22 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Phe Gly Gly Thr Phe Ser Asp Asn 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Lys Pro Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Methionine Valine Leucine Serine Serine Leucine Arginine Serine Glutamic Acid Aspartic Acid Threonine Alanine Valine Tyrosine Tyrosine Cysteine 85 90 95 Alanine Arginine Threonine Methionine Valine Arginine Glycine Phenylalanine Leucine Glycine Valine Methionine Aspartic Acid Valine Tryptophan Glycine 100 105 110 Glutamine Glycine Threonine Threonine Valine Threonine Valine Serine Serine 115 120 <210> 23 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Variable Light Chain of Ab-32 <400> 23 gatattgtga tgacccagac tccatccttc ctgtccgcat ccataggaga cagagtcacc 60 atcacttgcc gggccagtca gggcattggc agttatttag cctggtatca gcaaagacca 120 ggggaagccc ctaagctcct gatctatgct gcatcgactt tgcaaagtgg agtcccatca 180 aggttcagcg gcagtggatc tgggacggac ttcactctca caatcagcaa cctgcagcct 240 gaagattttg caacttatta ctgtcaacag cttaataatt acccgatcac cttcggccaa 300 gggacacgac tggagattaa a 321 <210> 24 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-32 <400> twenty four Asp Ile Val Met Thr Gln Thr Pro Ser Phe Leu Ser Ala Ser Ile Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Gly Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Arg Pro Gly Glu Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Asn Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 25 <211> 375 <212> DNA <213> Artificial sequence <220> <223> Variable heavy chain of Ab-38 <400> 25 caggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcag 300 ttcgttacga tttttggagt gccaagatac ggtatggacg tctggggcca agggaccacg 360 gtcaccgtct cctca 375 <210> 26 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-38 <400> 26 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gln Phe Val Thr Ile Phe Gly Val Pro Arg Tyr Gly Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 27 <211> 315 <212> DNA <213> Artificial sequence <220> <223> Variable light chain of Ab-38 <400> 27 cagtctgccc tgactcagcc accctcagtg tccgtgtccc caggacagac agccaacatc 60 ccctgctctg gagataaatt ggggaataaa tatgcttact ggtatcagca gaagccaggc 120 cagtcccctg tactgctcat ctatcaagat atcaagcggc cctcaaggat ccctgagcga 180 ttctctggct ccaactctgc ggacacagcc actctgacca tcagcgggac ccaggctatg 240 gatgaggctg actattactg tcagacgtgg gacaacagcg tggtcttcgg cggcgggacc 300 aagctgaccg tcctc 315 <210> 28 <211> 105 <212> PRT <213> Artificial sequence <220> <223> Variable light chain of Ab-38 <400> 28 Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Asn Ile Pro Cys Ser Gly Asp Lys Leu Gly Asn Lys Tyr Ala 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Leu Ile Tyr 35 40 45 Gln Asp Ile Lys Arg Pro Ser Arg Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Ala Asp Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Asp Asn Ser Val Val Phe 85 90 95 Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 29 <211> 360 <212> DNA <213> Artificial sequence <220> <223> Variable heavy chain of Ab-42 <400> 29 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc 120 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr cctggacaag ggcttgagtg gatgggaggg atcatcccta tctttggtac agcaaactac 180 Pro Trp Thr Lys Gly Leu Ser Trp Gly Gly Ile Ile Pro Ile Phe Gly Thr Gln Asn Tyr gcacagaagt tccagggcag agtcacgatt accgcggaca aatccacgag cacagcctac 240 Ala Gln Lys Phe Gln Gly Gln Ser Thr Ile Thr Ala Gly Asn Ser His Glu His Ser Tyr atggagctga gcagcctgag atctgaggac acggccgtct attactgtgc gagagggcgt 300 Met Glu Leu Ser Gln Pro Glu Ile Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg caaatgttcg gtgcgggaat tgatttctgg ggcccgggca ccctggtcac cgtctcctca 360 Gln Met Phe Gly Ala Gly Asn Asp Phe Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser <210> 30<210> 30 <211> 120<211> 120 <212> PRT<212> PRT <213> 人工序列<213> Artificial Sequence <220><220> <223> Ab-42的可变重链 <223> Variable heavy chain of Ab-42 <400> 30 <400> 30 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Gln Met Phe Gly Ala Gly Ile Asp Phe Trp Gly Pro 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 31 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Variable light chain of Ab-42 <400> 31 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60 tcctgcaccc gcagcagtgg cagcattgac agcaactatg tgcagtggta ccagcagcgc 120 ccgggcagcg cccccaccac tgtgatctat gaggataacc aaagaccctc tggggtccct 180 gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240 ctgaagactg aggacgaggc tgactactac tgtcagtctt atgatagcaa caatcgtcat 300 gtgatattcg gcggagggac caagctgacc gtccta 336 <210> 32 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-42 <400> 32 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Asp Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Asn Asn Arg His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 33 <211> 354 <212> DNA <213> Artificial sequence <220> <223> Variable heavy chain of Ab-46 <400> 33 gaggtgcagc tggtggagtc tggggctgaa gtaaagaagc ctgggtcctc ggtgaaagtc 60 tcctgcaagg tttcaggagg cacattcggc acctatgctc tcaactgggt gcgccaggcc 120 cctggacaag ggcttgagtg gatgggaagg atcgtccctc tcattggtct agtaaactac 180 gcacataact ttgagggcag aatctcgatt accgcggaca agtccacggg cacagcctac 240 atggaactga gcaacctgag atctgacgac acggccgtgt attactgtgc gagagaggtc 300 tacggtggta actccgacta ctggggccag ggaaccctgg tcaccgtctc ctca 354 <210> 34 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Variable heavy chain of Ab-46 <400> 34 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Gly Thr Phe Gly Thr Tyr 20 25 30 Ala Leu Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Val Pro Leu Ile Gly Leu Val Asn Tyr Ala His Asn Phe 50 55 60 Glu Gly Arg Ile Ser Ile Thr Ala Asp Lys Ser Thr Gly Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Asn Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Tyr Gly Gly Asn Ser Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 35 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Variable light chain of Ab-46 <400> 35 aattttatgc tgactcagcc ccactcagtg tcggagtctc cggggaagac ggtaaccatc 60 tcctgcactc gcagtagtgg caacattggc accaactatg tgcagtggta ccagcagcgc 120 ccgggcagtg cccccgtcgc tttgatctac gaggattatc gaagaccctc tggggtccct 180 gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcat catctctgga 240 ctgaagcctg aggacgaggc tgactactac tgtcagtctt atcatagcag cggttgggaa 300 ttcggcggag ggaccaagct gaccgtcctc 330 <210> 36 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-46 <400> 36 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Asn Ile Gly Thr Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Val Ala Leu 35 40 45 Ile Tyr Glu Asp Tyr Arg Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Ile Ile Ser Gly 65 70 75 80 Leu Lys Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr His Ser 85 90 95 Ser Gly Trp Glu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 37 <211> 360 <212> DNA <213> Artificial sequence <220> <223> Variable heavy chain of Ab-50 <400> 37 caggtgcagc tggtgcagtc tggaggtgag gtgaagaagc cgggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta caccttgagc agtcatggta taacctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcagcgctc acaatggtca cgctagcaat 180 gcacagaagg tggaggacag agtcactatg actactgaca catccacgaa cacagcctac 240 atggaactga ggagcctgac agctgacgac acggccgtgt attactgtgc gagagtacat 300 gctgccctct actatggtat ggacgtctgg ggccaaggaa ccctggtcac cgtctcctca 360 <210> 38 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Variable heavy chain of Ab-50 <400> 38 Gln Val Gln Leu Val Gln Ser Gly Gly Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu Ser Ser His 20 25 30 Gly Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala His Asn Gly His Ala Ser Asn Ala Gln Lys Val 50 55 60 Glu Asp Arg Val Thr Met Thr Thr Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val His Ala Ala Leu Tyr Tyr Gly Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 39 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Variable light chain of Ab-50 <400> 39 cagtctgtgc tgactcagcc accctcggtg tcagtggccc caggacagac ggccaggatt 60 acctgtgggg gaaacaacat tggaagtaaa ggtgtgcact ggtatcagca gaagccaggc 120 caggcccctg tactggtcgt ctatgatgat agtgaccggc cctcagggat ccctgagcga 180 ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240 gatgaggccg actattactg tcaggtgtgg gatagtagta gtgatcattg ggtgttcggc 300 ggagggacca agctgaccgt ccta 324 <210> 40 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-50 <400> 40 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Gly Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 41 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-52 <400> 41 caggtgcagc tgcaggagtc ggggggaggc gtggtgcagc ctgggaggtc cctgagactc 60 tcctgttcag cctctggatt caccttcagc agacatggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtg atatcacatg atggaagtgt aaaatactat 180 gcagactcca tgaagggccg attcagcatc tccagagaca attccaacaa cacactgtat 240 ctccaaatgg acagcctgag agctgacgac acggccgttt attactgtgc gagaggactg 300 tcgtaccagg tgtcggggtg gttcgacccc tggggccagg gcaccctggt caccgtctcc 360 tca 363 <210> 42 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-52 <400> 42 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Arg His 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser His Asp Gly Ser Val Lys Tyr Tyr Ala Asp Ser Met 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Asn Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Ser Tyr Gln Val Ser Gly Trp Phe Asp Pro Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 43 <211> 333 <212> DNA <213> Artificial sequence <220> <223> Variable light chain of Ab-52 <400> 43 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60 tcctgcaccc gcagcagtgg cagcattgcc agcaactatg tgcagtggta ccagcagcgc 120 ccgggcagtg cccccaccac tgtgatctat gaggataacc aaagaccctc tggggtccct 180 gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240 ctgaagactg aggacgaggc tgactactac tgtcagtctt atgatagcac caccccttcg 300 gtgttcggcg gcgggaccaa gctgaccgtc cta 333 <210> 44 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-52 <400> 44 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Thr Thr Pro Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 45 <211> 360 <212> DNA <213> Artificial sequence <220> <223> Variable heavy chain of Ab-55 <400> 45 caggtgcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta cacctttacc agctatggta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg accagccctc ataatggtct cacagcattt 180 gcacagatcc tagagggccg agtcaccatg accacagaca catccacgaa cacagcctac 240 atggaattga ggaacctgac atttgatgac acggccgttt atttctgtgc gaaagtacat 300 cctgtcttct cttatgcgtt ggacgtctgg ggccaaggca ccctggtcac cgtctcctca 360 <210> 46 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Variable heavy chain of Ab-55 <400> 46 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Thr Ser Pro His Asn Gly Leu Thr Ala Phe Ala Gln Ile Leu 50 55 60 Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Asn Leu Thr Phe Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Val His Pro Val Phe Ser Tyr Ala Leu Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 47 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Variable light chain of Ab-55 <400> 47 aattttatgc tgactcagcc ccactctgtg tcggagtccc cggggaagac ggtaaccatc 60 tcctgcaccc gcagcagtgg cagcattgcc agcaactatg tacagtggta ccagcagcgc 120 ccgggcagtt cccccaccac tgtgatctat gaagataacc aaagaccctc tggggtccct 180 gatcggttct ctggctccat cgacacctcc tccaactctg cctccctcac catctctgga 240 ctgaagacta aggacgaggc ggactactac tgtcagtctt atgatggcat cactgtgatt 300 ttcggcggag ggaccaagtt gaccgtccta 330 <210> 48 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-55 <400> 48 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Thr Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Lys Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly 85 90 95 Ile Thr Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 49 <211> 354 <212> DNA <213> Artificial sequence <220> <223> Variable heavy chain of Ab-56 <400> 49 gaggtgcagc tggtggagtc tggagctgag gtgatgaacc ctgggtcctc ggtgagggtc 60 tcctgcaggg gttctggagg cgacttcagt acctatgctt tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaagg atcatcccta tccttggtat agcaaactac 180 gcacagaagt tccagggcag ggtcacgatt accgcggaca aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgacgat acggccgtgt attactgtgc gagagatggc 300 tatggttcgg acccggtgct atggggccag ggcaccctgg tcaccgtctc ctca 354 <210> 50 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Variable heavy chain of Ab-56 <400> 50 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Met Asn Pro Gly Ser 1 5 10 15 Ser Val Arg Val Ser Cys Arg Gly Ser Gly Gly Asp Phe Ser Thr Tyr 20 25 30 Ala Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Tyr Gly Ser Asp Pro Val Leu Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 51 <211> 333 <212> DNA <213> Artificial sequence <220> <223> Variable light chain of Ab-56 <400> 51 aattttatgc tgactcagcc ccactctgtg tcggggtctc cggggaagac ggtaaccctc 60 ccctgcaccc gcagcagtgg cagcattgcc agccactatg tccagtggta ccagcagcgc 120 ccgggcagtg cccccaccac tgtgatctat gaggataaca agagaccctc tggggtccct 180 gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcag catctctgga 240 ctgaagactg aggacgaggc tgactactac tgtcagtctt atgatagcag caatcgttgg 300 gtgttcggcg gagggaccaa gctgaccgtc cta 333 <210> 52 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-56 <400> 52 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Gly Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Leu Pro Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser His 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Ser Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Ser Asn Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 53 <211> 354 <212> DNA <213> Artificial sequence <220> <223> Variable heavy chain of Ab-65 <400> 53 gaggtgcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta cacctttacc aactatggta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcagcgctt acaatggtaa cacaaactat 180 gcacagaagg tccagggcag agtcaccatg accacagaca catccacgag cacaggctac 240 atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagaggagat 300 tttcggaaac cctttgacta ctggggccag ggaaccctgg tcaccgtctc ctca 354 <210> 54 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Variable heavy chain of Ab-65 <400> 54 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Val 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Gly Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Phe Arg Lys Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 55 <211> 345 <212> DNA <213> Artificial sequence <220> <223> Variable light chain of Ab-65 <400> 55 ctgcctgtgc tgactcagcc ggcttccctc tctgcatccc ccggagcatc agccagtctc 60 acctgcacct tacgcagtgg cctcaatgtt ggttcctaca ggatatactg gtaccagcag 120 aagccaggga gtcgtcccca gtatctcctg aactacaaat cagactcaaa taaacagcag 180 gcctctggag tccccagccg cttctctgga tccaaggatg cttcggccaa tgcagggatt 240 ttactcatct ccgggctcca gtctgaggat gaggctgact attactgtat gatttggtac 300 agcagcgctg tggtattcgg cggagggacc aagctgaccg tccta 345 <210> 56 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-65 <400> 56 Leu Pro Val Leu Thr Gln Pro Ala Ser Leu Ser Ala Ser Pro Gly Ala 1 5 10 15 Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Leu Asn Val Gly Ser 20 25 30 Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Arg Pro Gln Tyr 35 40 45 Leu Leu Asn Tyr Lys Ser Asp Ser Asn Lys Gln Gln Ala Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile 65 70 75 80 Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 85 90 95 Met Ile Trp Tyr Ser Ser Ala Val Val Phe Gly Gly Gly Thr Lys Leu 100 105 110 Thr Val Leu 115 <210> 57 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR1 consensus region, Ab-42 <400> 57 Ser Tyr Ala Ile Ser 1 5 <210> 58 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR1 of Ab-14 and Ab-55 <400> 58 Ser Tyr Gly Ile Ser 1 5 <210> 59 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR1 of Ab-16 <400> 59 Ser Tyr Ala Leu Ser 1 5 <210> 60 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Ab-22 non-human heavy chain CDR1 <400> 60 Asp Tyr Ala Met His 1 5 <210> 61 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR1 of Ab-30 <400> 61 Asn Tyr Asp Met Ser 1 5 <210> 62 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR1 of Ab-31 <400> 62 Gly Leu Tyr Ile His 1 5 <210> 63 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR1 of Ab-32 <400> 63 Asp Asn Ala Ile Ser 1 5 <210> 64 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR1 of Ab-38 <400> 64 Ser Tyr Ala Met Ser 1 5 <210> 65 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR1 of Ab-46 <400> 65 Thr Tyr Ala Leu Asn 1 5 <210> 66 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR1 of Ab-50 <400> 66 Ser His Gly Ile Thr 1 5 <210> 67 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR1 of Ab-52 <400> 67 Arg His Gly Met His 1 5 <210> 68 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR1 of Ab-56 <400> 68 Thr Tyr Ala Phe Ser 1 5 <210> 69 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR1 of Ab-65 <400> 69 Asn Tyr Gly Ile Ser 1 5 <210> 70 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 consensus region <400> 70 Trp Ile Ser Pro Ile Gly Gly Ser Thr Asn Tyr Ala Gln Lys Val Gln 1 5 10 15 Gly <210> 71 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 of Ab-14 <400> 71 Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu Gln 1 5 10 15 Gly <210> 72 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 of Ab-16 <400> 72 Ala Ile Ser Gly Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Asp <210> 73 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 of Ab-22 <400> 73 Leu Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Asp <210> 74 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 of Ab-30 <400> 74 Arg Val Asn Trp Asn Gly Gly Ser Thr Thr Tyr Ala Asp Ala Val Lys 1 5 10 15 Asp <210> 75 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 of Ab-31 <400> 75 Trp Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Glu 1 5 10 15 Asp <210> 76 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 of Ab-32 <400> 76 Trp Ile Ile Pro Ile Phe Gly Lys Pro Asn Tyr Ala Gln Lys Phe Glu 1 5 10 15 Asp <210> 77 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 of Ab-38 <400> 77 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Asp <210> 78 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 of Ab-42 <400> 78 Trp Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Glu 1 5 10 15 Asp <210> 79 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 of Ab-46 <400> 79 Arg Ile Val Pro Leu Ile Gly Leu Val Asn Tyr Ala His Asn Phe Glu 1 5 10 15 Asp <210> 80 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 of Ab-50 <400> 80 Trp Ile Ser Ala His Asn Gly His Ala Ser Asn Ala Gln Lys Val Glu 1 5 10 15 Asp <210> 81 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 of Ab-52 <400> 81 Val Ile Ser His Asp Gly Ser Val Lys Tyr Tyr Ala Asp Ser Met Lys 1 5 10 15 Asp <210> 82 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 of Ab-55 <400> 82 Trp Thr Ser Pro His Asn Gly Leu Thr Ala Phe Ala Gln Ile Leu Glu 1 5 10 15 Asp <210> 83 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 of Ab-56 <400> 83 Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe Glu 1 5 10 15 Asp <210> 84 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 of Ab-65 <400> 84 Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Val Glu 1 5 10 15 Asp <210> 85 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 consensus region <220> <221> Other features <222> (3)..(17) <223> Xaa can be any naturally occurring amino acid <400> 85 Gly Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Asp Val <210> 86 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 of Ab-14 <400> 86 Ala Leu Pro Ser Gly Thr Ile Leu Val Gly Gly Trp Phe Asp Pro 1 5 10 15 <210> 87 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 of Ab-16 <400> 87 Asp Val Phe Pro Glu Thr Phe Ser Met Asn Tyr Gly Met Asp Val 1 5 10 15 <210> 88 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 of Ab-22 <400> 88 Val Leu Leu Pro Cys Ser Ser Thr Ser Cys Tyr Gly Ser Val Gly Ala 1 5 10 15 Phe Asp Ile <210> 89 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 of Ab-30 <400> 89 Glu Phe Val Gly Ala Tyr Asp Leu 1 5 <210> 90 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 of Ab-31 <400> 90 Gly Leu Arg Trp Gly Ile Trp Gly Trp Phe Asp Pro 1 5 10 <210> 91 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 of Ab-32 <400> 91 Thr Met Val Arg Gly Phe Leu Gly Val Met Asp Val 1 5 10 <210> 92 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 of Ab-38 <400> 92 Asp Gln Phe Val Thr Ile Phe Gly Val Pro Arg Tyr Gly Met Asp Val 1 5 10 15 <210> 93 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 of Ab-42 <400> 93 Gly Arg Gln Met Phe Gly Ala Gly Ile Asp Phe 1 5 10 <210> 94 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 of Ab-46 <400> 94 Glu Val Tyr Gly Gly Asn Ser Asp Tyr 1 5 <210> 95 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 of Ab-50 <400> 95 Val His Ala Ala Leu Tyr Tyr Gly Met Asp Val 1 5 10 <210> 96 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 of Ab-52 <400> 96 Gly Leu Ser Tyr Gln Val Ser Gly Trp Phe Asp Pro 1 5 10 <210> 97 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 of Ab-55 <400> 97 Val His Pro Val Phe Ser Tyr Ala Leu Asp Val 1 5 10 <210> 98 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 of Ab-56 <400> 98 Asp Gly Tyr Gly Ser Asp Pro Val Leu 1 5 <210> 99 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 of Ab-65 <400> 99 Gly Asp Phe Arg Lys Pro Phe Asp Tyr 1 5 <210> 100 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR1 consensus region <400> 100 Thr Arg Ser Ser Gly Ser Ile Gly Ser Asn Tyr Val Gln 1 5 10 <210> 101 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR1 of Ab-14 <400> 101 Thr Arg Ser Ser Gly Asn Ile Ala Ser Asn Tyr Val Gln 1 5 10 <210> 102 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR1 of Ab-16 <400> 102 Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser 1 5 10 <210> 103 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR1 of Ab-22 <400> 103 Gly Gly Ser Asp Ile Gly Arg Lys Ser Val His 1 5 10 <210> 104 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR1 of Ab-30 <400> 104 Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser 1 5 10 <210> 105 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR1 of Ab-31 <400> 105 Arg Ala Ser Gln Ser Ile Gly Asn Ser Leu Ala 1 5 10 <210> 106 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR1 of Ab-32 <400> 106 Arg Ala Ser Gln Gly Ile Gly Ser Tyr Leu Ala 1 5 10 <210> 107 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR1 of Ab-38 <400> 107 Ser Gly Asp Lys Leu Gly Asn Lys Tyr Ala Tyr 1 5 10 <210> 108 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR1 of Ab-42 <400> 108 Thr Arg Ser Ser Gly Ser Ile Asp Ser Asn Tyr Val Gln 1 5 10 <210> 109 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR1 of Ab-46 <400> 109 Thr Arg Ser Ser Gly Asn Ile Gly Thr Asn Tyr Val Gln 1 5 10 <210> 110 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR1 of Ab-50 <400> 110 Gly Gly Asn Asn Ile Gly Ser Lys Gly Val His 1 5 10 <210> 111 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR1 of Ab-52 <400> 111 Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn Tyr Val Gln 1 5 10 <210> 112 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR1 of Ab-55 <400> 112 Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn Tyr Val Gln 1 5 10 <210> 113 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR1 of Ab-56 <400> 113 Thr Arg Ser Ser Gly Ser Ile Ala Ser His Tyr Val Gln 1 5 10 <210> 114 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR1 of Ab-65 <400> 114 Thr Leu Arg Ser Gly Leu Asn Val Gly Ser Tyr Arg Ile Tyr 1 5 10 <210> 115 <211> 7 <212> PRT <213> Artificial sequence <220> <223> The light chain CDR2 consensus region of Ab-14, Ab-42, Ab-52, and Ab-55 <400> 115 Glu Asp Asn Gln Arg Pro Ser 1 5 <210> 116 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR2 of Ab-16 <400> 116 Gly Lys Asn Asn Arg Pro Ser 1 5 <210> 117 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR2 of Ab-22 <400> 117 Ser Asp Arg Asp Arg Pro Ser 1 5 <210> 118 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR2 of Ab-30 <400> 118 Asp Val Ser Asn Arg Pro Ser 1 5 <210> 119 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR2 of Ab-31 <400> 119 Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 120 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR2 of Ab-32 <400> 120 Ala Ala Ser Thr Leu Gln Ser 1 5 <210> 121 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR2 of Ab-38 <400> 121 Gln Asp Ile Lys Arg Pro Ser 1 5 <210> 122 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR2 of Ab-46 <400> 122 Glu Asp Tyr Arg Arg Pro Ser 1 5 <210> 123 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR2 of Ab-50 <400> 123 Asp Asp Ser Asp Arg Pro Ser 1 5 <210> 124 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR2 of Ab-56 <400> 124 Glu Asp Asn Lys Arg Pro Ser 1 5 <210> 125 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR2 of Ab-65 <400> 125 Tyr Lys Ser Asp Ser Asn Lys Gln Gln Ala Ser 1 5 10 <210> 126 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR3 consensus region <400> 126 Gln Ser Tyr Asp Ser Ser Thr Trp Val 1 5 <210> 127 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR3 of Ab-14 <400> 127 Gln Ser Tyr Asp Ser Ser Asn Leu Trp Val 1 5 10 <210> 128 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR3 of Ab-16 <400> 128 Asn Ser Arg Asp Ser Ser Gly Asn His Tyr Val 1 5 10 <210> 129 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR3 of Ab-22 <400> 129 Gln Val Trp Asp Asn Asn Ser Asp His Tyr Val 1 5 10 <210> 130 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR3 of Ab-30 <400> 130 Ser Ser Tyr Thr Ser Ser Thr Leu Pro 1 5 <210> 131 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR3 of Ab-31 <400> 131 Gln Gln His Thr Ile Pro Thr Phe Ser 1 5 <210> 132 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR3 of Ab-32 <400> 132 Gln Gln Leu Asn Asn Tyr Pro Ile Thr 1 5 <210> 133 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR3 of Ab-38 <400> 133 Gln Thr Trp Asp Asn Ser Val Val 1 5 <210> 134 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR3 of Ab-42 <400> 134 Gln Ser Tyr Asp Ser Asn Asn Arg His Val Ile 1 5 10 <210> 135 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR3 of Ab-46 <400> 135 Gln Ser Tyr His Ser Ser Gly Trp Glu 1 5 <210> 136 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR3 of Ab-50 <400> 136 Gln Val Trp Asp Ser Ser Ser Asp His Trp Val 1 5 10 <210> 137 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR3 of Ab-52 <400> 137 Gln Ser Tyr Asp Ser Thr Thr Pro Ser Val 1 5 10 <210> 138 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR3 of Ab-55 <400> 138 Gln Ser Tyr Asp Gly Ile Thr Val Ile 1 5 <210> 139 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR3 of Ab-56 <400> 139 Gln Ser Tyr Asp Ser Ser Asn Arg Trp Val 1 5 10 <210> 140 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR3 of Ab-65 <400> 140 Met Ile Trp Tyr Ser Ser Ala Val Val 1 5 BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 .Amino acid sequences of anti-PD-L1 scFv-phage clones (14 clones). Framework regions 1-4 (FW1-4), complementary determining regions 1-3 (CDR1-3) and family nomenclature for both IGHV and IGLV / IGKV are shown. Kabat numbering is used. Key table: "." AA matching the consensus region, "X" non-consensus region AA and "-" is blank (i.e. no AA).

