Bispecific antibodies against human PD-l1 and PD-l2 and methods of use thereof

Abstract

Provided are bispecific antibodies that bind to both PD-L1 and PD-L2, and methods of using the antibodies to treat cancers (e.g., cancers that express or overexpress PD-L).SOLUTION: Provided is an antibody or antibody fragment that specifically binds to PD-L1 and PD-L2, comprising: A heavy chain CDRH1, CDRH2 and CDRH3 having the sequences indicated in certain three amino acid sequences, respectively, and a light chain CDRL1, CDRL2 and CDRL3 having the sequences indicated in certain three amino acid sequences, respectively. Preferably, the antibody is an IgG.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Priority claims This application claims the benefit of priority to U.S. Provisional Application No. 62 / 647,407, filed March 23, 2018, and U.S. Provisional Application No. 62 / 755,408, filed November 2, 2018, each of which is incorporated herein by reference in its entirety.

[0002] Incorporation of sequence listings The sequence listing contained in the 49 KB (as measured in Microsoft Windows®) file entitled "UTFC_P1338WO_ST25", created on March 14, 2019, has been submitted herewith by electronic submission and is incorporated herein by reference.

[0003] 1. Field This disclosure relates generally to the fields of medicine, oncology, and immunology. More specifically, this disclosure relates to human bispecific antibodies that bind to PL-L1 and PD-L2 and their use in cancer treatment. [Background technology]

[0004] 2. Description of Related Art Blockade of the interaction between the T cell co-inhibitory receptor PD-1 and its ligand, PD-L1, has become a mainstay of modern oncology, now available even in the first-line setting for a subset of melanoma and lung cancer patients (Boussiotis, 2016). While numerous antibodies targeting PD-1 or PD-L1 are currently FDA-approved or in clinical trials, no drugs targeting the second PD-1 ligand, PD-L2, are currently under clinical investigation. PD-L2 binds to PD-1 with approximately three-fold higher affinity than PD-L1 and, like PD-L1, generates inhibitory signals that attenuate T cell function (Cheng et al., 2013; Latchman et al., 2001; Lee et al., 2016; Li et al., 2017; Youngnak et al., 2003). Historically, PD-L2 was largely considered an inducible co-inhibitory molecule, with expression restricted to the tumor stroma; however, improved PD-L2 detection reagents have revealed widespread PD-L2 expression both in the tumor microenvironment and on the surface of tumor cells themselves (Baptista et al., 2016; Danilova et al., 2016; Derks et al., 2015; Dong et al., 2016; Howitt et al., 2016; Kim et al., 2015; Kim et al., 2015; Nomi et al., 2007; Obeid et al., 2016; Ohigashi et al., 2005; Roemer et al., 2016; Shi et al., 2014; Shin et al., 2015; Xu et al., 2016). Recently, PD-L2 has been shown to be an independent predictor of responsiveness to the PD-1 antibody pembrolizumab in multiple cancers ( Yearley et al., 2017 ).

[0005] First described in many cases of classical Hodgkin lymphoma (cHL), amplification of chromosome region 9p24.1 leads to direct upregulation of PD-L1 and PD-L2 (located there) as well as indirect induction via enhanced JAK2 activity (Roemer et al., 2016; Shi et al., 2014; Green et al., 2010; Van Roosbroeck et al., 2016). In addition to cHL, this genetic driver of high PD-L1 / PD-L2 coexpression is also found in a large proportion of primary mediastinal large B-cell lymphoma (PMBL), T-cell lymphoma, and various histiocytic and dendritic cell malignancies. Not surprisingly, many of these cancers have been shown to respond to PD-1 blockade. More recently, amplification of 9p24.1 has been demonstrated in solid tumors such as triple-negative breast cancer (TNBC) ( Howitt et al., 2016 ; Barrett et al., 2015 ). Relatively high co-expression of PD-L1 and PD-L2 has also been observed in numerous other cancers, including gastric cancer, melanoma, lung, head and neck, cervical and vulvar squamous carcinoma, bladder cancer, and hepatocellular carcinoma, among others (Baptista et al., 2016; Danilova et al., 2016; Derks et al., 2015; Dong et al., 2016; Howitt et al., 2016; Kim et al., 2015; Nomi et al., 2007; Obeid et al., 2016; Xu et al., 2016; Yearley et al., 2017; Van Roosbroeck et al., 2016; Barrett et al., 2015; Shin et al., 2016; Inoue et al., 2016; Wang et al., 2016). 2011). For many of these tumors, stromal and endothelial expression of PD-L2 has also been reported in addition to expression by the tumor itself (Yearley et al., 2017). These findings suggest limitations to the therapeutic potential of PD-L1 blockade in these cancers.

[0006] The PD-1 co-inhibitory receptor is primarily expressed by activated T cells and NK cells and can therefore be best targeted with antibodies that bind to the receptor and prevent binding by PD ligands. PD-L1, in contrast, is expressed by tumor cells and suppressive stromal populations and can be targeted with antibodies capable of cytotoxic effector function. While the theoretical benefits of these antibody-dependent cellular cytotoxicity (ADCC)-capable PD-L1 antibodies can be demonstrated in vitro, there are no patient data demonstrating actual effector function in patients or improved outcomes compared to purely blocking variants (Boyerinas et al., 2015).

[0007] PD-L1 and PD-L2 share only approximately 40% identity, and each binds to an additional receptor distinct from PD-1 (Latchman et al., 2001). PD-L1 also binds to B7-1 in an additional negative T cell regulatory interaction (Butte et al., 2007; Butte et al., 2008). In mice, PD-L2 can bind to RGMb on either myeloid cells or T cells to regulate tolerance to inhaled antigens (Xiao et al., 2014; Nie et al., 2017). The role of PD-L2 binding to RGMb in tumors remains to be described, as does the relevance of this interaction in humans. Having bispecific antibodies against PD-L1 and PD-L2 could be extremely advantageous from a therapeutic standpoint. Summary of the Invention

[0008] overview In this context, the present disclosure provides antibodies or antibody fragments that selectively bind to both PD-L1 and PD-L2 and have the heavy and light chain CDR sequence pairs of clones from Tables 3 and 4, respectively. The antibodies or antibody fragments may be encoded by the variable region sequence pairs of the clones shown in Table 1, or may be encoded by light and heavy chain variable region sequences that are 70%, 80%, or 90% identical to the variable region sequence pairs of the clones shown in Table 1, or may be encoded by light and heavy chain variable region sequences that are 95% or higher identical to the sequence pairs of the clones shown in Table 1. The antibodies or antibody fragments may comprise light and heavy chain variable region sequences according to the sequence pairs of the clones from Table 2, or may comprise light and heavy chain variable region sequences that are 70%, 80%, or 90% identical to the sequence pairs of the clones from Table 2, or may comprise light and heavy chain variable region sequences that are 95% or higher identical to the sequence pairs of the clones from Table 2.

[0009] Also provided are methods of treating cancer in a subject, comprising contacting PD-L1 or PD-L2 positive cancer cells in the subject with an antibody as described above. The PD-L1 or PD-L2 positive cancer cells may be solid tumor cells, such as lung cancer cells, brain cancer cells, head and neck cancer cells, breast cancer cells, skin cancer cells, liver cancer cells, pancreatic cancer cells, gastric cancer cells, colon cancer cells, rectal cancer cells, uterine cancer cells, cervical cancer cells, ovarian cancer cells, testicular cancer cells, skin cancer cells, esophageal cancer cells, lymphoma cells, or renal cell carcinoma cells, or leukemia or myeloma, such as acute myeloid leukemia, chronic myeloid leukemia, or multiple myeloma.

[0010] The method may further include contacting the PD-L1 or PD-L2 positive cancer cells with a second anti-cancer agent or therapy, e.g., chemotherapy, radiation therapy, immunotherapy, hormone therapy, or toxin therapy. The second anti-cancer agent or therapy may inhibit the function of PD-L1 or PD-L2 in the cells. The second anti-cancer agent or therapy may be administered simultaneously with the first agent, or before and / or after the first agent. The PD-L1 or PD-L2 positive cancer cells may be metastatic cancer cells, multi-drug resistant cancer cells, or recurrent cancer cells.

[0011] The antibody may be a single-chain antibody, a single-domain antibody, a chimeric antibody, or a Fab fragment. The antibody may be a human antibody, a mouse antibody, an IgG, a humanized antibody, or a humanized IgG. The antibody or antibody fragment may further comprise a label (e.g., a peptide tag, an enzyme, a magnetic particle, a chromophore, a fluorescent molecule, a chemiluminescent molecule, or a dye). The antibody or antibody fragment may further comprise an anti-tumor drug linked thereto (e.g., linked to the antibody or antibody fragment via a photolabile linker or an enzyme-cleavable linker). The anti-tumor drug may be a toxin, a radioisotope, a cytokine, or an enzyme. The antibody or antibody fragment may be conjugated to a nanoparticle or a liposome.

[0012] In another aspect, a method of treating cancer in a subject is provided, comprising delivering to the subject an antibody or antibody fragment having a heavy chain and light chain CDR sequence pair of a clone from Table 3 and Table 4, respectively. The antibody fragment may be a recombinant scFv (single-chain variable region fragment) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. The antibody may be an IgG. The antibody may be a chimeric antibody. Delivery may include administration of the antibody or antibody fragment, or gene delivery using an RNA or DNA sequence or vector encoding the antibody or antibody fragment.

[0013] The antibody or antibody fragment may be encoded by the light and heavy chain variable region sequence pair of a clone shown in Table 1, or may be encoded by the light and heavy chain variable region sequence pair of a clone having 95% identity to that shown in Table 1, or may be encoded by the light and heavy chain variable region sequences having 70%, 80%, or 90% identity to the sequence pair of a clone from Table 1. The antibody or antibody fragment may comprise the light and heavy chain variable region sequences as per the sequence pair of a clone from Table 2, or may comprise the light and heavy chain variable region sequences having 70%, 80%, or 90% identity to the sequence pair of a clone from Table 2, or may comprise the light and heavy chain variable region sequences having 95% identity to the sequence pair of a clone from Table 2.

[0014] Also provided are monoclonal antibodies, wherein the antibody or antibody fragment is characterized by the heavy and light chain CDR sequence pairs of a clone from Table 3 and Table 4, respectively. The antibody fragment may be a recombinant scFv (single-chain variable region fragment) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. The antibody may be a chimeric antibody or an IgG.

[0015] The antibody or antibody fragment may be encoded by the light and heavy chain variable region sequence pair of a clone shown in Table 1, or may be encoded by the light and heavy chain variable region sequence pair of a clone having 95% identity to that shown in Table 1, or may be encoded by the light and heavy chain variable region sequences having 70%, 80%, or 90% identity to the sequence pair of a clone from Table 1. The antibody or antibody fragment may comprise the light and heavy chain variable region sequences as per the sequence pair of a clone from Table 2, or may comprise the light and heavy chain variable region sequences having 70%, 80%, or 90% identity to the sequence pair of a clone from Table 2, or may comprise the light and heavy chain variable region sequences having 95% identity to the sequence pair of a clone from Table 2.

[0016] In yet another embodiment, there is provided a hybridoma or engineered cell encoding an antibody or antibody fragment, wherein the antibody or antibody fragment is characterized by the heavy and light chain CDR sequence pairs of a clone from Table 3 and Table 4, respectively. The antibody fragment may be a recombinant scFv (single-chain variable region fragment) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. The antibody may be a chimeric antibody or an IgG.

[0017] The antibody or antibody fragment may be encoded by the light and heavy chain variable region sequence pair of a clone shown in Table 1, or may be encoded by the light and heavy chain variable region sequence pair of a clone having 95% identity to that shown in Table 1, or may be encoded by the light and heavy chain variable region sequences having 70%, 80%, or 90% identity to the sequence pair of a clone from Table 1. The antibody or antibody fragment may comprise the light and heavy chain variable region sequences as per the sequence pair of a clone from Table 2, or may comprise the light and heavy chain variable region sequences having 70%, 80%, or 90% identity to the sequence pair of a clone from Table 2, or may comprise the light and heavy chain variable region sequences having 95% identity to the sequence pair of a clone from Table 2.