[0031] Figure 2 Binding analysis of huPD-L1 antibodies to human PD-L1 (hPD-L1) expressing cells by FACS. Four types of cells were tested, including the parental cell line 300.9 and 300.9 cells transfected with hPD-L1, hPD-L2, or human C-type lectin domain family 2 member (hCLEC2D). GF1538 is a humanized antibody against hPD-L1. GF1757 is a humanized antibody against hPD-L2. The secondary antibody was PE-goat anti-human IgG.

[0032] Figure 3 Inhibition of hPD-1 binding to hPD-L1 by anti-PD-L1 phage-antibodies in competitive FACS analysis All anti-hPD-L1 antibodies in phage-scFv format were tested for inhibition of binding of hPD1-hFc fusion protein to hPD-L1 expressing 293T cells. 12 pfu of phage-scFv was mixed with about 0.25 μg / mL of soluble hPD1-hFc and added to 293T cells transfected with hPD-L1 expression plasmid. After washing, the cells were incubated with FITC-anti-human IgG antibody to measure the binding of hPD1-hFc to hPD-L1 on the cell surface.

[0033] Figure 4 . Inhibition of hPD-1 binding to hPD-L1 by anti-PD-L1 soluble antibodies in competitive FACS analysis. All anti-hPD-L1 antibodies were pre-incubated with 300.9 cells transfected with hPD-L1 expression plasmid at the indicated concentrations for 30 minutes, followed by the addition of 0.125 μg of hPD-1-mouse IgG2a to each reaction and incubation for an additional 30 minutes. After washing, PE-goat-anti-mouse IgG2a Ab was added followed by washing and FACS analysis. GF1538 is a humanized Ab against hPD-L1. GF1757 is a humanized Ab against hPD-L2.

[0034] Figure 5 .Design and generation of bispecific antibodies.

[0035] Figure 6. Determination of bispecific antibody (bsAb) constructs. A) Schematic representation of a bispecific antibody recognizing CAIX and PD-L1 and the "knob into hole" approach to connect the CH3 domains. B) Schematic representation of three types of bsAb constructs with different mutations in the CH2 domain to alter ADCC activity.

[0036] Figure 7. Generation of bispecific antibodies and their function. A) Protein gel showing the dissociation of the engineered (G37KIHA) antibody under reducing conditions compared to conjugated (non-reduced) control IgG, parental G37 (WT), and bispecific (G37 KIHA + PD-L1 KIHB) antibodies. B) Protein gel showing the dissociation of the engineered (PD-L1 KIHB) antibody under reducing conditions compared to control IgG, parental PDL-1 (WT), and bispecific (G37 KIHA + PD-L1KIHB) antibodies. C) Bispecific antibody and CAIX by flow cytometry + PDL-1 - Analysis of binding of SKRC-52 cells.

[0037] Figure 8 .Functional characterization of PD-L1-specific mAb42. PBMCs from four healthy donors (D1-D4) were cultured in the presence of αPDL1 (mAb42) or control isotype antibody, stimulated with 0.1 μg / ml SEB for 48 hours, and TNFα production was measured by MSD device. Data are presented in triplicate *, p<0.0005.

[0038] Details

[0039] The present invention provides a humanized monoclonal antibody specific for PD-L1, also referred to as B7H1. The antibody is identified by a phage display antibody library selection method using proteoliposome-coupled-PD-L1 as the library selection target. These antibodies represent a new class of human monoclonal antibodies against PD-L1.

[0040] These anti-PD-L1 human monoclonal antibodies are referred to herein as "huPD-L1 antibodies."

[0041] The binding of PD-L1 to PD-1 negatively regulates T cell antigen-specific responses, which is extremely important for tolerance and prevention of autoimmunity and immunopathology. However, excessive PD-L1 / PD-1 interactions, which can be caused by chronic antigen stimulation, can lead to suppression of T cell antigen-specific responses and loss of T cells, which is a characteristic of T cell exhaustion. T cell exhaustion is a state of abnormal T cell function that can occur in chronic infections and cancer. It is defined as: weak effector function, persistent expression of inhibitory receptors, and a transcriptional state that is different from that of functional effector or memory T cells. Exhaustion prevents the management of infection and tumor progression.

[0042] Overexpression of PD-L1 has been detected in different cancers. For example, in breast cancer, PD-L1 is overexpressed and associated with high-risk prognostic factors. In renal cell carcinoma, PD-L1 is upregulated and increased PD-1 expression has also been found in tumor-infiltrating leukocytes. Anti-PD-L1 and anti-PD-1 antibodies have shown some clinical efficacy in renal cell carcinoma in Phase I clinical trials. Therapeutic agents that can be bound to PD-1 or PD-L1 can be used to specifically target tumor cells. Agents that can block the interaction of PD-1 / PD-L1 may be even more useful in treating cancers that have induced T cell exhaustion to escape anti-tumor T cell activity. The use of such agents (used alone or in combination with other anti-cancer therapies) can effectively target tumor cells that overexpress PD-L1 and increase anti-tumor T cell activity, thereby improving the immune response to target tumor cells.

[0043] PD-1 and PD-L1 can also be upregulated by T cells after chronic antigen stimulation (e.g., by chronic infection). During chronic HIV infection, HIV-specific CD8+T cells are functionally impaired, showing reduced ability to produce cytokines and effector molecules and weakened proliferation capacity. PD-1 is highly expressed on HIV-specific CD8+T cells in HIV-infected individuals. Therefore, blocking this pathway can enhance the ability of HIV-specific T cells to proliferate and produce cytokines in response to stimulation with HIV peptides, thereby improving the immune response to HIV. Other chronic infections can also benefit from the use of PD-1 / PD-L1 blockers, such as chronic viral infections, bacterial infections, or parasitic infections.

[0044] The present invention provides human monoclonal antibodies that specifically bind to PD-L1 proteins. The binding of the antibodies of the present invention to PD-L1 interferes with the ability of the ligand to bind to its receptor PD1. Through various mechanisms, huPD-L1 antibodies prevent negative feedback mechanisms that inhibit T cell responses. In some cases, huPD-L1 antibodies prevent, inhibit or reverse T cell exhaustion. Administration of huPD-L1 antibodies can result in increased T cell proliferation, increased antigen-specific T cell activity and increased production of effector cytokines. In some instances, huPD-L1 antibodies promote or enhance antigen-specific immune responses. This immune response can be mediated by effector T cells.

[0045] The huPD-L1 antibody is monovalent or divalent and comprises a single chain or a double chain. Functionally, the binding affinity of the huPD-L1 antibody is in the range of 10 -5 M-10 -12 For example, the binding affinity of huPD-L1 antibody is 10 -6 M-10 -12 M, 10 -7 M-10 -12 M, 10 -8 M-10 -12 M, 10 -9 M-10 -12 M, 10 -5 M-10 -11 M, 10 -6 M-10 -11 M, 10 -7 M-10 -11 M, 10 -8 M-10 -11 M, 10 -9 M-10 -11 M, 10 -10 M-10 -11 M, 10 -5 M-10 -10 M, 10-6 M-10 -10 M, 10 -7 M-10 -10 M, 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 M.

[0046] Additionally, the antibodies of the present invention encompass therapeutic agents including, but not limited to, toxins, radiolabels, siRNAs, or cytokines.

[0047] The huPD-L1 antibody is capable of inducing cell death. Cell death is induced by direct or indirect mechanisms. For example, binding of PD-L1 by the huPD-L1 antibody can lead to complement-dependent cytotoxicity (CDC). Alternatively, the huPD-L1 antibody binds to PD-L1 and leads to the recruitment of a second cell type, which kills PD-L14-expressing target cells. Exemplary mechanisms by which huPD-L1 antibodies mediate cell death by recruiting a second cell type include, but are not limited to, antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Target PD-L1-expressing cell types include tumor cells and T cells (e.g., activated T cells).

[0048] Fourteen unique monoclonal huPD-L1 antibodies were identified. These include Ab-14, Ab-16, Ab-22, Ab-30, Ab-31, Ab-32, Ab-38, Ab-42, Ab-46, Ab-50, Ab-52, Ab-55, Ab-56, and Ab-65.

[0049] The nucleic acid and amino acid sequences of the monoclonal huPD-L1 antibody are provided below:

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058] The amino acid sequences of the heavy and light chain complementarity determining regions of the huPD-L1 antibody are shown below in Tables 15A and 15B.

[0059] Table 15A. Amino acid sequences of the heavy chain complementarity determining regions.

[0060]

[0061]

[0062] Table 15B. Amino acid sequences of the light chain complementarity determining regions.

[0063]

[0064]

[0065] The huPD-L1 antibodies described herein bind to PD-L1. In one aspect, the huPD-L1 antibody has high affinity and high specificity for PD-L1. In another aspect, the huPD-L1 antibody can bind to the PD-1 receptor and prevent, inhibit or block the ligand PD-L1 from binding to its receptor PD-1. In some instances, the huPD-L1 antibody may have some cross-reactivity with PD-L2. In some instances, the huPD-L1 antibody does not show any cross-reactivity with PD-L2. In some instances, the huPD-L1 antibody binds to PD-L1 with higher affinity and / or higher specificity than binding to PD-L2.

[0066] The present invention also describes the characteristics of the following antibodies, which have a specific percentage of identity or similarity with the amino acid or nucleotide sequence of the huPD-L1 antibodies described herein. For example, when compared to a specific region or the full length of any of the huPD-L1 antibodies described herein, the antibody may have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity. Sequence identity or similarity with the nucleic acids and proteins of the present invention can be determined by sequence comparison and / or alignment performed by methods known in the art. For example, the percent sequence identity or similarity to the nucleic acids and proteins of the present invention can be determined using a sequence comparison algorithm (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

[0067] With respect to amino acid sequences, those skilled in the art will readily recognize that single substitutions, deletions or additions to nucleic acid, peptide, polypeptide or protein sequences 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 alterations result 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. Such conservatively modified variants of the huPD-L1 antibodies disclosed herein may exhibit enhanced cross-reactivity to PD-L2 compared to unmodified huPD-L1 antibodies.

[0068] Antibody

[0069] As used herein, the term "antibody" refers 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 with" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides. Antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, dAbs (domain antibodies), single-chain antibodies, F ab 、F ab’ and F (ab')2 Fragments, scFv and F ab Expression library.

[0070] Single-chain Fv ("scFv") polypeptide molecules are covalently linked V H :V L Heterodimers that are self-contained V and V connected by a peptide-encoded linker H - and V L-expression of gene fusions encoding genes (see Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). Many methods have been described to resolve chemical structures for converting naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules (which can fold into a three-dimensional structure that is substantially similar to the structure of the antigen-binding site). See, e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,946,778.

[0071] Very large natural human scFv libraries have been and can be created 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 89:3175-79 (1992)).

[0072] Generally, antibody molecules obtained from humans refer to any of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other in the nature of the heavy chains present in the molecule. Certain classes also have subclasses, such as IgG1, IgG2, and others. In addition, in humans, the light chain may be a kappa chain or a lambda chain. The term "antigen-binding site" or "binding portion" refers to a portion of an immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues in the N-terminal variable ("V") region of the heavy ("H") chain and the light ("L") chain. Three highly different stretches, referred to as "hypervariable regions" in the V region of the heavy and light chains, are inserted between more conservative flanking stretches, referred to as "framework regions" or "FRs". Therefore, the term "FR" refers to the amino acid sequence of the hypervariable regions naturally present in immunoglobulins and adjacent to the hypervariable regions in 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 bound antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity determining regions" or "CDRs". Figure 2 The CDRs of the VH and VL regions of the scFv antibody are shown in FIG.

[0073] The term "epitope" as used herein includes any protein determinant that can specifically bind to an immunoglobulin, scFv or T-cell receptor. Epitope determinants are usually composed of chemically active surface clusters of molecules (e.g., amino acids or sugar side chains), and usually have specific three-dimensional structural characteristics, and specific charge characteristics. For example, antibodies can be generated against the N-terminal or C-terminal peptide of a polypeptide.

[0074] As used herein, the terms "immunobinding" and "immunobinding properties" 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 the immunobinding interaction can be measured by the dissociation constant (K) of the interaction. d ) indicates that the smaller K d =K indicates 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 formation and dissociation of antigen-binding site / antigen complexes, where those rates depend on the concentration of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates equally in both directions. Thus, the "binding rate constant" (K on ) and the "dissociation rate constant" (K off ) Both can be determined by calculation of the concentration and the actual rates of association and dissociation. (See Nature 361: 186-87 (1993)). off / K on The ratio of makes it possible to remove all parameters not related to affinity and is equal to the dissociation constant K d (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473). d ) is ≤10 μM, preferably ≤10 nM, more preferably ≤10 nM, and most preferably ≤100 pM to about 1 pM (as measured by an assay such as a radioligand binding assay or similar assays known to those skilled in the art).

[0075] The PD-L1 protein of the present invention or its derivative, fragment, analog, homologue or orthologue can be used as an immunogen in the production of antibodies (which immunologically specifically bind to these protein components). The PD-L1 protein coupled to proteoliposomes or its derivative, fragment, analog, homologue or orthologue can be used as an immunogen in the production of antibodies (which immunologically specifically bind to these protein components).

[0076] Those skilled in the art will recognize that by ascertaining whether one human monoclonal antibody prevents the human monoclonal antibody of the invention from binding to PD-L1, it is possible to determine (without undue experimentation) whether the former has the same specificity as the latter. If the tested human monoclonal antibody competes with the human monoclonal antibody of the invention (as indicated by a reduction in binding produced by the human monoclonal antibody of the invention), then it is likely that the two monoclonal antibodies bind to the same or a closely related epitope.

[0077] Another method to determine whether a human monoclonal antibody has the specificity of the human monoclonal antibodies of the present invention is to pre-incubate the human monoclonal antibodies of the present invention with PD-L1 protein (which is usually reactive with PD-L1 protein) and then add the tested human monoclonal antibody to determine whether the tested human monoclonal antibody is inhibited in its ability to bind to PD-L1. If the tested human monoclonal antibody is inhibited, it is likely that it has the same or functionally equivalent epitope specificity as the monoclonal antibody of the present invention. Screening of human monoclonal antibodies of the present invention can also be implemented by using PD-L1 and determining whether the tested monoclonal antibody can neutralize PD-L1.

[0078] A variety of methods known in the art can be used to prepare polyclonal or monoclonal antibodies directed against the proteins of the invention or against derivatives, fragments, analogs, homologs or orthologs thereof (see, e.g., Antibody: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0079] Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which primarily provides the IgG fraction of immune serum. Subsequently or alternatively, the specific antigen (which is the target of the immunoglobulin being sought) or its epitope can be immobilized on a column to purify the immunospecific antibodies by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0080] The term "monoclonal antibody" or "MAb" or "monoclonal antibody composition" as used herein refers to a population of antibody molecules containing only one molecular species of antibody molecules, which are composed of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibodies are identical in all molecules of the population. MAbs contain an antigen binding site that is capable of immunoreacting with a specific antigenic epitope (which is characterized by a unique binding affinity for the antigenic epitope).

[0081] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, mice, hamsters or other suitable host animals are usually immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0082] The immunizing agent generally includes a protein antigen, a fragment thereof, or a fusion protein thereof. Typically, peripheral blood lymphocytes are used if human cells are required, or spleen cells or lymph node cells are used if non-human mammalian cells are required. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: principles and practice , Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are used. Hybridoma cells can be cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parent cell lacks the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas usually contains hypoxanthine, aminopterin and thymidine ("HAT medium"), which prevents the growth of HGPRT-deficient cells.

[0083] Preferred immortalized cell lines are those that effectively fuse, support stable high-level expression of antibodies produced by selected antibody-producing cells, and are sensitive to culture media such as HAT culture media. Preferred immortalized cell lines are mouse myeloma lines, which can be obtained, for example, 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 (monoclonal antibody production techniques and applications), Marcel Dekker, Inc., New York, (1987) 51-63 pages)).

[0084] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is 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 the 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 application of monoclonal antibodies, it is important to identify antibodies with high specificity and high binding affinity to the target antigen.

[0085] 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 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, hybridoma cells can be grown in vivo as ascites of a mammal.

[0086] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, for example, protein A agarose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0087] Monoclonal antibodies can also be produced by recombinant DNA methods (such as those described in U.S. Patent No. 4,816,567). DNA encoding the monoclonal antibodies of the present invention can be easily separated and sequenced using conventional methods (for example, by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of mouse antibodies). The hybridoma cells of the present invention are preferred sources of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into a host cell such as a monkey COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell (which does not otherwise produce immunoglobulins) to obtain the synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, for example, by replacing homologous mouse sequences with coding sequences of human heavy and light chain constant domains (see U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of the antigen-binding site of an antibody of the invention to create a chimeric bivalent antibody.

[0088] Full human antibodies are antibody molecules in which the entire sequence of both the light chain and the heavy chain (including CDR) is produced by human genes. Such antibodies are referred to herein as "humanized antibodies", "human antibodies" or "full human antibodies". Human monoclonal antibodies can be prepared using tri-hybridoma technology, human B cell hybridoma technology (see Kozbor et al., 1983 Immunol Today 4: 72) and EBV hybridoma technology for preparing human monoclonal antibodies (see Cole et al., 1985 In: monoclonal antibodies and cancer therapy (Monoclonal Antibodies and Cancer Therapy), Alan R. Liss, Inc., 77-96 pages). Human monoclonal antibodies can be used and prepared by using human hybridomas (see Cote et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells with Epstein Barr virus in vitro (see Cole et al., 1985 In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[0089] In addition, human antibodies can also be prepared 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 endogenous immunoglobulin genes are partially or completely inactivated). Upon stimulation, human antibody production is observed, which is closely similar to that observed in humans, including all aspects of gene rearrangement, assembly, and antibody repertoire. This method is described, for example, in U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016 and in Marks et al., Bio / Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 1365-93 (1995).

[0090] Human antibodies can also be prepared using transgenic non-human animals that are modified so that they produce fully human antibodies rather than the endogenous antibodies of the animal in response to antigenic stimulation. (See PCT Publication WO94 / 02602). The endogenous genes encoding heavy and light immunoglobulin chains in the non-human host have been disabled, and the loci encoding the activity of human heavy and light chain immunoglobulins are inserted into the host's genome. Human genes are incorporated, for example, using yeast artificial chromosomes containing the desired human DNA fragments. Subsequently, an animal providing all the desired modifications is obtained by hybridizing an intermediate transgenic animal (which contains less than all the modified supplements) as a progeny. A preferred embodiment of this type of non-human animal is a mouse, and is referred to as a Xenomouse TM(As disclosed in PCT publications WO 96 / 33735 and WO 96 / 34096). This animal produces B cells that secrete fully human immunoglobulins. Antibodies can be obtained directly from the animal after immunization with the desired immunogen (e.g., as a preparation of polyclonal antibodies) or alternatively from immortalized B cells derived from the animal (e.g., hybridomas that produce monoclonal antibodies). In addition, genes encoding immunoglobulins with human variable regions can be recovered and expressed to obtain antibodies directly, or can be further modified to obtain analogs of the antibodies, e.g., single-chain Fv (scFv) molecules.

[0091] An example of a method for producing a non-human host (e.g., mouse) lacking expression of endogenous immunoglobulin heavy chains is disclosed in U.S. Pat. No. 5,939,598. It can be obtained by the following method, the method comprising: deleting a J segment gene from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and the formation of transcripts of the rearranged immunoglobulin heavy chain locus, the deletion being achieved by a targeting vector containing a gene encoding a selectable marker; and generating a transgenic mouse from an embryonic stem cell whose somatic and germ cells contain a gene encoding a selectable marker.

[0092] A method for preparing an antibody of interest (e.g., a human antibody) is disclosed in U.S. Pat. No. 5,916,771. This method comprises introducing an expression vector containing a nucleotide sequence encoding a heavy chain into a mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing a heavy chain and a light chain.

[0093] In a further improvement on this approach, methods for identifying clinically relevant epitopes on an immune source and related methods for selecting antibodies that immunospecifically bind to the relevant epitopes with high affinity are disclosed in PCT Publication No. WO 99 / 53049.

[0094] The antibodies can be expressed by vectors containing the DNA fragment encoding the single-chain antibodies described above.

[0095] These may include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, etc. Vectors include chemical conjugates such as those described in WO 93 / 64701 (which have a targeting portion (e.g., a ligand for a cell surface receptor) and a nucleic acid binding portion (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 containing a targeting portion (e.g., an antibody specific for a target cell) and a nucleic acid binding portion (e.g., protamine)), plasmids, phages, etc. The vector may be chromosomal, non-chromosomal, or artificial.

[0096] Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus. DNA viral vectors are preferred. These vectors include pox vectors, such as orthopox or fowlpox vectors, herpes virus vectors, such as herpes simplex virus I (HSV) vectors (see 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 (see Kaplitt, MG et al., Nat. Genet. 8:148 (1994)).

[0097] Poxvirus vectors introduce genes into the cell cytoplasm. Fowlpox virus vectors only result in short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors are preferred for introducing nucleic acids into neural cells. Adenovirus vectors (about 2 months) result in shorter-term expression compared to adeno-associated virus (about 4 months), which in turn has a shorter expression period than HSV vectors. The specific vector selected will depend on the target cells and the conditions being treated. The introduction can be carried out by standard techniques, such as infection, transfection, transduction, or transformation. Examples of gene transfer patterns include, for example, naked DNA, CaPO4 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0098] Vectors can be used to target essentially any desired target cell. For example, stereotaxic injection can be used to direct vectors (e.g., adenovirus, HSV) to the desired location. In addition, particles can be delivered by intracerebroventricular (icv) perfusion using a micropump perfusion system (e.g., SynchroMed perfusion system). Bulk flow-based methods (referred to as convection) have also been shown to be effective in delivering macromolecules to extended areas of the brain and can be useful in 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 catheters, intravenous, parenteral, intraperitoneal and subcutaneous injections, and oral or other known routes of administration.

[0099] These vectors can be used to express large amounts of antibodies, which 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 try to bind to and interfere with PD-L1 activity.

[0100] The techniques can be modified to prepare single chain antibodies specific for the antigenic proteins of the invention (see, e.g., U.S. Pat. No. 4,946,778). In addition, the methods can be modified to construct F ab Expression libraries (see, e.g., Huse et al., 1989 Science 246:1275-1281) are used to allow rapid and efficient identification of monoclonal F3k4 ... ab Fragments. Antibody fragments containing idiotypes directed against protein antigens can be prepared by techniques known in the art, including but not limited to: (i) F fragments prepared by digestion of antibody molecules with pepsin; (ab')2 fragment; (ii) by (ab')2 F ab(iii) F fragments produced by treating the antibody molecule with papain and a reducing agent ab Fragment and (iv)F v Fragment.

[0101] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (see U.S. Patent No. 4,676,980) and for the treatment of HIV infection (see WO 91 / 00360; WO 92 / 200373; EP 03089). It is expected that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry (including those comprising cross-linking agents). For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyric imide esters and those disclosed, for example, in U.S. Patent No. 4,676,980.

[0102] For effector functions, in order to enhance the effect of antibodies in treating cancer, for example, it may be desirable to modify the antibodies of the present invention. For example, cysteine ​​residues may be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. The homodimeric antibodies thus generated can enhance internalization capacity and / or increase 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 to have dual Fc regions, and can thereby have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Caner Drug Design, 3: 219-230 (1989)).

[0103] The invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (eg, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof) or a radioactive isotope (ie, a radioconjugate).

[0104] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitory factor, curculin, crotonin, phytoncidin inhibitory factor, gelonin, milincomycin, restrictocin, phenomycin, neomycin and tricothecenes. A variety of radionuclides are available for the preparation of radioconjugated antibodies. Examples include 212 Bi, 131 I. 131 In, 90 Y and 186 Re.

[0105] Conjugates of antibodies and cytotoxic agents are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiocyclopentane (IT), bifunctional derivatives of iminoesters (e.g., dimethyl adipimidate hydrochloride), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., tolyl 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin 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 chelator for conjugating radionucleotides to antibodies (see WO94 / 11026).

[0106] Those of ordinary skill in the art will recognize that a wide variety of possible moieties can be coupled to the resulting antibodies or other molecules of the invention. (See, e.g., "Conjugate Vaccins", 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).