[0018] Further embodiments include cancer vaccines comprising one or more antibodies or antibody fragments characterized by heavy and light chain CDR sequence pairs of clones from Tables 3 and 4, respectively. At least one antibody fragment may be a recombinant scFv (single-chain variable region fragment) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. At least one of the antibodies may be a chimeric antibody or an IgG. At least one antibody or antibody fragment may be encoded by a light and heavy chain variable region sequence pair of a clone shown in Table 1, a light and heavy chain variable region sequence pair of a clone having 95% identity to those shown in Table 1, or a light and heavy chain variable region sequence having 70%, 80%, or 90% identity to a sequence pair of a clone from Table 1. At least one antibody or antibody fragment may comprise light and heavy chain variable region sequences according to a sequence pair of a clone from Table 2, or may comprise light and heavy chain variable region sequences that have 70%, 80% or 90% identity to a sequence pair of a clone from Table 2, or may comprise light and heavy chain variable region sequences that have 95% identity to a sequence pair of a clone from Table 2.

[0019] In another aspect, a method for detecting cells expressing PD-L1 or PD-L2 in a subject is provided, comprising contacting a sample from the subject with an antibody or antibody fragment characterized by the heavy and light chain CDR sequence pairs of clones from Table 3 and Table 4, respectively, and detecting PD-L1- or PD-L2-expressing cells in the sample by allowing the antibody or antibody fragment to bind to cells in the sample. The sample may be a body fluid or tissue sample. The cells may be cancer cells, such as lymphoma cells, breast cancer cells, or renal cell carcinoma cells. The cells may be cells associated with immunosuppression. The cells associated with immunosuppression may be non-cancerous cells in the tumor microenvironment, such as stromal cells or endothelial cells. Detection may include ELISA, RIA, or Western blot. The method may further include performing the method a second time and determining a change in orthopoxvirus antigen levels compared to the first assay. The antibody or antibody fragment may be encoded by the light and heavy chain variable region sequence pair of a clone shown in Table 1, or may be encoded by the light and heavy chain variable region sequence pair of a clone having 95% identity to that shown in Table 1, or may be encoded by the light and heavy chain variable region sequences having 70%, 80%, or 90% identity to the sequence pair of a clone from Table 1. The antibody or antibody fragment may comprise the light and heavy chain variable region sequences as per the sequence pair of a clone from Table 2, or may comprise the light and heavy chain variable region sequences having 70%, 80%, or 90% identity to the sequence pair of a clone from Table 2, or may comprise the light and heavy chain variable region sequences having 95% identity to the sequence pair of a clone from Table 2.

[0020] It is contemplated that any method or composition described herein may be implemented with respect to any other method or composition described herein. Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the present disclosure, are given by way of illustration only, and that various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from the detailed description herein.

[0021] As used herein, "essentially free" with respect to a specified component means that none of the specified components are intentionally incorporated into the composition and / or are present only as contaminants or in trace amounts. The total amount of the specified components resulting from any unintentional contamination of the composition is preferably less than 0.01%. Most preferred are compositions in which no amount of the specified component can be detected using standard analytical methods.

[0022] As used in the specification and claims of this application, "a" or "an" can mean one or more. As used in the specification and claims of this application, when used in conjunction with the word "comprising," "a" or "an" can mean one or more than one. As used in the specification and claims of this application, "another" or "further" can mean at least a second or more.

[0023] As used in the specification and claims of this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists in the study subjects.

[0024] [The present invention 1001] An antibody or antibody fragment comprising the heavy and light chain CDR sequence pairs of the clones from Tables 3 and 4, respectively. [The present invention 1002] 1001. An antibody or antibody fragment of the invention, encoded by light and heavy chain variable region sequences as per the sequence pairs of clones from Table 1. [The present invention 1003] 1001. An antibody or antibody fragment of the invention, encoded by light and heavy chain variable region sequences having at least 70%, 80%, or 90% identity to the sequence pair of a clone from Table 1. [The present invention 1004] 1001. An antibody or antibody fragment of the invention, encoded by light and heavy chain variable region sequences having at least 95% identity to the sequence pair of a clone from Table 1. [The present invention 1005] 1001. An antibody or antibody fragment of the invention comprising light and heavy chain variable region sequences according to the sequence pairs of clones from Table 2. [The present invention 1006] 1001. An antibody or antibody fragment of the invention comprising light and heavy chain variable region sequences having 70%, 80% or 90% identity to the sequence pair of a clone from Table 2. [The present invention 1007] 1001. An antibody or antibody fragment of the invention comprising light and heavy chain variable region sequences having 95% identity to the sequence pair of a clone from Table 2. [The present invention 1008] The antibody or antibody fragment of any one of claims 1001 to 1007, wherein the antibody fragment is a recombinant scFv (single chain variable region fragment) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. [The present invention 1009] The antibody or antibody fragment of any one of claims 1001 to 1007, wherein the antibody is a chimeric antibody. [The present invention 1010] The antibody or antibody fragment of any one of claims 1001 to 1009 of the present invention, wherein the antibody is an IgG. [The present invention 1011] The antibody or antibody fragment of any of claims 1001 to 1010, further comprising a cell-penetrating peptide and / or being an intrabody. [The present invention 1012] The antibody or fragment of any one of 1001 to 1011 of the present invention, which is a human antibody. [The present invention 1013] The antibody or fragment of any one of claims 1001 to 1011 of the present invention, which is a humanized antibody. [The present invention 1014] A method of treating a subject having cancer, comprising delivering to the subject an antibody or antibody fragment having a heavy chain and light chain CDR sequence pair of a clone from Table 3 and Table 4, respectively. [The present invention 1015] The method of claim 10, wherein said antibody or antibody fragment is encoded by a pair of light and heavy chain variable region sequences of a clone shown in Table 1. [The present invention 1016] 1016. The method of claim 1014 or 1015, wherein said antibody or antibody fragment is encoded by light and heavy chain variable region sequences having at least 70%, 80%, or 90% identity to the sequence pair of a clone from Table 1. [The present invention 1017] 1016. The method of claim 1014 or 1015, wherein said antibody or antibody fragment is encoded by a pair of light and heavy chain variable region sequences of a clone having at least 95% identity to a pair of sequences of a clone from Table 1. [The present invention 1018] 1014. The method of claim 10, wherein said antibody or antibody fragment comprises light and heavy chain variable region sequences according to the sequence pair of a clone from Table 2. [The present invention 1019] 1014. The method of claim 10, wherein said antibody or antibody fragment comprises light and heavy chain variable region sequences having 70%, 80% or 90% identity to the sequence pair of a clone from Table 2. [The present invention 1020] 1014. The method of claim 10, wherein the antibody is encoded by light and heavy chain variable region sequences having 95% identity to the sequence pair of a clone from Table 2. [The present invention 1021] The method of any one of claims 1014 to 1020, wherein the antibody fragment is a recombinant scFv (single chain variable region fragment) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. [The present invention 1022] The method of any one of claims 1014 to 1021, wherein the antibody is an IgG. [The present invention 1023] The method of any one of claims 1014 to 1020, wherein the antibody is a chimeric antibody. [The present invention 1024] 1024. The method of any of claims 1014 to 1023, wherein the delivery comprises administration of an antibody or antibody fragment, or gene delivery using an RNA or DNA sequence or vector encoding said antibody or antibody fragment. [The present invention 1025] A hybridoma or engineered cell encoding an antibody or antibody fragment, wherein the antibody or antibody fragment is characterized by the heavy and light chain CDR sequence pairs of a clone from Table 3 and Table 4, respectively. [The present invention 1026] 1025. The hybridoma or engineered cell of the present invention, wherein said antibody or antibody fragment is encoded by light and heavy chain variable region sequences according to the sequence pair of a clone from Table 1. [The present invention 1027] 1025. The hybridoma or engineered cell of the present invention, wherein said antibody or antibody fragment is encoded by light and heavy chain variable region sequences having at least 70%, 80%, or 90% identity to a pair of variable region sequences of a clone from Table 1. [The present invention 1028] 1025. The hybridoma or engineered cell of the present invention, wherein said antibody or antibody fragment is encoded by light and heavy chain variable region sequences having 95% identity to the variable region sequence pair of a clone from Table 1. [The present invention 1029] 1025. The hybridoma or engineered cell of the present invention, wherein said antibody or antibody fragment comprises light and heavy chain variable region sequences according to the sequence pair of a clone from Table 2. [The present invention 1030] 1025. The hybridoma or engineered cell of the present invention, wherein the antibody or antibody fragment is encoded by light and heavy chain variable region sequences having at least 70%, 80%, or 90% identity to a pair of variable region sequences of a clone from Table 2. [The present invention 1031] 1025. The hybridoma or engineered cell of the present invention, wherein said antibody or antibody fragment comprises light and heavy chain variable region sequences having 95% identity to a sequence pair of a clone from Table 2. [The present invention 1032] The hybridoma or engineered cell of any of claims 1025 to 1031, wherein the antibody fragment is a recombinant scFv (single chain variable region fragment) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. [The present invention 1033] The hybridoma or engineered cell of any of claims 1025 to 1032, wherein the antibody is a chimeric antibody. [The present invention 1034] The hybridoma or engineered cell of any of claims 1025 to 1032, wherein the antibody is an IgG. [This invention 1035] The hybridoma or engineered cell of any of claims 1025 to 1034, wherein the antibody or antibody fragment further comprises a cell-penetrating peptide and / or is an intrabody. [The present invention 1036] A vaccine formulation comprising one or more antibodies or antibody fragments characterized by the heavy and light chain CDR sequence pairs of clones from Tables 3 and 4, respectively. [This invention 1037] 1036. The vaccine formulation of the invention, wherein at least one antibody or antibody fragment is encoded by light and heavy chain variable region sequences according to a sequence pair of a clone from Table 1. [The present invention 1038] 1036. The vaccine formulation of the present invention, wherein at least one antibody or antibody fragment is encoded by light and heavy chain variable region sequences having at least 70%, 80%, or 90% identity to a sequence pair of a clone from Table 1. [This invention 1039] 1038. The vaccine formulation of the invention, wherein at least one antibody or antibody fragment is encoded by light and heavy chain variable region sequences having at least 95% identity to a sequence pair of a clone from Table 1. [The present invention 1040] 1036. The vaccine formulation of the invention, wherein at least one antibody or antibody fragment comprises light and heavy chain variable region sequences according to the sequence pair of a clone from Table 2. [This invention 1041] 1036. The vaccine formulation of the invention, wherein at least one antibody or antibody fragment comprises light and heavy chain variable region sequences having 95% identity to a sequence pair of a clone from Table 2. [The present invention 1042] 2. The vaccine formulation of any one of claims 1036 to 1041, wherein at least one antibody fragment is a recombinant scFv (single chain variable region fragment) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. [This invention 1043] The vaccine preparation of any of claims 1036 to 1041, wherein at least one antibody is a chimeric antibody. [This invention 1044] The vaccine preparation of any of claims 1036 to 1043, wherein at least one antibody is an IgG. [This invention 1045] The vaccine formulation of any of claims 1036 to 1044, wherein at least one antibody or antibody fragment further comprises a cell-penetrating peptide and / or is an intrabody. [The present invention 1046] 1. A method for detecting cells expressing PD-L1 or PD-L2 in a subject, comprising: (a) contacting a sample from the subject with an antibody or antibody fragment having a heavy chain and light chain CDR sequence pair of a clone from Table 3 and Table 4, respectively; and (b) detecting cells in the sample that express PD-L1 or PD-L2 by binding of the antibody or antibody fragment to cells in the sample. A method comprising: [This invention 1047] 1046. The method of claim 1046, wherein the sample is a body fluid. [This invention 1048] 1048. The method of any one of claims 1046 to 1047, wherein said sample is a tissue sample. [This invention 1049] 1048. The method of claim 1046 or 1047, wherein the detection comprises ELISA, RIA, or Western blot. [The present invention 1050] Any of the methods of claims 1046 to 1049, further comprising performing steps (a) and (b) a second time and determining a change in orthopoxvirus antigen levels compared to the first assay. [This invention 1051] The method of any of claims 1046 to 1050, wherein said antibody or antibody fragment is encoded by a pair of variable region sequences of a clone shown in Table 1. [This invention 1052] Any of the methods of claims 1046 to 1050, wherein the antibody or antibody fragment is encoded by light chain and heavy chain variable region sequences having 70%, 80%, or 90% identity to the variable region sequence pairs of the clones shown in Table 1. [This invention 1053] 1050. The method of any of claims 1046 to 1050, wherein said antibody or antibody fragment is encoded by light and heavy chain variable region sequences having 95% identity to the sequence pairs of the clones shown in Table 1. [This invention 1054] 1050. The method of any of claims 1046 to 1050, wherein said antibody or antibody fragment comprises light and heavy chain variable region sequences according to the sequence pair of the clone from Table 2. [This invention 1055] 1050. The method of any of claims 1046 to 1050, wherein said antibody or antibody fragment comprises light and heavy chain variable region sequences having 70%, 80% or 90% identity to the sequence pair of a clone from Table 2. [This invention 1056] 1050. The method of any of claims 1046 to 1050, wherein said antibody or antibody fragment comprises light and heavy chain variable region sequences having 95% identity to the sequence pair of a clone from Table 2. [This invention 1057] The method of any one of claims 1046 to 1056, wherein the antibody fragment is a recombinant scFv (single chain variable region fragment) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. [This invention 1058] The method of any one of claims 1046 to 1057, wherein the cells are cancer cells. [This invention 1059] 1058. The method of claim 1058, wherein said cancer cells are lymphoma cells, breast cancer cells, or renal cell carcinoma cells. [The present invention 1060] The method of any one of claims 1046 to 1057, wherein the cells are cells associated with immunosuppression. [This invention 1061] The method of claim 1060, wherein said cells associated with immunosuppression are non-cancerous cells in the tumor microenvironment. [This invention 1062] The method of claim 1061, wherein the non-cancerous cells in the tumor microenvironment are stromal cells or endothelial cells. [This invention 1063] A method for treating immunosuppression in a tumor microenvironment, comprising delivering to a subject an antibody or antibody fragment having a heavy chain and light chain CDR sequence pair of a clone from Table 3 and Table 4, respectively. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating certain particular embodiments of the invention, are given by way of illustration only, and that various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description herein. [Brief explanation of the drawings]