[0107] Coupling can be implemented by any chemical reaction that combines two molecules as long as the antibody and other parts maintain their respective activities. This connection may include many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordination bonding and complexing. However, preferred bonding is covalent bonding. Covalent bonding can be completed by direct condensation of the side chains present or by the incorporation of external bridging molecules. Many divalent or multivalent linking agents are useful in coupling protein molecules (such as antibodies of the present invention) to other molecules. For example, representative coupling agents may include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene and hexamethylenediamines. This list is not intended to be an exhaustive list of various types of coupling agents known in the art, but rather an example of a more common coupling agent. (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)). Preferred linkers are described in the following literature (see, e.g., 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 acetyl hydrazide derivatives coupled to antibodies by means of oligopeptide linkers. Particularly preferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., Cat# 21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio)-propionamido] hexanoate (Pierce Chem. Co. Cat. # 2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. # 2165-G) conjugated to EDC. Chem. Co., Cat. #24510).

[0108] The linkers described above contain components with different properties, resulting in conjugates with different physiochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-esters containing linkers are more insoluble than sulfo-NHS esters. In addition, the linker SMPT contains sterically hindered disulfide bonds and can form conjugates with increased stability. Disulfide bonds are generally more unstable than other bonds because disulfide bonds are cleaved in vitro, resulting in less conjugates being available. Sulfo-NHS can especially enhance the stability of carbodiimide coupling. Carbodiimide coupling (e.g., EDC), when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than a single carbodiimide coupling reaction.

[0109] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies are prepared by methods known in the art (e.g., 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 Nos. 4,485,045 and 4,544,545). Liposomes with increased circulation time are disclosed in U.S. Patent No. 5,013,556.

[0110] Particularly useful liposomes can be generated by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of defined pore size to produce liposomes having the desired diameter. Fab' fragments of the antibodies of the invention can be conjugated to liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) by disulfide exchange reactions.

[0111] Uses of Anti-PD-L1 Antibodies

[0112] The antibodies or fragments thereof that specifically bind to the PD-L1 protein of the present invention can be administered for the treatment of cancer or other proliferative disorders. Many cancers overexpress PD-L1 and the upregulation of PD-L1 is associated with high-risk prognostic factors. Overexpression of PD-L1 in tumor cells may also indicate a mechanism by which tumor cells evade anti-tumor immunity, such as by inducing T cell exhaustion. Such cancers include renal cell carcinoma and breast cancer. Other exemplary cancers are those that are associated with T cell exhaustion or utilize T cell exhaustion to escape anti-tumor T cell activity. The use of the antibodies of the present invention can enhance the ability of tumor antigen-specific T cells to proliferate and produce cytokines in response to stimulation with tumor antigen peptides, thereby increasing T cell activity or anti-tumor immune responses.

[0113] The antibodies or fragments thereof that specifically bind to the PD-L1 protein of the present invention can be administered for the treatment of chronic infections. Such chronic infections include, for example, viral infections, bacterial infections, and parasitic infections. An exemplary chronic viral infection is HIV. During chronic HIV infection, HIV-specific CD8+T cells are functionally impaired, showing 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. The use of the antibodies of the present invention can enhance the ability of HIV-specific T cells to proliferate and produce cytokines in response to stimulation with HIV peptides, thereby increasing T cell activity or anti-viral immune responses.

[0114] The antibodies of the present invention (including bispecific, polyclonal, monoclonal, humanized and fully human antibodies) can be used as therapeutic agents. These agents can be generally used to treat or prevent cancer in a subject, increase vaccine efficacy or enhance natural immune response. Antibody preparations (preferably those with high specificity and high affinity for their target antigens) are administered to a subject and generally have an effect due to their binding to the target. Administration of antibodies can eliminate or inhibit or interfere with the activity of the PD-L1 protein.

[0115] The antibodies of the present invention are capable of inducing cell death. Cell death is induced by direct or indirect mechanisms. For example, binding of PD-L1 by huPD-L1 antibodies can lead to complement-dependent cytotoxicity (CDC). Alternatively, huPD-L1 antibodies bind to PD-L1 and lead to the recruitment of a second cell type, which kills PD-L14-expressing target cells. Exemplary mechanisms (according to which huPD-L1 antibodies mediate cell death by the recruitment of a second cell type) include, but are not limited to, antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Target PD-L1-expressing cell types include tumor cells and T cells, e.g., activated T cells.

[0116] The antibody or fragment thereof that specifically binds to the PD-L1 protein of the present invention can be administered in the form of a pharmaceutical composition for the treatment of cancer or chronic infection. Principles and considerations related to the preparation of therapeutic compositions (which include antibodies), and guidelines for selecting components are provided in, for example, Remington: The Science And Practice Of Pharmacy 19th Edition (Alfonso R. Gennaro et al., ed.) Mack Pub. Co., Easton, Pa., 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends (drug absorption enhancement: concepts, possibilities, limitations and trends), Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And protein Drug Delivery (peptide and protein drug delivery) (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0117] The therapeutically effective amount of the antibody of the present invention generally relates to the amount required to achieve the therapeutic goal. As noted above, this can be a binding interaction between the antibody and its target antigen, which in some cases interferes with the function of the target. In addition, the required amount depends on the binding affinity of the antibody to its specific antigen, and also depends on the rate at which the administered antibody is exhausted from other subjects to whom the free volume of the antibody is administered. The conventional range of effective dosing for the treatment of the antibody or antibody fragment of the present invention (in the form of a non-limiting example) can be about 0.1 mg / kg body weight-about 50 mg / kg body weight. Common administration frequencies can be, for example, in the range of twice a day to once a week.

[0118] In the case of using antibody fragments, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the variable region sequence of the antibody, a peptide molecule can be designed that retains the ability to bind to the target protein sequence. Such peptides can be chemically synthesized and / or prepared by recombinant DNA technology. (See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The preparation may also contain more than one active compound according to the needs of the specific indication for treatment, preferably those with complementary activities that do not adversely affect each other. Alternatively or in addition, the composition may include an agent that enhances its function, for example, a cytotoxic agent, a cytokine, a chemotherapeutic agent or a growth inhibitor. Such molecules are suitable for being present in an effective amount combination for the intended purpose.

[0119] The active ingredients can also be entrapped in microcapsules (e.g. prepared by coacervation techniques or by interfacial polymerization, e.g. hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.

[0120] Formulations for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0121] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies 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 γ-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene vinyl acetate and lactic acid-glycolic acid enable sustained release of molecules for more than 100 days, certain hydrogels release proteins for shorter periods of time.

[0122] The antibodies of the present invention can be used as agents for detecting the presence of PD-L1 (or protein or protein fragment thereof) in a sample. Preferably, the antibody contains a detectable label. The antibody can be polyclonal or more preferably monoclonal. The whole antibody or its fragment (e.g., F ab , scFv or F (ab)2). The term "labeled" about a probe or antibody is intended to include direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody, and indirect labeling of a probe or antibody by reactivity with another directly labeled reagent. Examples of indirect labeling include detection of a first antibody using a fluorescently labeled second antibody and end-labeling a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids separated from a subject, as well as tissues, cells, and fluids present in a subject. Therefore, blood and blood fractions or components (which include serum, plasma, or lymph) are included in the use of the term "biological sample". That is, the detection method of the present invention can be used for detecting analyte mRNA, protein, or genomic DNA in a biological sample in vitro or in vivo. For example, in vitro techniques for detecting analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detecting analyte protein include enzyme-linked immunosorbent assay (ELISA), western blotting, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting analyte genomic DNA include Southern hybridization. Methods for conducting immunoassays are described, for example, in "ELISA: Theory and Practice: methods in Molecular Biology", Vol. 42, JR 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 Immunoassay", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. In addition, in vivo techniques for detecting analyte proteins include introducing labeled anti-analyte protein antibodies into a subject. For example, the antibody can be labeled with a radioactive marker, the presence and location of which in the subject can be detected by standard imaging techniques.

[0123] Antibodies directed against PD-L1 proteins (or fragments thereof) can be used in methods known in the art that involve localization and / or quantification of PD-L1 proteins (e.g., for measuring the level of PD-L1 proteins in suitable physiological samples, for diagnostic methods, for protein imaging, etc.). In a given embodiment, antibodies specific for PD-L1 proteins or derivatives, fragments, analogs or homologs thereof (which contain antibody-derived antigen-binding domains) are utilized as pharmacologically active compounds (hereinafter referred to as "therapeutic agents").

[0124] The antibodies of the present invention that are specific for PD-L1 protein can be used to separate PD-L1 polypeptide by standard techniques, such as immunoaffinity, chromatography or immunoprecipitation. Antibodies (or fragments thereof) directed against PD-L1 protein can be used diagnostically to monitor the level of protein in tissues as part of a clinical trial procedure, for example, to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include a variety of 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; and examples of suitable radioactive materials include 125 I. 131 I. 35 S or 3 H.

[0125] Therapeutic Compositions

[0126] The antibodies or agents of the present invention (also referred to herein as "active compounds"), and their derivatives, fragments, analogs and homologues can be incorporated into pharmaceutical compositions suitable for administration. Such compositions generally include antibodies or agents and pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" as used herein is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are compatible with drug administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard reference text in this field, which is 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. Liposomes and non-aqueous media (e.g., fixed oils) can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except for the case where any conventional media or agents are incompatible with the active compound, their use in the composition is expected. Supplementary active compounds can also be incorporated into the composition.

[0127] The pharmaceutical composition of the present invention is formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, subcutaneous), oral (e.g., inhalation), transdermal (i.e., surface), transmucosal and rectal administration. Solutions or suspensions for parenteral, intradermal or subcutaneous applications may include the following components: sterile diluents (e.g., water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents); antibacterial agents (e.g., benzyl alcohol or methyl paraben); antioxidants (e.g., ascorbic acid or sodium bisulfite); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); buffers (e.g., acetate, citrate or phosphate), and agents (e.g., sodium chloride or glucose) for regulating tension. pH may be adjusted with acid or alkali (e.g., hydrochloric acid or sodium hydroxide). Parenteral preparations may be packaged in ampoules, disposable syringes or multiple dose bottles made of glass or plastic.

[0128] Pharmaceutical compositions suitable for injectable use 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 TM(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy injectability exists. It must be stable under manufacturing and storage conditions and must maintain antimicrobial (e.g., bacterial and fungal) contamination activity. The carrier can be a solvent or dispersion medium, which includes, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating (e.g., lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. The activity of microorganisms can be prevented by a variety of antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it is preferred to include isotonic agents (e.g., sugars, polyols such as mannitol, sorbitol, sodium chloride) in the composition. The extended absorption of the injectable composition can occur by including an agent (e.g., aluminum monostearate and gelatin) that delays absorption into the composition.

[0129] Sterile injectable solutions can be prepared by incorporating the active compound in the desired amount into a suitable solvent with one or a combination of ingredients listed above, as required, followed by sterilization by filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and lyophilization, which produce a powder of the active ingredient plus any additional required ingredients from a previously sterile filtered solution thereof.

[0130] Oral compositions generally include an inert diluent or edible carrier. They can be encapsulated in a gelatin capsule or compressed into tablets. For the purpose of oral treatment administration, the active compound can be blended with an excipient and used in the form of a tablet, lozenge or capsule. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is orally administered and gargled and spit out or swallowed. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar nature: a binder (e.g., microcrystalline cellulose, tragacanth gum or gelatin); an excipient (e.g., starch or lactose); a disintegrant (e.g., alginic acid, Primogel or corn starch); a lubricant (e.g., magnesium stearate or Sterotes); a glidant (e.g., colloidal silicon dioxide); a sweetener (e.g., sucrose or saccharin); or a flavoring agent (e.g., mint, methyl salicylate or orange flavoring).

[0131] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0132] Systemic administration can also be by transmucosal or transdermal mode.For transmucosal or transdermal administration, the penetrant that is suitable for the barrier to be penetrated is used for preparation.This type of penetrant is generally known in the art, and for transmucosal administration, includes for example detergent, bile salt and fusidic acid derivative.Transmucosal administration can be completed by using nasal spray or suppository.For transdermal administration, active compound is formulated into ointment, salves, gel or cream as generally known in the art.

[0133] The compounds may 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.

[0134] In one embodiment, the active compound is prepared with a carrier (e.g., a controlled release formulation, including implants and microencapsulated delivery systems) that protects the compound from rapid elimination from the body. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such preparations are obvious to those skilled in the art. The materials can also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art (e.g., methods described in U.S. Patent No. 4,522,811).

[0135] It is particularly advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physiologically discrete units suitable as unitary dosages for subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the present invention are dictated by and directly dependent on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the limitations inherent in the art of formulating such active compounds for individual treatment.

[0136] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0137] Diagnostic assays

[0138] The huPD-L1 antibodies of the invention, when linked to a detectable moiety, provide methods for detecting "cancerous tissue" or tissue that is prone to abnormal cell proliferation and is therefore at risk for cancer. In addition to tissue that becomes cancerous due to in situ neoplasms, for example, the antibody-detectable moiety conjugates also provide methods for detecting cancerous metastatic tissue present in distant organs and / or tissues. Such tissues can thus be detected by contacting tissue suspected of being cancerous with the antibody-detectable moiety under appropriate conditions to cause the detectable moiety to be detected in the cancerous tissue, thereby detecting the presence of the cancerous tissue.