[0025] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0026] [Figure 1A] Figures 1A-1C: Identification of bispecific PD-L1 / PD-L2 (BiPDL) antibodies that block PD-L1 / PD-L2 binding to PD-1. Antibody candidates identified as described in the Examples were tested for their ability to bind to PD-L1 or PD-L2 and block their binding to PD-1. 5 μg / mL of antibody was bound to CHO-PD-L1 or CHO-PD-L2 cells, and then recombinant PD-1 labeled with Alexafluor 532 was added for 1 hour. (Figure 1A) The percentage of PD-1+ cells is shown. (Figure 1B) The maximum fluorescence intensity of Alexafluor 532-labeled PD-1 was measured, and blockade of PD-L1 or PD-L2 was determined as a reduction in Alexa 532 fluorescence in FACS analysis. BiPDL antibody clones 1–4 (ADI-16413, -16414, -16415, and -16418) were evaluated against unstained cells, a control antibody, durvalumab (anti-PD-L1), and a commercially available anti-PD-L2 antibody (Figure 1C). Candidate BiPDL monoclonal antibodies were assayed using the Promega PD-L1:PD-1 blockade system. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 2A]Figures 2A-2D: Identification of bispecific antibody subclones that bind to PD-L1 and PD-L2. Subclones of bispecific antibodies BiPDL3 or BiPDL4 were assayed for binding to CHO-PD-L1 (Figures 2A and 2C) or CHO-PD-L2 cells (Figures 2B and 2D). The indicated concentrations of BiPDL (human IgG1) antibody were added to the indicated cells, and binding was detected by the addition of a PE-conjugated anti-human IgG1 secondary antibody. Reactions were performed on a ForteBio Octet® platform. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 3] Evaluation of bispecific antibodies compared with FDA-approved antibody therapeutics. Candidate BiPDL monoclonal antibodies were assayed using both the Promega PD-L1:PD-1 blockade system and the PD-L2:PD-1 blockade system. Varying concentrations of antibody were added to CHO-PD-L1 or CHO-PD-L2 (CHO-PD-L1 / 2) cells, which are capable of stimulating Jurkat T cells stably expressing PD-1. When co-cultured, the interaction between CHO-PD-L1 / 2 cells and Jurkat T cells inhibits luminescence. Luminescence is induced when their activation is blocked by the addition of PD-L1, PD-L2, PD-1, or BiPDL antibodies. Jurkat T cells were incubated with the indicated concentrations of the indicated antibodies and CHO-PD-L1 / 2 cells for 6 hours. Results were generated using the Bio-Glo™ Assay Kit (Promega). [Figure 4A]Figures 4A-4F: Evaluation of second-, third-, and fourth-generation BiPDL antibodies. Candidate second-generation (Figures 4A-B), third-generation (Figures 4C-D), and fourth-generation (Figures 4E-F) BiPDL monoclonal antibodies were again assayed using the Promega PD-L1 / PD-L2:PD-1 blockade system. Keytruda was used as a positive control anti-PD-L2 antibody (Figures 4B-D), and darvalumab was used as a control anti-PD-L1 antibody (Figures 4C-D). Promega's control mAb against PD-1 was also used as a control (Figures 4C-D). [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E] See legend to Figure 4A. [Figure 4F] See legend to Figure 4A. [Figure 5A] Figures 5A-5C: Candidate BiPDL antibodies are active in multiple human mixed lymphocyte reactions. Candidate, FDA-approved, or control antibodies were evaluated in the presence of induced dendritic cells and T cells from separate donors, and IL-2 or IFN-γ production was assessed by ELISA. (Figure 5A) Magnetically selected iDCs were analyzed by flow cytometry for human PD-L1 and PD-L2 expression. (Figure 5B) Keytruda (anti-PD-1), atezolizumab (anti-PD-L1), a fourth-generation BiPDL antibody, and an isotype control were added to IDCs before addition to CD4+ T cells. (Figure 5C) Different fourth-generation BiPDL antibodies were evaluated alongside atezolizumab and an isotype control. Each was added to IDCs before addition to CD4+ T cells. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 6-1]BiPDL antibody mediates effective ADCC against human PD-L1 / PD-L2+ lymphoma. Various concentrations of BiPDL antibody (mouse IgG2a) were added to calcein-labeled U2940 PMBL cells at an effector-to-target ratio of 15:1, along with in vitro expanded mouse NK cells. % specific lysis was calculated as the difference between experimental and spontaneous release of calcein, as measured by a fluorescent plate reader. [Figure 6-2] This is a continuation of Figure 6-1. [Figure 7A] Figures 7A-7C: Candidate antibodies with ADCC are highly active against human U2940 lymphoma in vivo. PBML xenograft tumors were established in SCID mice and allowed to reach 150 mm3. (Figure 7A) Cells were analyzed for PD-L1 or PD-L2 expression by flow cytometry. (Figures 7B-C) Mice were treated with either mIgG2a control antibody, Herceptin, Rituxan, or the indicated BiPDL antibody at 10 mg / kg twice weekly for 3 weeks, and tumor volume was assessed by caliper on the indicated days. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 8A] Figures 8A-8B: Candidate antibodies are active against MDA-MB-231. MDA-MB-231 triple-negative breast cancer xenograft tumors were established in SCID mice and allowed to reach 150 mm3. (Figure 8A) Cells were analyzed for PD-L1 or PD-L2 expression by flow cytometry. (Figure 8B) Mice were then treated with the indicated antibodies at 10 mg / kg twice weekly for 3 weeks. Data shown are from a single experiment with 9 mice per group, and tumor volume was assessed on the indicated days. [Figure 8B] See legend to Figure 8A. [Figure 9A]Figures 9A-9C: BiPDL antibodies treat syngeneic MC38-PD-L2 colon cancer more effectively than PD-L1 antibodies. 5x105 MC38-PDL2 tumor cells were implanted subcutaneously into C57BL / 6J mice. Mice were treated with 100µg of the indicated antibody on days 3, 6, 9, 12, and 15. (Figure 9A) Survival is shown for a single experiment with 10 mice per group (p-values ​​by Gehran-Breslow-Wilcoxon test). (Figure 9B) Growth of MC-38-PD-L2 tumors in the flank was measured using calipers, and tumor volumes are shown for all groups until any tumor measured ≥1000mm3. (Figure 9C) The presence of PD-L1 and PD-L2 was assessed by flow cytometry. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 10A] Figures 10A-10B: BiPDL antibodies with antibody-dependent cell-mediated cytotoxicity (ADCC) generate favorable CD8 to Treg ratios in MC38-PD-L2 mice. C57BL / 6J mice were implanted subcutaneously with 1.5 x 10 MC38-PDL2 tumor cells in 30% Matrigel (Corning) and treated with 100 μg of the indicated antibodies by intraperitoneal injection on days 7, 10, and 13. Tumors were harvested on day 15. (Figure 10A) The presence of PD-L1 and PD-L2 was assessed by flow cytometry. (Figure 10B) The ratio of infiltrating CD8 T cells to FoxP3+ Tregs was measured by flow cytometry. [Figure 10B] See legend to Figure 10A. [Figure 11A]Figures 11A-11B: BiPDL antibodies treat systemic EL4 T-cell lymphoma more effectively than PD-L1 or PD-L2 blockade. Systemic disease was established in C57BL / 6J mice by implantation of 1.5 x 10 EL4-PD-L2 cells, which also express luciferase to facilitate bioluminescence imaging. Mice were injected with 100 μg of the indicated antibody on days 3, 6, 9, 12, and 15. (Figure 11A) Flow cytometry was used to assess the presence of PD-L1 and PD-L2. (Figure 11B) Survival is shown for one to three independent experiments (depending on group) with five mice per group. Survival statistics were calculated using the Gehan-Breslow-Wilcoxon test. [Figure 11B] See legend to Figure 11A. [Figure 12] Equivalence to and superiority to PD-1 blockade in the Promega PD-L1 / PD-L2 assay. BiPDL3-14 completely blocks PD-1 binding. Keytruda, Tecentriq, and BiPDL were tested for their ability to block PD-1-mediated T cell suppression using a Promega PD-1 assay consisting of CHO cells expressing a T cell activation element and human PD-L1 and PD-L2, and Jurkat T cells with an NFAT-luciferase reporter. [Figure 13-1] F16-F10-PDL2 data suggest promise for the effector BiPDL3 antibody in "cold" tumors. BiPDL3 can cure PD-1-resistant B16 melanoma. Mice implanted with 5x104 B16.F10 melanoma tumors in the flank were treated with the indicated antibodies i.p. on days 3, 6, 9, and 12. GASDIE = hIgG1 with T236A, S239D, and I332E mutations that enhance ADCC / P. [Figure 13-2] A continuation of Figure 13-1 is shown. DETAILED DESCRIPTION OF THE INVENTION

[0027] Description of exemplary embodiments The present inventors have generated monoclonal antibodies with binding specificity for both human PD-L1 and PD-L2 proteins. These antibodies have been demonstrated to bind to both PD-L1 and PD-L2, providing an opportunity to block the binding of either PD-L1 or PD-L2 to PD-1. They can also be used to deliver therapeutic payloads to cancer cells that express PD-L1 or PD-L2. These and other aspects of the present disclosure are described in greater detail below.

[0028] I. PD-L1 A. Structure Programmed cell death ligand 1 (PD-L1) is a protein encoded by the CD274 gene. PD-L1 is a 40 kDa type 1 transmembrane protein that may play a major role in immunosuppression during various events, such as pregnancy, tissue allotransplantation, autoimmune diseases, cancer, and other disease states. Human PD-L1 protein is encoded by the amino acid sequence shown below. TIFF2026035716000001.tif30149

[0029] B. Function PD-L1 is the ligand for its receptor, PD-1. PD-1 can be found on activated T cells, B cells, and myeloid cells. Binding of PD-L1 to PD-1 modulates the activation or inhibition of T and B cells. It transmits an inhibitory signal that reduces the proliferation of antigen-specific CD8+ T cells and CD4+ helper T cells. Binding of PD-L1 to PD-1 also induces apoptosis. This reduction in CD8+ T cells and CD4+ helper T cells is thought to help PD-L1-expressing cancer cells evade antitumor immunity (Dong et al., 2002). Upregulation of PD-L1 has been associated with evasion of the host immune system and is thought to be responsible for increased tumor aggressiveness (Thompson et al., 2004). PD-L1's role in evading antitumor immunity makes it an attractive target for therapeutic intervention.

[0030] II. PD-L2 A. Structure Programmed cell death ligand 2 (PD-L2) is a protein encoded by the CD273 gene. PD-L2 is a 31 kDa protein that may play a major role in immunosuppression during various events, such as pregnancy, tissue allografts, autoimmune diseases, cancer, and other disease states. Human PD-L2 protein is encoded by the amino acid sequence shown below. TIFF2026035716000002.tif30149

[0031] PD-L2 is initially produced with a signal peptide corresponding to amino acids 1-19 of SEQ ID NO:2, which is then removed to yield the mature protein. The mature PD-L2 protein, corresponding to amino acids 20-273 of SEQ ID NO:2, contains an Ig-like V domain, an Ig-like C2-type domain, a transmembrane domain, and a cytoplasmic tail.

[0032] B. Function PD-L2 is the ligand for its receptor, PD-1. PD-1 can be found on activated T cells, B cells, and myeloid cells. Binding of PD-L2 to PD-1 initiates an immunological cascade that impairs T cell proliferation, cytokine production, cytolytic function, and survival. PD-1 transmits inhibitory signals that reduce the proliferation of antigen-specific CD8+ T cells and CD4+ helper T cells. PD-L2 has also been shown to be an independent predictor of responsiveness to the PD-1 antibody pembrolizumab in multiple cancers (Yearley et al., 2017).

[0033] III. Monoclonal Antibodies and Their Generation A. General method Antibodies to PD-L1 and PD-L2 can be generated by standard methods known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; US Patent 4,196,265). Methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as methods for preparing polyclonal antibodies. The first step in both of these methods is the immunization of a suitable host or the identification of a subject who is immune due to a previous natural infection. As is well known in the art, a given composition for immunization can vary in its immunogenicity. Therefore, it is often necessary to boost the host immune system, which can be achieved by linking a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins, such as ovalbumin, mouse serum albumin, or rabbit serum albumin, can also be used as carriers. Means for conjugating polypeptides to carrier proteins are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide, and bisdiazotized benzidine. As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of nonspecific stimulators of the immune response known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a nonspecific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant.

[0034] The amount of immunogen composition used in the production of polyclonal antibodies varies depending on the nature of the immunogen as well as the animal used for immunization. Various routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous, and intraperitoneal). Polyclonal antibody production can be monitored by sampling the immunized animal's blood at various times after immunization. A second booster injection may be given. The process of boosting and titration is repeated until a suitable titer is achieved. When a desired level of immunogenicity is achieved, the immunized animal can be bled and the serum isolated and stored, and / or the animal can be used to generate monoclonal antibodies.

[0035] After immunization, somatic cells with the potential to produce antibodies, specifically B lymphocytes (B cells), are selected for use in mAb production protocols. These cells can be obtained from biopsied spleens or lymph nodes, or from circulating blood. Antibody-producing B lymphocytes from the immunized animal are then fused with immortal myeloma cells, generally cells of the same species as the immunized animal, or human or human / mouse chimeric cells. Myeloma cell lines suitable for use in hybridoma production fusion procedures are preferably non-antibody-producing, have high fusion efficiency, and possess enzyme deficiencies that render them unable to grow in certain selective media that support the growth of only the desired fused cells (hybridomas).

[0036] As known to those skilled in the art, any of a number of myeloma cells can be used (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). For example, when the animal to be immunized is a mouse, P3-X63 / Ag8, X63-Ag8.653, NS1 / 1.Ag 4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG 1.7, and S194 / 5XX0 Bul can be used; when the animal to be immunized is a rat, R210.RCY3, Y3-Ag 1.2.3, IR983F, and 4B210 can be used; and U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6 are all useful in connection with human cell fusions. One particular mouse myeloma cell is the NS-1 myeloma cell line (also referred to as P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by submitting cell line repository number GM3573. Another mouse myeloma cell line that can be used is the 8-azaguanine-resistant mouse myeloma SP2 / 0 non-producer cell line. More recently, additional fusion partner lines for use with human B cells have been described, including KR12 (ATCC CRL-8658); K6H6 / B5 (ATCC CRL-1823); SHM-D33 (ATCC CRL-1668); and HMMA2.5 (Posner et al., 1987). The antibodies in this disclosure were generated using the SP2 / 0 / mIL-6 cell line, an IL-6-secreting derivative of the SP2 / 0 line.

[0037] Methods for generating hybrids between antibody-producing spleen or lymph node cells and myeloma cells typically involve mixing somatic cells with myeloma cells in a 2:1 ratio, although the ratio can vary from about 20:1 to about 1:1, respectively, in the presence of one or more agents (chemical or electrical) that promote cell membrane fusion. A fusion method using Sendai virus is described by Kohler and Milstein (1975; 1976), and a fusion method using polyethylene glycol (PEG), e.g., 37% (v / v) PEG, is described by Gefter et al. (1977). Electrically induced fusion methods are also suitable (Goding, pp. 71-74, 1986).

[0038] The fusion procedure typically involves approximately 1 x 10 -6 ~1×10 -8 This produces viable hybrids at a low frequency. However, this is not a problem because viable fused hybrids differentiate from parental unfused cells (especially unfused myeloma cells, which usually continue to divide indefinitely) by culturing in a selective medium. Selective media are generally media containing agents that block de novo synthesis of nucleotides in tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, while azaserine blocks only purine synthesis. When aminopterin or methotrexate is used, hypoxanthine and thymidine are added to the medium as a source of nucleotides (HAT medium). When azaserine is used, hypoxanthine is added to the medium. If the B cell source is an Epstein-Barr virus (EBV) transformed human B cell line, ouabain is added to remove EBV transformed cells that have not fused to myeloma cells.

[0039] The preferred selective medium is HAT or HAT containing ouabain. Only cells capable of operating the nucleotide salvage pathway can survive in HAT medium. Myeloma cells lack key enzymes in the salvage pathway, such as hypoxanthine phosphoribosyltransferase (HPRT), and cannot survive. B cells can operate this pathway, but they have a limited lifespan in culture and generally die within about two weeks. Therefore, the only cells that can survive in selective medium are hybrids formed from myeloma and B cells. When the source of B cells used for fusion is an EBV-transformed B cell line, as in this case, ouabain is also used for drug selection of the hybrid, since EBV-transformed B cells are sensitive to drug killing, while the myeloma partner used is selected to be ouabain-resistant.

[0040] Culture provides a population of hybridomas from which specific hybridomas are selected. Hybridoma selection is typically performed by culturing the cells by dilution of single clones in microtiter plates and then testing the supernatants of individual clones (after approximately 2-3 weeks) for the desired reactivity. The assay should be sensitive, simple, and rapid, such as radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, or dot immunobinding assay.

[0041] The selected hybridomas are then serially diluted or single cells are selected by flow cytometry sorting and cloned to produce individual antibody-producing cell lines, which can then be propagated indefinitely to provide mAbs. Cell lines can be exploited for MAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into an animal (e.g., a mouse). Optionally, the animal is primed with a hydrocarbon, particularly an oil such as pristane (tetramethylpentadecane), prior to injection. When human hybridomas are used in this method, they are best injected into immunocompromised mice, such as SCID mice, to prevent tumor rejection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. Animal body fluids, such as serum or ascites fluid, can then be harvested to provide mAbs in high concentrations. Individual cell lines can also be cultured in vitro, where mAbs are naturally secreted into the culture medium, from which they can be readily obtained in high concentrations. Alternatively, human hybridoma cell lines can be used in vitro to produce immunoglobulins in the cell supernatant. To optimize the ability to recover highly pure human monoclonal immunoglobulins, the cell lines can be adapted for growth in serum-free medium.

[0042] Monoclonal antibodies produced by either means may be further purified, if necessary, using filtration, centrifugation, and various chromatographic methods, such as FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the present disclosure can be obtained from the purified monoclonal antibodies by methods including digestion with enzymes, such as pepsin or papain, and / or by breaking down disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

[0043] It is also contemplated that a molecular cloning approach may be used to generate monoclonal antibodies. For this purpose, RNA is isolated from hybridoma strains, antibody genes are obtained by RT-PCR, and cloned into immunoglobulin expression vectors. Alternatively, a combinatorial immunoglobulin phagemid library is prepared from RNA isolated from cell strains, and phagemids expressing suitable antibodies are selected by panning using viral antigens. The advantage of this approach over traditional hybridoma technology is that it allows approximately 10 4 The advantages of this approach are that twice as many antibodies can be produced and screened, and that new specificities can be generated by combining heavy and light chains, further increasing the chances of finding a suitable antibody.

[0044] Yeast-based antibody libraries may be rationally designed, and antibodies may be selected and / or isolated from such yeast-based antibody display libraries, as disclosed, for example, in WO2012 / 009568, WO2009 / 036379, WO2010 / 105256, WO2003 / 074679, U.S. Patent No. 8,691,730, and U.S. Patent No. 9,354,228. Antibodies may be expressed and purified as full-length IgG from any desired cell type, as disclosed above.

[0045] Other U.S. patents that teach the production of antibodies useful in the present disclosure, each of which is incorporated herein by reference, include U.S. Pat. No. 5,565,332, which describes the production of chimeric antibodies using a combinatorial approach; U.S. Pat. No. 4,816,567, which describes recombinant immunoglobulin preparations; and U.S. Pat. No. 4,867,973, which describes antibody-therapeutic agent conjugates.

[0046] B. Antibodies of the Present Disclosure Antibodies according to the present disclosure can be defined, in a first instance, by their binding specificity (i.e., binding to PD-L1 and PD-L2). One of skill in the art can determine whether a given antibody falls within the scope of the claims of this application by assessing the binding specificity / affinity of such an antibody using techniques well known to those of skill in the art. In one aspect, monoclonal antibodies are provided having the heavy and light chain CDR pairs of the clones set forth in Tables 3 and 4, respectively. Such antibodies may be produced by the clones discussed below in the Examples section using the methods described herein.

[0047] In a second aspect, antibodies may be defined by their variable region sequences, including additional "framework" regions. These are provided in Tables 1 and 2, which encode or represent full-length variable regions. Furthermore, antibody sequences may optionally differ from these sequences using methods discussed in more detail below. For example, nucleic acid sequences may differ from those set forth above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology, if the nucleic acid is cleaved under low salt and / or high temperature conditions (e.g., about 0.02 M to about 0.15 M NaCl at a temperature of about 50° C. to about 70° C.). (e) amino acids may differ from those set forth above by a given percentage of homology, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or (f) amino acids may differ from those set forth above by allowing for conservative substitutions (discussed below). Each of the foregoing applies to the nucleic acid sequences set forth as Table 1 and the amino acid sequences of Table 2.

[0048] C. Antibody Sequence Modifications In various embodiments, one may choose to modify the sequence of an identified antibody for various reasons, such as improved expression, improved cross-reactivity, or reduced off-target binding. Below is a general discussion of relevant techniques for antibody engineering.

[0049] After culturing the hybridomas, cells can be lysed and total RNA extracted. After generating cDNA copies of the RNA using random hexamers in RT, PCR can be performed using a multiplexed mixture of PCR primers expected to amplify all human variable gene sequences. The PCR products can be cloned into the pGEM-T Easy vector and then sequenced by automated DNA sequencing using standard vector primers. Antibodies recovered from hybridoma supernatants and purified by FPLC using a Protein G column can be used for binding and neutralization assays.