[0139] The huPD-L1 antibodies of the invention, when linked to a detectable moiety, provide methods for detecting T cell exhaustion in a subject suffering from cancer or chronic infection. For example, the huPD-L1 antibodies can be used to detect the level of PD-L1 in a subject, wherein the level compared to a reference level can indicate whether the subject is suffering from T cell exhaustion. Thus, this method can also be used to determine whether a treatment (which uses huPD-L1 antibodies to improve immune response by reversing or inhibiting T cell exhaustion) will benefit a subject.

[0140] The detectable moiety can be conjugated directly to the antibody or fragment, or indirectly by using, for example, a fluorescent secondary antibody. Direct conjugation can be accomplished by, for example, standard chemical coupling of a fluorophore to an antibody or antibody fragment or by genetic engineering. Chimeras or fusion proteins can be constructed containing an antibody or antibody fragment coupled to a fluorescent or bioluminescent protein. For example, Casadei et al. describe methods for preparing a vector construct capable of expressing a fusion protein of aequorin and an antibody gene in mammalian cells.

[0141] The term "labeled" used herein with respect to a probe or antibody is intended to include direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody, and indirect labeling of the probe or antibody by reactivity with another reagent directly labeled. Examples of indirect labeling include detection of a first antibody using a fluorescently labeled second antibody and end-labeling of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids (e.g., biopsy samples) separated from a subject, as well as tissues, cells, and fluids present in a subject. That is, the detection method of the present invention can be used to detect cancer, cancer cells, or cancer-related cells (e.g., stromal cells associated with tumors or cancer cells) in vitro and in vivo in biological samples. For example, in vitro techniques for detecting PD-L1 include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. In addition, in vivo techniques for detecting PD-L1 include introducing labeled anti-PD-L1 antibodies into a subject. For example, antibodies can be labeled with radioactive markers, the presence and location of which in a subject can be detected by standard imaging techniques. In an embodiment, the present invention provides a non-invasive method for detecting tumors or cancer cells in a subject. The subject is administered an antibody or scFv antibody of the present invention, and when the antibody is linked to a detectable moiety (i.e., any moiety capable of being detected by, for example, fluorescence, chemistry, chemiluminescence, radioactivity, or other methods known in the art), the antibody is allowed to localize in the tumor and then detected by observing the detectable moiety.

[0142] In the case of a "targeted" conjugate, i.e., a conjugate containing a targeting moiety (a molecule or feature designed to localize the conjugate at one or more specific sites in a subject or animal), localization refers to the state when an equilibrium between bound "localized" entities and unbound "free" entities within the subject has been substantially reached. The rate at which such an equilibrium is reached depends on the route of administration. For example, a conjugate administered by intravenous injection to localize a thrombus may achieve localization or accumulation in the thrombus within a few minutes of injection. On the other hand, a conjugate administered orally to localize an infection in the intestine may take several hours to achieve localization. Alternatively, localization may simply refer to the location of the entity in a subject or animal within a selected time period after administration of the entity. By way of another example, localization is achieved when the moiety becomes dispersed after administration.

[0143] In all of the above cases, reasonable estimates of the time to achieve localization can be made by those skilled in the art. In addition, the localization state as a function of time can be followed by imaging the detectable portion (e.g., light-emitting conjugate) according to the methods of the present invention (e.g., using a light detection device). The "light detection device" used should have a high enough sensitivity to be able to image the weak light from within a mammal in a reasonable amount of time, and use the signal from such a device to construct an image.

[0144] In cases where it is possible to use particularly bright light-generating moieties and / or detect light-generating fusion proteins that are positioned near the surface of the subject or animal being imaged, a pair of "night vision" goggles or a standard high-sensitivity video camera, such as a Silicon Intensified Tube (SIT) camera (e.g., from Hammamatsu Photonic Systems, Bridgewater, NJ) can be used. More often, however, a more sensitive light detection method is desired.

[0145] At extremely low light levels, the photon flux per unit area becomes so low that the imaged scene no longer appears continuous. Instead, it is represented by individual photons that are far apart in time and space. Viewed on a display, such images appear as flickering points of light, each representing a single detected photon. An image is obtained and constructed by accumulating these detected photons over time in a digital image processor. 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 does not carry any meaning. The goal is to detect only the presence of a signal (photon) and count the occurrence of the signal with respect to its position over time.

[0146] At least two types of light detection devices (described below) can detect individual photons and produce signals that can be analyzed by an image processor. Noise reduction photon detection devices achieve sensitivity by reducing background noise in the photon detector rather than amplifying the photon signal. Noise is reduced primarily by cooling the detector array. Devices include charge coupled device (CCD) cameras, which are referred to as "post-thinned", cooled CCD cameras. In more sensitive devices, cooling is achieved using, for example, liquid nitrogen (which brings the temperature of the CCD array to about -120°C). "Post-thinning" refers to an ultra-thin back plate that shortens the path length (along which photons travel to be detected), thereby enhancing quantum efficiency. A particularly sensitive post-thinned cryogenic CCD camera is the "TECH512", a series 200 camera available from Photometrics, Ltd. (Tucson, Ariz.).

[0147] "Photon amplification devices" amplify photons before they strike a detection screen. This category includes CCD cameras with intensifiers, such as microchannel intensifiers. Microchannel intensifiers typically contain an array of metal channels perpendicular and coextensive with the camera detection screen. The microchannel array is placed between the sample, subject or animal to be imaged and the camera. Most photons entering the channels of the array contact one side of the channel before exiting. A voltage applied across the array results in the release of many electrons from the photon collisions. The electrons from such collisions leave the channels from which they originated in a "shotgun" manner and are detected by the camera.

[0148] Even greater sensitivity can be achieved by placing an array of enhanced microchannels 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 enhancement in sensitivity is achieved at the expense of spatial resolution, which decreases with each additional stage of amplification. An exemplary microchannel intensifier-based single-photon detection device is the C2400 series available from Hamamatsu.

[0149] The image processor processes the signals generated by the light detection device, which counts photons to construct an image (which can, for example, be displayed on a display or printed on an image printer). Such image processors are typically sold as part of a system (which includes the sensitive photon counting camera described above) and are therefore available from the same source. The image processor is typically connected to a personal computer (e.g., an IBM-compatible PC or Apple Macintosh (Apple Computer, Cupertino, Calif.)), which may or may not be included as part of the purchased imaging system. Once the images are in the form of digital files, they can be manipulated (e.g., "ADOBE PHOTOSHOP", Adobe Systems, Adobe Systems, Mt. View, Calif.) and printed by a variety of image processing programs.

[0150] In one embodiment, the biological sample contains protein molecules from a test subject.A preferred biological sample is a peripheral blood leukocyte sample, which is isolated from a subject by conventional methods.

[0151] The present invention also includes a kit for detecting the presence of PD-L1 or PD-L1-expressed cells in a biological sample. For example, the kit may include: a compound or agent (e.g., anti-PD-L1 scFv or monoclonal antibody) capable of detecting a marker of cancer or tumor cells in a biological sample, a tool for detecting the amount of PD-L1 in a sample, and a method for comparing the amount of PD-L1 in a sample with a standard. In some embodiments, the standard is a non-cancerous cell or a cell extract thereof. The compound or agent may be packaged in a suitable container. The kit may further include instructions for using the kit to detect cancer in a sample.

[0152] Bispecific Antibodies

[0153] Bispecific antibodies (bsAbs) are antibodies that include two variable domains or scFv units that allow the resulting antibodies to recognize two different antigens. The present invention provides bispecific antibodies that recognize PD-L1 and a second antigen. Exemplary second antigens include tumor-associated antigens, cytokines, and cell surface receptors. In some embodiments, the second antigen may be CAIX (carbonic anhydrase IX or G250), IL-10, or CCR4. In some embodiments, the second antigen may be a cell surface receptor, wherein the cell surface receptor is CCR4, IL21R, BTLA, HVEM, or TIM3.

[0154] The bispecific antibodies of the present invention include a heavy chain and light chain combination or scFv of the huPD-L1 antibody disclosed herein.

[0155] Construction of bispecific antibodies

[0156] The bispecific antibodies of the present invention can be constructed using methods known in the art. In some embodiments, the bispecific antibody is a single polypeptide, wherein two scFv fragments are connected to form an antibody by a long linker polypeptide (its length is sufficient to allow intramolecular association between two scFv units). In other embodiments, the bispecific antibody is more than one polypeptide connected by covalent or non-covalent bonds.

[0157] In another embodiment, bispecific antibodies are constructed using the "knob-into-hole" approach (Ridgway et al., Protein Eng 7:617-621 (1996)). In this approach, the Ig heavy chains of two different variable domains are reduced to selectively disrupt heavy chain pairing while retaining heavy chain-light chain pairing. Two heavy chain-light chain heterodimers that recognize two different antigens are mixed to promote heterojunction pairing, which is mediated by the engineered "knob-into-hole" of the CH3 domain (e.g., in Figure 5 and 6A ).

[0158] In another embodiment, bispecific antibodies can be constructed as follows: heavy chain-light chain dimers from two or more different antibodies are exchanged to produce hybrid antibodies, wherein the first heavy chain-light chain dimer recognizes PD-L1 and the second heavy chain-light chain dimer recognizes the second antigen. The mechanism of heavy chain-light chain dimers is similar to the formation of human IgG4 (which also functions as a bispecific molecule). The dimerization of IgG heavy chains is driven by intramolecular forces (e.g., intramolecular forces of the CH3 domains and disulfide bridges of each heavy chain). The presence of specific amino acids in the CH3 domain (R409) has been shown to promote the construction and dimer exchange of IgG4 molecules. Heavy chain pairing is also further stabilized by disulfide bridges between heavy chains in the hinge region of the antibody. Specifically, in IgG4, the hinge region contains the amino acid sequence Cys-Pro-Ser-Cys at amino acids 226-230 (compared to the stable IgG1 hinge region containing the sequence Cys-Pro-Pro-Cys). This sequence difference at serine 229 has been associated with the propensity of IgG4 to form new intrachain disulfide bonds in the hinge region (Van der Neut Kolfschoten, M. et al., 2007, Science 317: 1554-1557 and Labrijn, AF et al., 2011, Journal of immunol 187: 3238-3246).

[0159] Therefore, the bispecific antibodies of the present invention can be produced by introducing the R409 residue in the CH3 domain of the antibody that recognizes PD-L1 or a second antigen and introducing the Cys-Pro-Ser-Cys sequence in the hinge region, so that the heavy chain-light chain dimer is exchanged to produce an antibody molecule having a heavy chain-light chain dimer that recognizes PD-L1 and a second heavy chain-light chain dimer that recognizes a second antigen, wherein the second antigen is any antigen disclosed herein. Preferably, the bispecific antibody contains an anti-PD-L1 heavy chain-light chain dimer conjugated to an anti-CAIX (carbonic anhydrase IX or 250) heavy chain-light chain dimer, as discussed in Examples 3 and 4. The heavy chain-light chain dimer exchange can also be enhanced by the addition of a reducing agent (e.g., reduced glutathione) to promote. Known IgG4 molecules can also be changed so that the heavy chain and light chain recognize PD-L1 or a second antigen as disclosed herein. This approach can be advantageous for constructing the bispecific antibodies of the invention due to the intrinsic properties of the IgG4 molecule, in which the Fc region differs from other IgG subtypes in that it interacts poorly with the effector systems of the immune response, such as Fc receptors and complement expressed by certain white blood cells. This property makes these IgG4-based bispecific antibodies attractive for therapeutic applications in which an antibody is required to bind to a target and functionally alter a signal transduction pathway associated with the target, but not trigger effector activity.

[0160] In some embodiments, mutations are introduced into the constant region of a bsAb in order to alter the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the bsAb. Such examples are described in Figure 6B In one aspect, bsAb contains a mutation on a scFv unit of a heterodimer bsAb, which reduces ADCC activity. In another aspect, bsAb contains mutations on both chains of a heterodimer bsAb, which completely eliminate ADCC activity. For example, the mutation introduced by one or two scFv units of bsAb is a LALA mutation in the CH2 domain. These bsAbs with variable ADCC activity can be optimized so that the bsAb shows the maximum selective killing of cells expressing an antigen recognized by the bsAb, but shows the minimum killing of the second antigen recognized by the bsAb.

[0161] Exemplary Second Antigen

[0162] The present invention provides a bispecific antibody that recognizes PD-L1 and a second antigen.