[0050] Recombinant full-length IgG antibodies may be produced by subcloning the heavy and light chain Fv DNA from the cloning vector into an IgG plasmid vector, transfected into 293 Freestyle cells or CHO cells, and the antibodies may be recovered and purified from the cell supernatant of the 293 or CHO cells.

[0051] The rapid availability of antibodies produced in the same host cell and cell culture method as the final cGMP manufacturing process has the potential to reduce the duration of method development programs. Lonza has developed a general method for the rapid production of small amounts (up to 50 g) of antibody in CHO cells using pooled transfectants grown in CDACF medium. While somewhat slower than true transient systems, advantages include higher product concentrations and the use of the same host and process as the production cell line. In an example of growth and productivity of a GS-CHO pool expressing a model antibody in a single-use bioreactor operated in fed-batch mode (5 L working volume), a harvested antibody concentration of 2 g / L was achieved within 9 weeks of transfection.

[0052] Antibodies, as well as antibody libraries from which such antibodies can be selected and / or isolated, may be rationally designed and synthesized, for example, by Adimab® technology as disclosed in WO2012 / 009568, WO2009 / 036379, WO2010 / 105256, WO2003 / 074679, U.S. Pat. No. 8,691,730, and U.S. Pat. No. 9,354,228. This method of synthetic antibody requires that a nucleotide sequence encoding the desired or engineered antibody be inserted into a vector for ectopic expression. The desired antibody may then be expressed and purified as a full-length IgG molecule.

[0053] Antibody molecules include, for example, fragments produced by proteolytic cleavage of mAbs (F(ab'), F(ab')2, etc.), or single-chain immunoglobulins that can be produced, for example, via recombinant means. Such antibody derivatives are monovalent. In one embodiment, such fragments can be combined with each other, or with other antibody fragments or receptor ligands, to form "chimeric" binding molecules. Notably, such chimeric molecules may contain substituents capable of binding to different epitopes of the same molecule.

[0054] In related embodiments, the antibody is a derivative of the disclosed antibodies, e.g., an antibody containing the same CDR sequences as those in the disclosed antibodies (e.g., a chimeric antibody or a CDR-grafted antibody). Alternatively, modifications, such as the introduction of conservative changes, into the antibody molecule may be desired. In making such modifications, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biological function to a protein is generally understood in the art (Kyte and Doolittle, 1982). It is recognized that the relative hydropathic characteristics of amino acids contribute to the secondary structure of the resulting protein, which in turn determines the interaction of the protein with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).

[0055] It is also understood in the art that substitutions of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with the biological properties of the protein. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values ​​are assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartic acid (+3.0±1), glutamic acid (+3.0±1), asparagine (+0.2), and glutamine (+0.2); hydrophilic nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4), sulfur-containing amino acids: cysteine ​​(-1.0) and methionine (-1.3); hydrophobic nonaromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5±1), alanine (-0.5), and glycine (0); Hydrophobic aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3).

[0056] It is understood that an amino acid can be substituted for another amino acid having a similar hydrophilicity to produce a biologically or immunologically modified protein, with substitution of amino acids whose hydrophilicity values ​​are within ±2 being preferred, those within ±1 being particularly preferred, and those within ±0.5 being even more particularly preferred.

[0057] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into consideration various of the above characteristics are well known to those of skill in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0058] The present disclosure also contemplates isotype modification. By modifying the Fc region to have a different isotype, different functions can be achieved. For example, changing to IgG1 can enhance antibody-dependent cellular cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve binding valency.

[0059] The modified antibodies can be prepared by any technique known to those skilled in the art, including expression through standard molecular biology techniques or chemical synthesis of the polypeptide. Methods of recombinant expression are covered elsewhere in this document.

[0060] D. Single chain antibody Single-chain variable fragments (scFvs) are fusions of the variable regions of immunoglobulin heavy and light chains linked together with a short (usually serine or glycine) linker. These chimeric molecules retain the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker peptide. This modification typically leaves the specificity unchanged. These molecules were historically generated to facilitate display by phage, where it is highly convenient to express the antigen-binding domain as a single peptide. Alternatively, scFvs can be generated directly from subcloned heavy and light chains derived from hybridomas. Single-chain variable fragments lack the constant Fc region found in intact antibody molecules and therefore the common binding site (e.g., protein A / G) used to purify antibodies. These fragments can often be purified / immobilized using protein L, as protein L interacts with the variable region of the kappa light chain.

[0061] Flexible linkers are generally composed of helix- and turn-promoting amino acid residues, such as alanine, serine, and glycine. However, other residues may also function. Tang et al. (1996) used phage display as a means to rapidly select specialized linkers for single-chain antibodies (scFv) from protein linker libraries. A random linker library was constructed in which genes for heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire (approximately 5 × 10 6 The tethers (10 distinct members) were displayed on filamentous phage and subjected to affinity selection with the hapten. The population of selected variants exhibited significantly increased binding activity while retaining considerable sequence diversity. Screening of 1054 individual variants subsequently yielded catalytically active scFvs that were efficiently produced in soluble form. Sequence analysis revealed the V as the only common feature of the selected tethers. H A conserved proline in two residues of the linker after the C-terminus of and numerous arginines and prolines in other positions were revealed.

[0062] The recombinant antibodies of the present disclosure may also include sequences or moieties that allow receptor dimerization or multimerization. Such sequences include sequences derived from IgA that allow for the formation of multimers in combination with the J chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with agents that allow the combination of two antibodies, such as biotin / avidin.

[0063] In another embodiment, single-chain antibodies can be produced by linking the light and heavy chains of the receptor using a non-peptide linker or chemical unit. Generally, the light and heavy chains are produced in separate cells, purified, and then linked together by a suitable method (i.e., the N-terminus of the heavy chain is attached to the C-terminus of the light chain via a suitable chemical crosslinker).

[0064] Cross-linking reagents, such as stabilizers and coagulants, are used to form molecular bridges that connect the functional groups of two different molecules.However, it is contemplated that the heteromeric complexes that are composed of the dimer or multimer of the same analog or different analogs can be produced.To link two different compounds in a step-by-step manner, heterobifunctional cross-linking agents can be used, which eliminates the formation of undesired homopolymers.

[0065] Exemplary heterobifunctional crosslinkers contain two reactive groups, one that reacts with primary amine groups (e.g., N-hydroxysuccinimide) and the other that reacts with thiol groups (e.g., pyridyl disulfide, maleimide, halogen, etc.). Through the primary amine reactive group, the crosslinker may react with a lysine residue of one protein (e.g., a selected antibody or fragment), and through the thiol reactive group, the crosslinker already attached to the first protein reacts with a cysteine ​​residue (free sulfhydryl group) of another protein (e.g., a selective agent).

[0066] It is preferable to use a crosslinker that has reasonable stability in blood.Many types of disulfide bond-containing linkers are known that can be successfully used to conjugate targeting agents and therapeutic / prophylactic agents.Linkers that contain sterically hindered disulfide bonds may provide greater stability in vivo and prevent the release of targeting peptides before reaching the site of action.Therefore, these linkers are a group of linking agents.

[0067] Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is "sterically hindered" by adjacent benzene rings and methyl groups. The steric hindrance of the disulfide bond is thought to function to protect the bond from attack by thiolate anions, such as glutathione, that may be present in tissues and blood, thereby helping to prevent decoupling of the conjugate prior to delivery of the bound agent to the target site.

[0068] Like many other known cross-linking reagents, the SMPT cross-linking reagent offers the ability to cross-link functional groups such as the SH of cysteine ​​or primary amines (e.g., the epsilon-amino group of lysine). Another possible class of cross-linking reagents includes heterobifunctional photoreactive phenyl azides containing a cleavable disulfide bond, such as sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino groups, and the phenyl azide reacts nonselectively (by photolysis) with any amino acid residue.

[0069] In addition to hindered cross-linkers, unhindered linkers can also be used in accordance with the present invention. Other useful cross-linkers that are not thought to contain or generate protected disulfides include SATA, SPDP, and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.

[0070] U.S. Patent No. 4,680,338 describes bifunctional linkers useful for preparing conjugates of ligands with amine-containing polymers and / or proteins, particularly for forming antibody conjugates with chelators, drugs, enzymes, detectable labels, and the like. U.S. Patent Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing labile bonds that can be cleaved under a variety of mild conditions. This linker is particularly useful in that the agent of interest can be directly attached to the linker, and cleavage can result in release of the active agent. Specific uses include adding free amino or free sulfhydryl groups to proteins, such as antibodies, or drugs.

[0071] U.S. Patent No. 5,856,456 provides peptide linkers for use in connecting polypeptide components to prepare fusion proteins, such as single-chain antibodies. The linkers are up to about 50 amino acids in length, contain at least one proline residue after a charged amino acid (preferably arginine or lysine), and are characterized by greater stability and reduced aggregation. U.S. Patent No. 5,880,270 discloses aminooxy-containing linkers useful in various immunodiagnostic and separation techniques.

[0072] E. Purification In certain embodiments, the antibodies of the present disclosure may be purified. The term "purified," as used herein, is intended to refer to a composition that can be isolated from other components and that has been purified to any degree relative to the state in which the protein can be obtained in nature. Thus, a purified protein also refers to a protein that has been released from the environment in which it may naturally occur. When the term "substantially purified" is used, this designation refers to a composition in which the protein or peptide forms the majority of the composition, for example, constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the protein in the composition.

[0073] Protein purification techniques are well known to those skilled in the art. At one level, these techniques involve crude fractionation of the cellular milieu into polypeptide and non-polypeptide fractions. Once the polypeptide has been separated from other proteins, chromatographic and electrophoretic techniques can be used to further purify the polypeptide of interest to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of pure peptides include ion exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies, or heat denaturation followed by centrifugation; gel filtration, reverse-phase, hydroxylapatite, and affinity chromatography; and combinations of such and other techniques.

[0074] In purifying the antibody of the present disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions.The polypeptide can be purified from other cellular components using an affinity column that binds to the tagged portion of the polypeptide.As is generally known in the art, the order in which various purification steps are performed may be changed, or certain steps may be omitted, and still result in a suitable method for preparing a substantially purified protein or peptide.

[0075] Typically, whole antibodies are fractionated using an agent that binds to the Fc portion of the antibody (i.e., protein A). Alternatively, the antigen may be used to simultaneously purify and select the appropriate antibodies. Such methods often utilize a selection agent bound to a support, such as a column, filter, or beads. The antibody is bound to the support, contaminants are removed (e.g., by washing), and the antibody is released by applying conditions (salt, heat, etc.).

[0076] Various methods for quantifying the degree of purification of a protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction or assessing the amount of polypeptide within a fraction by SDS / PAGE analysis. Another way to assess the purity of a fraction is to calculate the specific activity of the fraction and compare it to the specific activity of the initial extract, thereby calculating the degree of purity. The actual units used to express the amount of activity will, of course, depend on the particular assay technique chosen for purification and whether the expressed protein or peptide exhibits detectable activity.

[0077] It is known that the migration of polypeptides can vary, sometimes significantly, using different conditions of SDS / PAGE (Capaldi et al., 1977). Therefore, it will be understood that the apparent molecular weight of purified or partially purified expression products may vary under different electrophoretic conditions.

[0078] IV. Pharmaceutical Preparations and Cancer Treatment A. Cancer Cancer results from the proliferation of a clonal population of cells from a tissue. Cancer development, termed carcinogenesis, can be modeled and characterized in a number of ways. The association between cancer development and inflammation has long been recognized. The inflammatory response participates in host defense against microbial infection and also promotes tissue repair and regeneration. Considerable evidence points to a link between inflammation and cancer risk; chronic inflammation can lead to dysplasia.

[0079] Cancer cells to which the methods of the present disclosure can be applied generally include any cells that express PD-L1 or PD-L2, more specifically, any cells that overexpress either PD-L1 or PD-L2. Suitable cancer cells may be breast cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, stomach cancer, liver cancer, bone cancer, blood cancer (e.g., leukemia or lymphoma), neural tissue cancer, melanoma, ovarian cancer, testicular cancer, prostate cancer, cervical cancer, vaginal cancer, or bladder cancer cells. Additionally, the methods of the present disclosure can be applied to a wide range of species, such as humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Cancers may also be recurrent, metastatic, and / or multidrug resistant, and the methods of the present disclosure may be particularly applied to such cancers to render them resectable, to prolong or re-induce remission, to inhibit angiogenesis, to prevent or limit metastasis, and / or to treat multidrug resistant cancers. At the cellular level, the methods of the present disclosure may lead to the killing of cancer cells, the inhibition of cancer cell proliferation, or otherwise the reversal or reduction of the malignant phenotype of tumor cells.

[0080] B. Formulation and Administration The present disclosure provides pharmaceutical compositions comprising bispecific antibodies against PD-L1 and PD-L2 (BiPDL). In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in animals, more specifically in humans, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia. The term "carrier" refers to a diluent, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, saline, dextrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0081] The compositions can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and salts formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0082] The antibodies of the present disclosure may comprise classical pharmaceutical preparations. Administration of these compositions according to the present disclosure may be via any common route, as long as the target tissue is accessible via that route. This includes oral, nasal, buccal, rectal, vaginal, or topical administration. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such compositions are typically administered as pharmaceutically acceptable compositions as described above. Of particular interest are direct intratumoral administration, tumor perfusion, or local or regional administration to tumors, for example, in the local or regional vascular or lymphatic system, or in a resected tumor bed.

[0083] Active compound can also be administered parenterally or intraperitoneally.The solution of active compound as free base or pharmacologically acceptable salt can be prepared in water, suitably mixed with surfactant such as hydroxypropyl cellulose.Dispersion can also be prepared in glycerol, liquid polyethylene glycol, and their mixture and oil.Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

[0084] C. Combination Therapy In the context of the present disclosure, it is also contemplated that the bispecific antibodies to PD-L1 and PD-L2 (BiPDLs) described herein may similarly be used in combination with chemotherapy or radiotherapy intervention or other treatments. In particular, it may also be effective to combine bispecific antibodies to PD-L1 and PD-L2 with other therapies that target different aspects of PD-L1 or PD-L2 function (e.g., peptides and small molecules that target the cytoplasmic domain of PD-L1 or PD-L2).

[0085] The methods and compositions of the present disclosure are used to generally contact "target" cells with an anti-PD-L1 and anti-PD-L2 bispecific antibody of the present disclosure and at least one other agent to kill the cells, inhibit cell proliferation, inhibit metastasis, inhibit angiogenesis, or otherwise reverse or reduce the malignant phenotype of tumor cells. These compositions are provided in a combined amount effective to kill the cells or inhibit cell proliferation. The method may involve simultaneously contacting the cells with an anti-PD-L1 and anti-PD-L2 bispecific antibody of the present disclosure and the other agent or factor. This may be accomplished by contacting the cells with a single composition or pharmacological formulation containing both agents, or by simultaneously contacting the cells with two separate compositions or formulations, one composition containing the anti-PD-L1 and anti-PD-L2 bispecific antibody of the present disclosure and the other containing the other agent.

[0086] Alternatively, anti-PD-L1 and anti-PD-L2 bispecific antibody therapy may precede or follow treatment with the other agent by intervals ranging from minutes to weeks. In embodiments in which the other agent and the anti-PD-L1 and anti-PD-L2 bispecific antibody are administered to cells separately, care is typically taken to ensure that no significant period of time passes between each delivery, so that the agents and expression constructs can still exert their advantageously combined effect on the cells. In such instances, it is contemplated that the cells will be contacted with both modalities within about 12-24 hours of each other, more preferably within about 6-12 hours, with a delay of only about 12 hours being most preferred. However, in some circumstances, it may be desirable to significantly extend the duration of treatment, such that several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) pass between administrations.

[0087] It may be desirable to administer multiple doses of either the anti-PD-L1 and anti-PD-L2 bispecific antibody or other agents. Various combinations may be used, and are exemplified below, where the anti-PD-L1 and anti-PD-L2 bispecific antibody therapy of the disclosure is designated "A" and the other therapy is designated "B." TIFF2026035716000003.tif17128

[0088] The administration of the therapeutic agent of the present invention to patients will follow the general protocol for administering a specific second-line therapy, taking into account the toxicity, if any, of antibody therapy. It is expected that treatment cycles will be repeated as necessary. It is also contemplated that surgical intervention may be applied in addition to various standard therapies in combination with the described cancer treatments.

[0089] Those skilled in the art are directed to "Remington's Pharmaceutical Sciences," 15th Edition, Chapter 33, especially pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.

[0090] 1. Chemotherapy Cancer treatment also includes various combination therapies involving both chemical and radiation-based therapies. Combination chemotherapy includes, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosoureas, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatin, 5-fluorouracil, vincristine, vinblastine, and methotrexate, temozolomide (aqueous form of DTIC), or any analog or derivative variant of the above. The combination of biological therapy and chemotherapy is known as biochemotherapy. The present invention contemplates any chemotherapeutic agent that may be used or known in the art to treat or prevent cancer.

[0091] 2. Radiation therapy Other agents that cause DNA damage and have been used extensively include gamma rays, X-rays, and / or what is commonly known as tumor-cell-directed delivery of radioisotopes. Other forms of DNA-damaging agents, such as microwave and UV radiation, are also contemplated. All of these agents most likely result in widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50-200 roentgens over prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and its uptake by neoplastic cells.

[0092] The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the manner in which a therapeutic agent and a chemotherapeutic or radiotherapeutic agent are delivered to or placed in direct proximity with a target cell. To achieve cell killing or stasis, both agents are delivered to the cell in a combined amount effective to kill the cell or prevent cell division.

[0093] 3. Immunotherapy Immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific to a marker on the surface of tumor cells. The antibody may act alone as an effector of therapy or may localize other cells and actually kill the cells. Antibodies may also be conjugated to drugs or toxins (such as chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) or simply function as targeting agents. Alternatively, the effector may be a lymphocyte bearing a surface molecule that interacts directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells. The combination of therapeutic modalities, i.e., direct cytotoxic activity and inhibition or reduction of Fortilin, provides therapeutic benefits in cancer treatment.

[0094] Immunotherapy can also be used as part of a combination therapy. General approaches for combination therapy are discussed below. In one aspect of immunotherapy, tumor cells must have some marker that is suitable for targeting, i.e., not present on most other cells. Many tumor markers exist, any of which may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate-specific antigen, urinary tumor-associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is the anti-cancer effect associated with immune stimulatory effects. There are also immune stimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand. Combining immune stimulatory molecules, either as proteins or using gene delivery in combination with tumor suppressors such as mda-7, has been shown to enhance antitumor effects (Ju et al., 2000).

[0095] As previously discussed, examples of immunotherapies currently under consideration or in use include immune adjuvants (e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds) (U.S. Patent No. 5,801,005, U.S. Patent No. 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998), cytokine therapy (e.g., interferon, IL-1, GM-CSF, and TNF) (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998), gene therapy (e.g., TNF, IL-1, IL-2, p53) (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patent Nos. 5,830,880 and 5,846,945) and monoclonal antibodies (e.g., anti-ganglioside GM2, anti-HER-2, anti-p185) (Pietras et al., 1998; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). Herceptin (trastuzumab) is a chimeric (mouse-human) monoclonal antibody that blocks the HER2-neu receptor. It has antitumor activity and has been approved for use in the treatment of malignant tumors (Dillman, 1999). Combination cancer therapy using Herceptin and chemotherapy has been shown to be more effective than individual therapies. Therefore, it is contemplated that one or more anticancer therapies may be used in conjunction with the tumor-associated HLA-restricted peptide therapy described herein.

[0096] In adoptive immunotherapy, a patient's circulating or tumor-infiltrating lymphocytes are isolated in vitro, activated with lymphokines such as IL-2, or transduced with genes for tumor necrosis, and then re-administered (Rosenberg et al., 1988; 1989). To achieve this, an immunologically effective amount of activated lymphocytes is administered to an animal or human patient in combination with an adjuvant-incorporated antigenic peptide composition as described herein. The activated lymphocytes are most preferably the patient's own cells, previously isolated from blood or tumor samples and activated (or "expanded") in vitro. This form of immunotherapy has resulted in regression of melanoma and kidney cancer in some cases, although the proportion of responders is small compared to non-responders.

[0097] There are many different approaches for passive immunotherapy of cancer, which can be broadly classified as follows: injection of antibodies alone, injection of antibodies linked to toxins or chemotherapeutic agents, injection of antibodies linked to radioactive isotopes, injection of anti-idiotypic antibodies, and finally, purging of the bone marrow for tumor cells.

[0098] Human monoclonal antibodies are used in passive immunotherapy because they cause minimal or no side effects in patients. However, their application is somewhat limited by their rarity, and to date they have only been administered intralesionally. Human monoclonal antibodies against ganglioside antigens have been administered intralesionally to patients with cutaneous recurrent melanoma (Irie & Morton, 1986). After daily or weekly intralesional injections, regression was observed in 6 out of 10 patients. In another study, moderate success was achieved from intralesional injections of two human monoclonal antibodies (Irie et al., 1989). Possible therapeutic antibodies include anti-TNF, anti-CD25, anti-CD3, anti-CD20, CTLA-4-IG, and anti-CD28.

[0099] It may be advantageous to administer more than one monoclonal antibody targeting two different antigens, or even to administer an antibody with multiple antigen specificities. Therapeutic protocols may also include the administration of lymphokines or other immune enhancers, such as those described by Bajorin et al. (1988). The development of human monoclonal antibodies is described in more detail elsewhere herein.

[0100] 4. Gene Therapy In yet another embodiment, the secondary treatment is gene therapy, in which a therapeutic polynucleotide is administered before, after, or simultaneously with the administration of a tumor-associated HLA-restricted peptide. Delivery of a vector encoding a tumor-associated HLA-restricted peptide in combination with a second vector encoding one of the following gene products has a combined anti-hyperproliferative effect on the target tissue. Alternatively, a single vector encoding both genes may be used. Various proteins are encompassed by the present invention, some of which are described below. Various genes that can be targeted by some forms of gene therapy in combination with the present invention are well known to those skilled in the art, and may include any gene involved in cancer.

[0101] Cell proliferation inducers. Proteins that induce cell proliferation are further classified into various categories depending on their function. What these proteins all have in common is their ability to regulate cell proliferation. For example, the sis oncogene, a form of PDGF, is a secreted growth factor. Oncogenes rarely arise from genes encoding growth factors, and currently, sis is the only known naturally occurring oncogenic growth factor. In one embodiment of the present invention, antisense mRNA targeting a specific cell proliferation inducer is contemplated for use in preventing the expression of the cell proliferation inducer.

[0102] The proteins FMS, ErbA, ErbB, and neu are growth factor receptors. Mutations to these receptors result in the loss of regulatable function. For example, a point mutation affecting the transmembrane domain of the Neu receptor protein results in the neu oncogene. The erbA oncogene is derived from the intracellular receptor for thyroid hormone. It is believed that the altered, oncogenic ErbA receptor competes with the endogenous thyroid hormone receptor, causing uncontrolled growth.

[0103] The largest class of oncogenes includes signal transduction proteins (e.g., Src, Abl, and Ras). The protein Src is a cytoplasmic protein tyrosine kinase, and its conversion from a proto-oncogene to an oncogene occurs in some cases through a mutation at tyrosine residue 527. In contrast, the conversion of the GTPase protein ras from a proto-oncogene to an oncogene, in one example, is achieved by a mutation from valine to glycine at amino acid 12 in the sequence, reducing ras GTPase activity. The proteins Jun, Fos, and Myc are proteins that directly exert their effects on nuclear function as transcription factors.