[0163] In some embodiments, the second antigen is a tumor-associated antigen. In some embodiments, the tumor-associated antigen is carbonic anhydrase IX (CAIX). For example, a CAIX / PD-L1 bispecific antibody can be constructed that comprises a combination of one heavy chain and one light chain of the huPD-L1 antibody described herein and a combination of one heavy chain and one light chain that recognizes CAIX ( Fig. 6A ). CAIX has been described as a prognostic marker for disease progression and a target for immunotherapy using IL-2. CAIX is a tumor-associated antigen that is highly expressed in cancers (e.g., renal cell carcinoma). In some cases, activation of the PD1 / PD-L1 axis of tumor cells can induce T cell exhaustion against certain tumor-specific antigens (e.g., CAIX), whereby tumor cells expressing CAIX escape recognition by the immune system. BsAbs targeting both CAIX and PD-L1 are used as new cancer therapeutics. Treatment with CAIX / PD-L1bsAbs inhibits or reverses PD-1 / PD-L1-mediated T-cell exhaustion against CAIX, and promotes tumor monitoring and immune responses against CAIX-expressing tumor cells. For example, treatment with CAIX / PD-L1bsAbs promotes antigen-specific immune responses against tumor cells, wherein the targeted antigen is CAIX.

[0164] In some examples, mutations can be introduced into the CAIX or PD-L1 chain constant region (e.g., CH2 domain) to reduce ADCC activity ( Figure 6B ). In some examples, mutations can be introduced simultaneously into the constant regions of the CAIX and PD-L1 chains to completely eliminate ADCC activity. Mutated bsAbs with variable ADCC activity can be determined by methods known in the art for their specificity in killing tumor cells. Preferably, the CAIX / PD-L1 bsAb exhibits maximum selective killing of CAIX-expressing tumor cells, but only minimal killing of PD-L1-expressing endogenous peripheral blood mononuclear cells (PBMCs).

[0165] In some embodiments, the second antigen is a cell surface receptor, wherein the cell surface receptor is an interleukin 21 receptor (IL21R). For example, an IL21R / PD-L1 bispecific antibody can be constructed, which includes a combination of a heavy chain and a light chain of the huPD-L1 antibody described herein and a combination of a heavy chain and a light chain that binds to IL21R in a competitive manner. Cytokine IL21 is secreted by CD4+T helper cells and binds to IL21R to promote a variety of immune activation pathways, in particular, promotes the antigen-specific cytotoxicity of CTLs and the maturation, proliferation and cytotoxicity of NK cells (Sondergaard, H et al., 2009 Tissue antigens 74: 467-479; Kasaian, MT et al., 2002 Immunity 16: 559-569; and Coquet, JM et al., 2008 J Immunol 178: 2827-2834). In particular, IL21 has been shown to promote the activation and cytotoxicity of anti-melanoma antigens (Li, Y. et al., 2005, J Immunol 175: 2261-2269). Systemic administration of IL21 has been explored for the treatment of cancer and has shown some efficacy (Schmidt, H. et al., 2010 Clinical cancer research, 16: 5312-5319), but some data have also shown harmful and undesirable side effects (Grunwald, V. et al., 2011, Acta Oncol 50: 121-126).

[0166] The IL21R / PD-L1 bsAb of the present invention binds to IL21R in a competitive manner, thereby acting as a mimetic or substitute for cytokine IL21. The combination of IL21R / PD-L1 bsAb can lead to the activation of IL21R-mediated pathways and the subsequent promotion of antigen-specific cytotoxic immune responses against tumor cells (which have induced PD-L1-mediated T-cell exhaustion). A specific benefit of treatment with the bispecific antibody of the present invention is to locate IL21R activation to an area where PD-1 / PD-L1-mediated T cell exhaustion has occurred (e.g., in a tumor microenvironment, such as near or within a tumor where T cell exhaustion has been induced to escape anti-tumor immune responses). In this way, ILR / PD-L1 bsAb promotes anti-tumor immune responses by the following dual mechanisms: 1) by reversing or inhibiting PD-1 / PD-L1-mediated T cell exhaustion, and 2) by promoting the activation of IL21 / IL21R-mediated cytotoxic immune responses, thereby inducing antigen-specific or anti-tumor immune responses and cytotoxicity.

[0167] IL21R / PD-L1 bispecific antibodies can also be used in vaccines for immunization of subjects, or as vaccine adjuvants. In germinal center reaction (GCR) (wherein high-affinity antibody-secreting plasma cells and memory B cells are produced, which ensure continuous immune protection and rapid memory response against previously encountered foreign antigens), PD1 is expressed by follicular T helper cells (TFH) while PDL1 is expressed by germinal center B cells (Crotty, S. et al., 2011, Annual review of Immunology, 29: 621-623). Overexpression of PD1 or PD-L1 suppresses the expansion of antibody-producing B cells. The use of IL21R / PD-L1 bispecific antibodies (wherein the PD-L1 portion of the antibody suppresses PD1 / PD-L1 axis) can lead to the preferential expansion of only high-affinity antibodies. Since IL-21 strongly promotes the transformation of antigen-specific B cells to antibody-secreting plasma cells, and the development and maintenance of TFH activity (Crotty, S. et al., 2011, Annual review of Immunology, 29:621-623), anti-PDL1 bsAbs with IL21 substitution or competition activity can act as GCR-specific adjuvants for promoting the production of high-affinity antibodies against specific antigens (e.g., antigens administered by vaccines or antigens of infectious agents).

[0168] In some embodiments, the second antigen is BTLA (B and T lymphocyte attenuating subprotein) or HVEM (herpes virus entry mediator, also known as TNFRSF14). For example, a BTLA / PD-L1 bispecific antibody can be constructed, which includes a combination of a heavy chain and a light chain of the huPD-L1 antibody described herein and a combination of a heavy chain and a light chain that binds BTLA. For example, an HVEM / PD-L1 bispecific antibody can be constructed, which includes a combination of a heavy chain and a light chain of the huPD-L1 antibody described herein and a combination of a heavy chain and a light chain that binds HVEM (preferably, those regions of HVEM that mediate binding to BTLA). The binding of BTLA to HVEM results in T cell inhibition, which is similar to the PD1 / PD-L1 interaction. The BTLA / PD-L1 or HVEM / PD-L1 bispecific antibodies of the present invention inhibit or prevent the association between BTLA and HVEM, and prevent BTLA / HVE-mediated T cell inhibition. BTLA inhibition promotes tumor-specific T cell responses. Thus, treatment with the bispecific antibodies of the invention results in the simultaneous blockade of two different inhibitory pathways limiting T cell activity.

[0169] In some examples, the HVEM / PD-L1 bsAb antibody also blocks the association between HVEM and another HVEM ligand, CD160, which has been shown to be an agonist for the survival of B-cell chronic lymphocytic leukemia (BCLL) cells and strongly inhibits CD4+ T-cell activation and function. Treatment with the HVEM / PD-L1 bispecific antibodies of the invention, which also block HVEM / CD160 binding, results in simultaneous blockade of two inhibitory factor pathways, which limit T cell activity and inhibit CD160-mediated tumor survival pathways.

[0170] In some embodiments, the second antigen is TIM3 (T-cell immunoglobulin and mucin domain 3). For example, a TIM3 / PD-L1 bispecific antibody can be constructed, which includes a combination of a heavy chain and a light chain of the huPD-L1 antibody described herein and a combination of a heavy chain and a light chain that binds TIM3. In one aspect, the antibody of the present invention prevents or inhibits the binding of TIM3 to galectin-9 (GAL9). The interaction between TIM3 and GAL9 leads to the activation of macrophages and the inhibition of T cell function (Sakuishi, K. et al., 2010, The Journal of experimental medicine, 207: 2187-2194 and Zhang, Y. et al., 2012, Journal of leukocyte biology, 91: 189-196). TIM3 has also been shown to promote the activity of myeloid-derived suppressor cells (MDSC) (Dardalhon, V. et al., 2012 Journal of leukocyte biology, 185: 1383-1392). Therefore, treatment with the bispecific antibodies of the present invention (e.g., TIM3 / PD-L1bsAb) will reverse and prevent T-cell exhaustion, promote tumor monitoring and inhibit the generation of MDSC. The use of TIM3 / PD-L1bsAb may be particularly beneficial for the treatment of subjects suffering from cancers with TIM3 polymorphisms (e.g., renal cell carcinoma and metastatic renal cell carcinoma).

[0171] The bispecific antibodies disclosed herein may be useful in the treatment of a disease or medical condition (e.g., cancer). The bispecific antibodies of the present invention may be particularly useful in diseases or medical conditions associated with T cell exhaustion. In some cases, the bispecific antibodies disclosed herein may be used as vaccines for promoting antigen-specific immune responses. The bispecific antibodies of the present invention target tumors that inhibit T-cell exhaustion.

[0172] Treatment

[0173] The present invention provides a method for treating a subject at risk of (or susceptible to) cancer or other cell proliferation-related diseases or conditions. Such diseases or conditions include, but are not limited to, for example, those diseases or conditions associated with abnormal expression of PD-L1. For example, the method is used to treat, prevent or alleviate the symptoms of renal cell carcinoma or breast cancer. Alternatively, the method is used to treat, prevent or alleviate the symptoms of cancer (wherein PD-L1 plays a negative regulatory role in T cell response). Alternatively, the method is used to treat, prevent or alleviate the symptoms of solid tumors (such as breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer or gastric cancer). Alternatively, the method is used to treat, prevent or alleviate the symptoms of metastatic cancer.

[0174] The present invention provides a method for preventing and treating a subject at risk of (or susceptible to) chronic viral infection, bacterial infection or parasitic infection. In particular, the present invention provides a method for preventing and treating a subject at risk of (or susceptible to) HIV infection or AIDS.

[0175] The present invention also provides a method for treating a subject at risk of a disease or disorder or condition associated with T-cell failure or developing a method for preventing and treating a subject at risk of T-cell failure. The present invention also provides a method for treating a subject at risk of a disease or disorder or condition associated with T-cell failure or developing a method for preventing and treating a subject at risk of T-cell failure. Such diseases or conditions include, but are not limited to, HIV, AIDS, and chronic bacterial infections, viral infections, 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), Helicobacter pylori (H.pylori), or Toxoplasma gondii.

[0176] Thus, in one aspect, the present invention provides a method for preventing, treating or ameliorating the symptoms of cancer or a cell proliferative disease or disorder in a subject by administering the monoclonal antibody or scFv antibody of the present invention to the subject. For example, the huPD-L1 antibody can be administered in a therapeutically effective amount.

[0177] Subjects at risk for cancer or cell proliferation-related diseases or disorders include patients with a family history of cancer or subjects exposed to substances known or suspected to cause cancer. Administration of preventive drugs can occur prior to the manifestation of cancer, such that the disease is prevented or, alternatively, delayed in its progression.

[0178] In another aspect, the growth of tumor cells is inhibited or the activity of inhibitory factor T cells is reduced by contacting cells with the PD-L1 antibody of the present invention. The cells are any cells expressing PD-L1. For example, the cells are T cells.

[0179] Methods for increasing or enhancing the immune response to an antigen are also included in the present invention. The immune response is increased or enhanced by administering a monoclonal antibody or scFv antibody of the present invention to a subject. The immune response is, for example, increased by improving antigen-specific T effector function. The antigen is a viral (e.g., HIV) antigen, a bacterial antigen, a parasite antigen, or a tumor antigen. The immune response is a natural immune response. A natural immune response refers to an immune response that is the result of an infection. An infection is a chronic infection. Increasing or enhancing the immune response to an antigen can be measured by a variety of methods known in the art. For example, the immune response can be measured by measuring any of the following: T cell activity, T cell proliferation, T cell activation, the production of effector cytokines, and T cell transcriptional signatures.

[0180] Alternatively, the immune response is a response induced by vaccination. Therefore, in another aspect, the present invention provides a method for increasing vaccine efficacy by administering a monoclonal antibody or scFv antibody of the present invention and a vaccine to a subject. The antibody and the vaccine are administered sequentially or simultaneously. The vaccine is a tumor vaccine, a bacterial vaccine or a viral vaccine.

[0181] Combination Methods

[0182] The present invention provides for treating cancer in a patient by administering two antibodies that bind to the same epitope of a PD-L1 protein or, alternatively, two different epitopes of a PD-L1 protein. Alternatively, cancer is treated by administering a first antibody that binds to PD-L1 and a second antibody that binds to a protein other than PD-L1. For example, the other protein other than PD-L1 may include, but is not limited to, CAIX, CCR4, and IL-10. For example, the other protein other than PD-L1 is a tumor-associated antigen.

[0183] In some embodiments, the invention provides for administration of huPD-L1 antibodies alone or together with additional antibodies (which recognize another protein in addition to PD-L1), wherein cells are capable of effecting or enhancing an immune response. For example, these cells can be peripheral blood mononuclear cells (PBMCs) or any cell type present in PBMCs, for example, cytotoxic T cells, macrophages, and natural killer (NK) cells.

[0184] In addition, the present invention provides antibodies and anti-tumor agents that bind to PD-L1 proteins, such as small molecules, growth factors, cytokines or other therapeutic agents (including biomolecules such as peptides, peptide mimetics, peptoids, polynucleotides, lipid-derived mediators, small biologically derived 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-α. Small molecule libraries are known in the art. (See Lam, Anticancer Drug Des., 12: 145, 1997.).