[0104] Cell proliferation inhibitors. Tumor suppressor oncogenes function to inhibit excessive cell proliferation. Inactivation of these genes destroys their inhibitory activity, resulting in uncontrolled proliferation. The most common tumor suppressors are Rb, p53, p21, and p16. Other genes that can be used in accordance with the present invention include APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73, VHL, C-CAM, MMAC1 / PTEN, DBCCR-1, FCC, rsk-3, p27, p27 / p16 fusions, and p21 / p27 fusions.

[0105] Regulators of Programmed Cell Death. Apoptosis, or programmed cell death, is an essential process for normal embryonic development, maintaining homeostasis in adult tissues, and suppressing carcinogenesis (Kerr et al., 1972). Bcl-2 family proteins and ICE-like proteases have been demonstrated to be important regulators and effectors of apoptosis in other systems. Bcl-2 proteins, discovered in association with follicular lymphoma, play a prominent role in regulating apoptosis and promoting cell survival in response to diverse apoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, 1985; Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). The evolutionarily conserved Bcl-2 protein is now recognized as a member of a family of related proteins and can be classified as death agonists or death antagonists.

[0106] Subsequent to its discovery, Bcl-2 was shown to act to suppress cell death induced by a variety of stimuli. It is now clear that there is a family of Bcl-2 cell death regulatory proteins that share common structural and sequence homology. These different family members have similar functions to Bcl-2 (e.g., Bcl XL , Bcl W , Bcl S , Mcl-1, A1, Bfl-1) or antagonize Bcl-2 function to promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).

[0107] 5. Surgery Approximately 60% of people with cancer will undergo some type of surgery, which includes preventative surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery is a cancer treatment that may be used in combination with other therapies, such as the treatment of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies.

[0108] Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgical surgery (Mohs surgery). It is further contemplated that the present invention may be used in combination with the removal of superficial cancers, pre-cancers, or incidental amounts of normal tissue.

[0109] When cancer cells, cancer tissue, or tumors are partially or entirely removed, a cavity may be formed in the body.Treatment can be achieved by perfusion, direct injection, or local application of additional anti-cancer therapy to the area.Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.These treatments can also be at different dosages.

[0110] V. Antibody Conjugates An antibody can be linked to at least one drug to form an antibody conjugate. To enhance the effectiveness of an antibody molecule as a diagnostic or therapeutic agent, it is customary to link, covalently bind, or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules include molecules with desired activity, such as immunosuppressive / anti-inflammatory properties. Non-limiting examples of such molecules are listed above. Such molecules are optionally attached via a cleavable linker designed to allow the molecule to be released at or near the target site.

[0111] In contrast, a reporter molecule is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin.

[0112] Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostics generally fall into two categories: in vitro diagnostics, such as those used in various immunoassays, and those used in in vivo diagnostic protocols, commonly known as "antibody-directed imaging." Many suitable imaging agents are known in the art, as are methods for their attachment to antibodies (see, for example, U.S. Patent Nos. 5,021,236, 4,938,948, and 4,472,509). The imaging moieties used can be paramagnetic ions, radioactive isotopes, fluorescent dyes, NMR-detectable substances, and X-ray imaging agents.

[0113] In the case of paramagnetic ions, examples include ions such as chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III) and / or erbium(III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include, but are not limited to, lanthanum(III), gold(III), lead(II), and especially bismuth(III).

[0114] For radioactive isotopes for therapeutic and / or diagnostic applications, astatine 211 , 14 carbon, 51 chromium, 36 chlorine,57 cobalt, 58 Cobalt, Copper 67 , 152 Eu, gallium 67 , 3 Hydrogen, iodine 123 , iodine 125 , iodine 131 ,indium 111 , 59 iron, 32 Phosphorus, rhenium 186 ,rhenium 188 , 75 Selenium, 35 Sulfur, technicium 99m and / or yttrium 90 Examples include: 125 I is often preferred for use in certain embodiments, and technicium 99m and / or indium 111 are also often preferred due to their low energy and suitability for long-distance detection. Radiolabeled monoclonal antibodies can be prepared according to methods well known in the art. For example, monoclonal antibodies can be iodinated by contact with sodium and / or potassium iodide and a chemical oxidizing agent, such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies can also be iodinated with technetium by a ligand exchange method, for example, by reducing pertechnetate with a stannous solution, chelating the reduced technetium to a Sephadex column, and applying the antibody to the column. 99m Alternatively, direct labeling techniques may be used, for example, by incubating pertechnetate, a reducing agent such as SNCl2, a buffer solution such as sodium-potassium phthalate solution, and the antibody. The intermediate functional groups often used to attach radioisotopes to antibodies and present as metal ions are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0115] Fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, tetramethylrhodamine, and / or Texas Red.

[0116] Another type of antibody conjugate that is contemplated is primarily intended for in vitro use, in which the antibody is linked to an enzyme (enzyme tag) that produces a colored product upon contact with a secondary binding ligand and / or a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such labels is well known to those skilled in the art and is described, for example, in U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241.

[0117] Yet another known method for site-specific attachment of molecules to antibodies involves reacting the antibody with a hapten-based affinity tag. Essentially, the hapten-based affinity tag reacts with amino acids in the antigen-binding site, thereby disrupting this site and blocking specific antigen reaction. However, this can be disadvantageous, as it results in loss of antigen binding by the antibody conjugate.

[0118] Molecules containing azide groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates generated by low-intensity ultraviolet light (Potter and Haley, 1983). In particular, 2- and 8-azido analogs of purine nucleotides have been used as site-directed photoprobes to identify nucleotide-binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). 2- and 8-azido nucleotides have also been used to map nucleotide-binding domains in purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and can be used as antibody binders.

[0119] Several methods for attaching or conjugating antibodies to their conjugate moieties are known in the art. Some attachment methods involve, for example, the use of organic chelating agents attached to antibodies, such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or metal chelate complexes using tetrachloro-3α-6α-diphenylglycouril-3 (U.S. Patent Nos. 4,472,509 and 4,938,948). Monoclonal antibodies can also be reacted with enzymes in the presence of coupling agents, such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanates. In U.S. Pat. No. 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies, with a detectable imaging moiety attached to the antibody using a linker, such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

[0120] In another embodiment, derivatization of immunoglobulins by selectively introducing sulfhydryl groups into the Fc region of the immunoglobulin is contemplated, using reaction conditions that do not alter the antibody's combining site. Antibody conjugates produced according to this methodology have been disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Patent No. 5,196,066; incorporated herein by reference). Site-specific attachment of effector or reporter molecules, in which the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region, has also been disclosed in the literature (O'Shannessy et al., 1987). This approach has been reported to produce antibodies with diagnostic and therapeutic potential that are currently undergoing clinical evaluation.

[0121] VI. Immunodetection In yet further embodiments, there are immunodetection methods for binding to, purifying, removing, quantifying, and otherwise generally detecting PD-L1 or PD-L2 and their associated antigens. Some immunodetection methods include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), immunoradiometric assays, fluoroimmunoassays, chemiluminescent assays, bioluminescent assays, and Western blots, to name a few. Competitive assays for detecting and quantifying PD-L1 and PD-L2 antibodies, in particular, are also provided. Steps for various useful immunodetection methods are described in the scientific literature, for example, in Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). In general, immunobinding methods involve obtaining a sample and, optionally, contacting the sample with a first antibody according to an embodiment described herein under conditions effective to allow the formation of an immune complex.

[0122] Contacting a selected biological sample with an antibody under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) generally involves simply adding the antibody composition to the sample and incubating the mixture for a period of time sufficient for the antibody to form immune complexes, i.e., bind to any PD-L1 and PD-L2 present. After this time, the sample-antibody composition, e.g., tissue section, ELISA plate, dot blot, or Western blot, is generally washed to remove any non-specifically bound antibody species, allowing only the specifically bound antibody within the primary immune complexes to be detected.

[0123] Generally, the detection of immune complex formation is well known in the art and can be achieved through the application of many approaches.These methods are generally based on the detection of label or marker, for example, radioactive tag, fluorescent tag, biological tag and enzyme tag.The patents relating to the use of such labels include U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241.Of course, as known in the art, additional advantages can be found through the use of secondary binding ligand, for example, secondary antibody and / or biotin / avidin ligand binding configuration.

[0124] The antibody used for detection may itself be linked to a detectable label, in which case the amount of primary immune complexes in the composition can be determined simply by detecting this label. Alternatively, the first antibody bound within the primary immune complex may be detected by a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is often itself an antibody, in which case it may be referred to as a "secondary" antibody. The primary immune complexes are contacted with a labeled secondary binding ligand or antibody under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, after which the label remaining in the secondary immune complexes is detected.

[0125] Another method involves detecting primary immune complexes using a two-step approach. As described above, a second binding ligand, such as an antibody, having binding affinity for the antibody is used to form secondary immune complexes. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing the detection of the tertiary immune complexes thus formed. This system can provide signal amplification, if desired.

[0126] One method of immunodetection uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and then a second antibody is used to detect the biotin bound to the complexed biotin. In this method, the sample to be tested is first incubated in a solution containing the first-step antibody. If the target antigen is present, a portion of the antibody binds to the antigen, forming a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with each step adding an additional biotin moiety to the antibody / antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing a second-step antibody directed against biotin. This second-step antibody is labeled with an enzyme that can be used to detect the presence of the antibody / antigen complex, for example, by histoenzymology using a chromogenic substrate. Once suitably amplified, a macroscopically visible conjugate can be produced.

[0127] Another known method of immunodetection utilizes immuno-PCR (polymerase chain reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, but instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA / biotin / streptavidin / antibody complex is washed with a low pH or high salt buffer, which releases the antibody. The resulting wash solution is then used to perform a PCR reaction using suitable primers along with appropriate controls. At least in theory, the enormous amplification power and specificity of PCR can be utilized to detect single antigen molecules.

[0128] A. ELISA In the simplest sense, immunoassay is binding assay.Some preferred immunoassays are various types of enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) known in the art.Immunohistochemical detection using tissue section is also particularly useful.However, it is easy to understand that detection is not limited to such technology, and Western blotting, dot blotting, FACS analysis, etc. can also be used.

[0129] In one exemplary ELISA, antibodies of the present disclosure are immobilized to a selected surface exhibiting protein affinity, such as wells in a polystyrene microtiter plate. A test composition suspected of containing PD-L1 and / or PD-L2 is then added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen can be detected. Detection can be achieved by adding another bispecific antibody against PD-L1 and PD-L2 linked to a detectable label, or an anti-PD-L1 or anti-PD-L2 antibody linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection can also be achieved by adding a second bispecific antibody against PD-L1 and PD-L2, or an anti-PD-L1 or anti-PD-L2 antibody, followed by a third antibody linked to a detectable label that has binding affinity for the second antibody.

[0130] In another exemplary ELISA, samples suspected of containing PD-L1 and / or PD-L2 antigens are immobilized on a well surface and then contacted with the anti-PD-L1 and anti-PD-L2 bispecific antibody. After binding and washing to remove non-specifically bound immune complexes, the bound anti-PD-L1 and anti-PD-L2 bispecific antibody is detected. If the first anti-PD-L1 and anti-PD-L2 bispecific antibody is linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody (linked to a detectable label) that has binding affinity for the first anti-PD-L1 and anti-PD-L2 bispecific antibody.

[0131] Regardless of the format used, ELISAs have certain features in common, such as coating, incubation and binding, washing to remove non-specifically bound species, and detection of bound immune complexes, which are described below.

[0132] Coating a plate with either an antigen or an antibody generally involves incubating the wells of the plate with a solution of the antigen or antibody overnight or for a specified period of time. The wells are then washed to remove any incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with respect to the test antiserum. These include bovine serum albumin (BSA), casein, or a solution of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface, thus reducing the background caused by nonspecific binding of the antiserum to the surface.

[0133] In ELISA, rather than a direct procedure, it is probably more conventional to use secondary or tertiary detection. Thus, after binding a protein or antibody to a well, coating it with a non-reactive material to reduce background, and washing to remove unbound material, the biological sample to be tested is contacted with the immobilized surface under conditions effective to allow immune complex (antigen / antibody) formation. Next, detection of the immune complex requires a labeled secondary binding ligand or antibody, and the secondary binding ligand or antibody in combination with a labeled tertiary antibody or third binding ligand.