[0185] The present invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. Example

[0186] Example 1: Generation of human mAbs against PD-L1

[0187] Human mAbs against human PD-L1 were generated by panning a human scFv phage display library of 27 billion components. Using full-length PD-L1 in the form of paramagnetic proteoliposomes (PMPLs), which ensures the correct orientation of the extracellular domain of PD-L1 for display in the library, 14 unique scFv-phages that bind PD-L1 were identified. Human IgG constructs were constructed for these 14 unique scFv-phages: Ab-14, Ab-16, Ab-22, Ab-30, Ab-31, Ab-32, Ab-38, Ab-42, Ab-46, Ab-50, Ab-52, Ab-5, Ab-56, and Ab-65.

[0188] Example 2: Characterization of huPD-L1 mAb Binding to PD-L1

[0189] Binding analysis of huPD-L1 antibody was performed using PD-L1-expressing cells. Four types of cells were tested, including the parental cell line 300.19 and cell lines transfected to express human PD-L1 (hPD-L1), human PD-L2 (HPD-L2), and human C-type lectin domain family 2 member (hCLEC2D). Binding assays were performed in duplicate and the results are summarized in Tables 16-19 and Figure 2The antibody affinity was tested by using four antibody concentrations: 10 μg / ml, 1 μg / ml, 0.1 μg / ml and 0.01 μg / ml. GF1538 and GF1757 antibodies were used as controls: GF1538 is a humanized Ab against hPD-L1 and GF1757 is a humanized Ab against hPD-L2. The secondary antibody used was PE-goat anti-human IgG. All values ​​show mean fluorescence intensity (GMFI) (as detected by FAC analysis).

[0190] Results from the binding assays indicated that the tested huPD-L1 antibodies exhibited high affinity and specificity for binding to PD-L1. Using the parental cell line 300.19 (which does not express human PD-L1) as a control, basal or non-specific fluorescence was established (Tables 16 and Figure 2 , upper left). Staining of 300.19 cells expressing human PD-L1 by transfection with huPD-L1 antibody showed significantly higher mean fluorescence intensity (MFI), which demonstrated that the antibody can bind to PD-L1. The significant FMI value (even at the lowest dilution of 0.01 μg / ml of the antibody) demonstrated the high affinity of huPD-L1 for binding to PD-L1 protein (Tables 17A, 17B and Figure 2 , upper right). When HuPD-L1 antibody was expressed in 300.19 cells, it showed some ability to bind to human PD-L2 or CLEC2D (as shown in Tables 18A, 18B, 19A, 19B and Figure 2 , as shown in the two lower figures). However, the MFI values ​​obtained by staining PD-L2 and CLE2D were not as high as those obtained for PD-L1. In addition, at lower dilutions, the MFI values ​​were not significantly higher than the basal level, indicating that the huPD-L1 antibody does not have high affinity or specificity for PD-L2 or CLE2D. Although a few huPD-L1 antibodies (such as Ab-42) showed some cross-reactivity with PD-L2, this antibody had significantly higher affinity and specificity for PD-L1.

[0191] Table 16. Staining with huPD-L1 on non-transfected 300.19 cells

[0192]

[0193] Table 17A. Assay 1 results using huPD-L1 staining on hPD-L1-transfected 300.19 cells.

[0194]

[0195] Table 17B. Assay 2 results using huPD-L1 staining on hPD-L1-transfected 300.19 cells.

[0196]

[0197] Table 18A. Assay 1 results using huPD-L1 staining on hPD-L2-transfected 300.19 cells.

[0198]

[0199] Table 18B. Assay 2 results using huPD-L1 staining on hPD-L2-transfected 300.19 cells.

[0200]

[0201] Table 19A. Staining with huPD-L1 on hCLEC2D-transfected 300.19 cells, Assay 1.

[0202]

[0203] Table 19B. Staining with huPD-L1 on hCLEC2-transfected 300.19 cells, Assay 2.

[0204]

[0205] Example 2: Characterization of anti-PD-L1 phage-antibodies that block PD / PD-L1 binding Competitive FACS analysis was performed to characterize the inhibition of hPD1 binding to hPD-L1 by anti-PD-L1 phage antibodies. All anti-hPD-L1 antibodies were in phage-scFv format. 293T cells were transfected with a vector encoding human PD-L1 fused to a human Fc region (for expression of hPD-L1-Fc). In this assay, 10 12 Plaque forming units (pfu) of phage-scFv were mixed with approximately 0.25 mg / ml of soluble hPD-1-hFc fusion protein and then added to hPD-L1-expressing 293T cells. After washing, the cells were incubated with FITC-anti-human IgG antibody and analyzed by FACS to measure the binding of hPD1-hFc to hPD-L1 on the cell surface.

[0206] Fluorescence values ​​were obtained by FACS analysis and used to generate the percentage inhibition of hPD-1 binding to hPD-L1+ cells. These values ​​are shown in Figure 3Almost all anti-PD-L1 phage scFvs showed some inhibitory ability on the binding of hPD-1 to hPD-L1. In particular, Ab-14, Ab-16, Ab-30, Ab-31, Ab-42, Ab-50, Ab-52 and Ab-55 phage-scFvs showed significant inhibition of hPD-1 / hPD-L1 binding.

[0207] Example 3: Characterization of huPD-L1 soluble mAbs that block PD / PD-L1 binding Competitive FACS analysis was performed to characterize the inhibition of hPD1 binding to hPD-L1 by the soluble huPD-L1 antibodies of the present invention. All huPD-L1 antibodies were tested for their ability to inhibit the binding of hPD1-IgG fusion protein to hPD-L1-expressing 300.19 cells. In this assay, 50,000 cells expressing hPD-L1 were pre-incubated for 30 minutes with the following concentrations of huPD-L1 or control antibody: 10 μg / ml, 1 μg / ml, 0.1 μg / ml, and 0.01 μg / ml. After pre-incubation, 0.125 μg of human PD1 fused to mouse IgG2 was added to the cells and incubated for another 30 minutes. The cells were washed twice, and then 0.125 μg of goat anti-mouse IgG2-PE antibody was added to the cells. After incubation for 30 minutes, the cells were washed twice and then analyzed by FACS. The values ​​obtained from the FACS analysis were expressed as mean fluorescence intensity units (MFI) and are summarized in Table 20. The MFI values ​​from Table 20 were used to generate Figure 4 As shown herein, all huPD-L1 soluble antibodies tested exhibited almost complete inhibition of hPD1 binding to hPD-L1 at 10 μg / ml. At successively lower concentrations, most soluble antibodies still exhibited very good inhibition of PD1 / PD-L1 binding, particularly Ab-42 and Ab-50.

[0208] Table 20. Results of blocking PD-1 binding using huPD-L1

[0209]

[0210] Example 4: Formation of bispecific antibodies

[0211] Based on the role of the hinge in generating half-monomers of IgG4 molecules, it was hypothesized that introducing charged mutations in the hinge region of human IgG1 may not only promote half-monomer exchange but also potentially stabilize bispecific molecules. This example demonstrates that the combination of hinge mutations and CH3 mutations increases the formation of bispecific antibodies.

[0212] To further stabilize the formation of heterodimers, oppositely charged mutations are further replaced in the CH3 domain, which is the concept of "knob into hole". The formation of bispecific antibodies is achieved by the following steps: First, two parent antibodies carrying bispecific mutations are expressed and purified separately. The antibodies are then mixed in the presence of a mild reducing agent. The mild reduction of the antibodies causes the antibodies to dissociate into two monomers each having a variable heavy chain and light chain. The monomers are then mixed together, followed by an oxidation step, which causes the formation of a bispecific antibody molecule.

[0213] Bispecific antibodies recognizing PD-L1 and G250 (carbonic anhydrase IX) were generated. Anti-G250 parental (G37 wild type, G37WT) and engineered (G37 KIHA) antibodies were produced and purified by two independent vectors. G37 KIHA (which follows the "knob-into-hole" concept and carries a bispecific mutation) was altered in the sequence of the immunoglobulin hinge region.

[0214] To understand the dissociation activity of G37 KIHA, the antibody was mixed in the presence of a mild reducing agent to interfere with the formation of antibody monomers ( Fig. 7A Addition of increasing concentrations of reducing agent (GSH) causes the antibody to dissociate into monomers (e.g. Fig. 7A , as indicated by increased levels of low molecular weight species).

[0215] Similarly, anti-PD-L1 parent (PD-L1 wild-type, PDL-1WT) and engineered (PD-L1 KIHB) antibodies were expressed and purified by two independent vectors. Different concentrations of GSH were used to verify the appropriate conditions for obtaining anti-PD-L1 monomers ( Figure 7B ). The optimal condition of GSH concentration was selected and the resulting anti-PD-L1 monomers were incubated with G37 KIHA monomers. The formation of bispecific antibodies was observed at the same size as wild-type IgG, indicating that an antibody containing two heavy chain-light chain monomers (one PD-L1-specific monomer and one G250-specific monomer) was generated.

[0216] Example 5: Bispecific Antibody Function

[0217] The bispecific antibodies generated in Example 3 were then tested for their ability to recognize two antigens (e.g., PD-L1 and G250). The functionality of the PD-L1 and G250 bispecific antibodies was tested using flow cytometry. + PD-L1 -SKRC-52 expresses CAIX (250) but not PD-L1, and therefore it is expected that the cells can be recognized only by the anti-CAIX antibody G37, but not the anti-PD-L1 antibody. To avoid saturation of antibody binding, the concentration of the antibodies was low and in a dose-reducing manner. In fact, the parent anti-G37 recognized SKRC-52 cells, while the parent anti-PD-L1 did not (levels were the same as the control). The bispecific antibody containing conjugated anti-G37 and anti-PD-L1 monomers recognized SKRC-52 cells at a concentration reduced by half relative to the parent G37 antibody, thereby demonstrating the functionality of the bispecific antibodies generated using the methods and antibodies described herein.

[0218] Example 6: Functional Characterization of mAb42

[0219] Further functional characterization of the monoclonal antibody (mAb42) against PD-L1 was performed. Peripheral blood mononuclear cells (PBMCs) from 4 different healthy donors (D1-D4) were cultured. PBMCs were cultured in the presence of mAb42 or in the presence of a control isotype IgG antibody. PMBCs were stimulated with 0.1 μg / ml SEB (Staphylococcal Enterotoxin B) and the production of TNFα was measured using MSD (MesoScale Delivery). Sample analysis was performed in triplicate.

[0220] As in Figure 8 As shown in, culturing PBMCs in the presence of mAb42 resulted in increased production of TNFα in response to SEB in all four donor samples when cultured in the presence of anti-PD-L1 antibody (mAb42) compared to control antibody. Furthermore, the increase in TNFα production was statistically significant, p<0.0005. Thus, treatment with anti-PD-L1 antibodies improves immune responses in response to antigens or infections in humans.

[0221] Other Implementations

[0222] Although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

Claims

1. An isolated humanized monoclonal antibody comprising: A heavy chain having CDR1, CDR2, and CDR3 consisting of the amino acid sequences SYGIS (SEQ ID NO:58), WISAYNGNTNYAQKLQG (SEQ ID NO:71), and ALPSGTILVGGWFDP (SEQ ID NO:86), respectively; and a light chain having CDR1, CDR2, and CDR3 consisting of the amino acid sequences TRSSGNIASNYVQ (SEQ ID NO:101), EDNQRPS (SEQ ID NO:115), and QSYDSSNLWV (SEQ ID NO:127), respectively; wherein the antibody binds to human PD-L1.

2. An isolated monoclonal antibody comprising: V encoded by the nucleotide sequence of SEQ ID NO: 1 H The amino acid sequence and the V encoded by the nucleotide sequence of SEQ ID NO: 3 L Amino acid sequence.

3. An isolated monoclonal antibody comprising: SEQ ID NO: 2 V H The amino acid sequence and V of SEQ ID NO: 4 L Amino acid sequence.

4. The antibody of any one of claims 1-3, wherein the antibody is monovalent or bivalent.

5. The antibody of claim 1, wherein the antibody is a single chain antibody.

6. The antibody of any one of claims 1-3, wherein the antibody is a bispecific antibody that also binds to a tumor-associated antigen, a cytokine, or a cell surface receptor.

7. The antibody of claim 6, wherein the tumor-associated antigen is CAIX.

8. The antibody of claim 6, wherein the cytokine is IL-10.

9. The antibody of claim 6, wherein the cell surface receptor is CCR4, IL21R, BTLA, HVEM or TIM3.

10. The antibody of any one of claims 1-3, linked to a therapeutic agent.

11. The antibody of claim 10, wherein the therapeutic agent is a toxin, a radiolabel, a siRNA, or a cytokine.

12. The antibody of claim 10, wherein the therapeutic agent is a small molecule.

13. A cell producing the antibody of any one of claims 1-12.

14. Use of the antibody of any one of claims 1 to 12 in the preparation of a medicament for treating or alleviating the symptoms of cancer, wherein the cancer is renal cell carcinoma or breast cancer.

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