[0134] "Under conditions effective to allow immune complex (antigen / antibody) formation" means that the conditions preferably include diluting the antigen and / or antibody in a solution such as BSA, bovine gamma globulin (BGG), or phosphate buffered saline (PBS) / Tween. These added agents also tend to help reduce nonspecific background.

[0135] "Suitable" conditions also mean that the incubation is carried out at a temperature or for a period of time sufficient to allow effective binding. The incubation step is typically carried out for about 1 to 2 to 4 hours, preferably at a temperature of about 25°C to 27°C, or may be carried out overnight at about 4°C.

[0136] After all incubation steps in ELISA, the contacted surface is washed to remove uncomplexed material. A preferred washing procedure involves washing with a solution such as PBS / Tween or borate buffer. After the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the presence of even minute amounts of immune complexes can be determined.

[0137] To provide a means of detection, the second or third antibody has an associated label to allow detection. Preferably, this is an enzyme that generates color upon incubation with an appropriate chromogenic substrate. Thus, for example, it may be desirable to contact or incubate the first and second immune complexes with urease, glucose oxidase, alkaline phosphatase, or hydrogen peroxidase-conjugated antibodies for a period and under conditions that favor the formation of additional immune complexes (e.g., incubation in a PBS-containing solution, such as PBS-Tween, at room temperature for 2 hours).

[0138] After incubation with the labeled antibody and subsequent washing to remove unbound material, the amount of label is quantified, for example, by incubation with a chromogenic substrate, e.g., urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), or HO in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the extent of color development, e.g., using a visible spectrum spectrophotometer.

[0139] B. Western Blot Western blot (or protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by polypeptide length (denaturing conditions) or by the protein's 3D structure (native / non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF) and probed (detected) using an antibody specific for the target protein.

[0140] Samples can be taken from whole tissues or cell cultures. In most cases, solid tissues are first mechanically disrupted using a blender (for large sample volumes), a homogenizer (for small volumes), or sonication. Cells may also be disrupted by one of the mechanical methods mentioned above. However, it should be noted that bacterial, viral, or environmental samples can be sources of proteins, and Western blotting is not limited to cellular studies alone. A combination of detergents, salts, and detergents may be used to promote cell lysis and solubilize proteins. Protease and phosphatase inhibitors are often added to prevent digestion of the sample by its own enzymes. Tissue preparation is often performed at low temperatures to avoid protein denaturation.

[0141] Gel electrophoresis is used to separate proteins from a sample. Proteins can be separated by isoelectric point (pi), molecular weight, charge, or a combination of these factors. The nature of the separation depends on the sample treatment and the properties of the gel. This is a very useful method for determining proteins. Two-dimensional (2-D) gels can also be used, which spread proteins from a single sample across two dimensions. Proteins are separated according to isoelectric point (pH at which they have a neutral net charge) in the first dimension and according to molecular weight in the second dimension.

[0142] To make proteins accessible for antibody detection, they are transferred from the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter paper is placed on top of it. The entire stack is then placed in a buffer solution, which wicks upward toward the paper by capillary action, carrying the proteins with it. Another method of protein transfer, called electroblotting, uses an electric current to draw proteins from the gel into a PVDF or nitrocellulose membrane. The proteins migrate from the gel onto the membrane while maintaining their organization within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). Both types of membrane are chosen for their nonspecific protein-binding properties (i.e., they bind all proteins equally). Protein binding is based on charge interactions between the membrane and the protein, as well as hydrophobic interactions. Nitrocellulose membranes are less expensive than PVDF but are much more fragile and do not withstand repeated probing as well. The uniformity and overall effectiveness of protein transfer from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dye. Once the proteins have been transferred, they are detected using a labeled primary antibody, or an unlabeled primary antibody followed by indirect detection using labeled Protein A or a secondary labeled antibody that binds to the Fc region of the primary antibody.

[0143] C. Immunohistochemistry The antibodies can also be used in combination with either fresh-frozen and / or formalin-fixed, paraffin-embedded tissue blocks prepared for immunohistochemical (IHC) studies. Methods for preparing tissue blocks from these particulate specimens have been used successfully in previous IHC studies of various prognostic factors and are well known to those skilled in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990).

[0144] Briefly, frozen sections can be prepared by rehydrating 50 ng of frozen "ground" tissue in a small plastic capsule in phosphate-buffered saline (PBS) at room temperature, pelleting the particles by centrifugation, resuspending them in a viscous embedding medium (OCT), inverting the capsule and / or pelleting again by centrifugation, snap-cooling in -70°C isopentane, cutting the plastic capsule and / or removing the frozen tissue cylinder, mounting the tissue cylinder on a cryostat microtome chuck, and / or cutting 25-50 serial sections from the capsule. Alternatively, the entire frozen tissue sample can be used for serial sectioning.

[0145] Permanent sections can be prepared by a similar method involving rehydrating a 50 mg sample in a plastic microcentrifuge tube, pelleting, resuspending in 10% formalin and fixing for 4 hours, washing / pelleting, resuspending in warm 2.5% agar, pelleting, chilling in ice-cold water to solidify the agar, removing the tissue / agar block from the tube, infiltrating and / or embedding the block in paraffin, and / or cutting up to 50 serial permanent sections. Again, the entire tissue sample may be substituted.

[0146] D. Immunodetection Kits In a still further aspect, there is an immunodetection kit for use with the immunodetection methods described above. The immunodetection kit thus comprises, in suitable container means, a first bispecific antibody that binds to PD-L1 and / or PD-L2 antigens, and optionally an immunodetection reagent.

[0147] In certain embodiments, the bispecific antibody against PD-L1 and PD-L2 may be pre-bound to a solid support, such as a column matrix and / or a well of a microtiter plate. The immunodetection reagent of the kit can be in any one of a variety of forms, such as a detectable label associated with or linked to a given antibody. Detectable labels associated with or linked to a secondary binding ligand are also contemplated. An exemplary secondary ligand is a secondary antibody that has binding affinity for the first antibody.

[0148] Further suitable immunodetection reagents for use in the kits of the invention include two-component reagents that include a second antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, where the third antibody is linked to a detectable label. As noted above, numerous exemplary labels are known in the art, and all such labels can be used in connection with the embodiments described herein.

[0149] The kit may further comprise suitably aliquoted compositions of PD-L1 antigen and PD-L2 antigen, which may be labeled or unlabeled, for use in generating standard curves for detection assays. The kit may contain the antibody-label conjugates in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The kit components may be packaged either in aqueous media or in lyophilized form.

[0150] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the antibody may be placed, or preferably suitably aliquoted. The kits will also include means, containers, for containing the antibody, antigen, and any other reagents in close confinement for commercial sale. Such containers may include injection- or blow-molded plastic containers for retaining the desired vials therein. [Example]

[0151] VII. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention and, as such, can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0152] Example 1 – Materials and Methods Antibody Selection, Generation, and Production Although additional details may be provided in the Examples that follow, the selection, generation, and production of the disclosed antibodies was generally carried out as follows.

[0153] Antigen preparation—Antigens were biotinylated using Pierce's EZ-Link Sulfo-NHS-Biotinylation Kit. Goat F(ab')2 anti-human kappa-FITC (LC-FITC), ExtrAvidin-PE (EA-PE), and streptavidin-AF633 (SA-633) were obtained from Southern Biotech, Sigma, and Molecular Probes, respectively. Streptavidin microbeads and MACS LC separation columns were purchased from Miltenyi Biotec. Goat anti-human IgG-PE (Human-PE) was obtained from Southern Biotech.

[0154] Naive Discovery - about 10 9Eight naive human synthetic yeast libraries, each with a diversity of 10, were grown as previously described (see, e.g., Xu et al., 2013, WO2009036379, WO2010105256, and WO2012009568). For the first two rounds of selection, magnetic bead sorting utilizing the Miltenyi MACS system was performed as previously described (see, e.g., Siegel et al., 2004). Briefly, yeast cells (approximately 10 10 The yeast (cells / library) were incubated with 3 ml of 10 nM biotinylated Fc fusion-antigen in wash buffer (phosphate-buffered saline (PBS) / 0.1% bovine serum albumin (BSA)) for 15 min at 30°C. After one wash with 40 ml of ice-cold wash buffer, the cell pellet was resuspended in 20 ml of wash buffer, and streptavidin microbeads (500 μl) were added to the yeast and incubated for 15 min at 4°C. The yeast were then pelleted, resuspended in 20 ml of wash buffer, and loaded onto a Miltenyi LS column. After loading 20 ml, the column was washed three times with 3 ml of wash buffer. The column was then removed from the magnetic field, and the yeast were eluted with 5 ml of growth medium before being grown overnight. Subsequent rounds of selection were performed using flow cytometry. Approximately 2 × 10 7Yeast cells were pelleted, washed three times with wash buffer, and incubated at 30°C under equilibrium conditions with either 10 nM Fc fusion antigen or, in later rounds, decreasing concentrations (100–1 nM) of biotinylated antigen. Alternatively, with 100 nM biotinylated antigen from a different species (mouse) to obtain species cross-reactivity, or with multispecific depletion reagent (PSR) to remove nonspecific antibodies from the selection. For PSR depletion, the library was incubated with a 1:10 dilution of biotinylated PSR reagent, as previously described (see, e.g., Xu et al., 2013). Yeast cells were then washed twice with wash buffer and stained with LC-FITC (diluted 1:100) and either SA-633 (diluted 1:500) or EAPE (diluted 1:50) secondary reagents for 15 min at 4°C. After washing twice with wash buffer, the cell pellet was resuspended in 0.3 mL of wash buffer and transferred to a sorting tube with a strainer cap. Sorting was performed using a FACS ARIA sorter (BD Biosciences), and sorting gates were determined to select antibodies with the desired characteristics. Selection rounds were repeated until a population with all of the desired characteristics was obtained.

[0155] To generate cross-reactive antibodies, alternate rounds of selection were performed independently with each target antigen, enriching for dual specificities. After the final round of selection, yeast were plated and individual colonies were picked for characterization.

[0156] Light chain batch shuffling (LCBS) - The primary discovery also included a light chain batch diversification protocol from heavy chain plasmids from naive selections: heavy chain plasmids from the naive round 4 selection output were extracted from yeast and 5 × 10 6 The resulting light chain library was transformed with a diversity of 1000. Selection was performed by one round of MACS and three rounds of FACS using the same conditions as for naive discovery.

[0157] Antibody Optimization - Antibody optimization was performed by introducing diversity into the heavy and light chain variable regions as described below. A combination of some of these approaches was used for each antibody.

[0158] Selection of CDRH1 and CDRH2 : 1×10 8 A single antibody CDRH3 was recombined into a pre-generated library containing CDRH1 and CDRH2 variants with a diversity of 1000, and selection was performed using one round of MACS and four rounds of FACS, as described for naive discovery. In the FACS rounds, the library was examined for PSR binding, species cross-reactivity, antigen cross-reactivity, and affinity pressure to obtain a population with the desired characteristics. For these selections, affinity pressure was applied either by downward titration of biotinylated monomeric antigen or by preincubating biotinylated antigen with the parent Fab for 30 minutes and then applying the precombined mixture to the yeast library for a period allowing the selection to reach equilibrium. Antibodies with higher affinities could then be selected.

[0159] VH Mut selection The heavy chain variable region (VH) was mutagenized by error-prone PCR. The mutagenized VH and heavy chain expression vectors were then transformed into yeast already containing the parental light chain plasmid to generate a library. Selection was performed as in the previous cycle using three rounds of FACS sorting. In the FACS rounds, the library was examined for cross-reactivity and affinity pressure, and selection was performed to obtain a population with the desired characteristics.

[0160] Selection of CDRL1, CDRL2, and CDRL3 Oligos were ordered from IDT, containing CDRL3 and diversified with NNK diversity. The CDRL3 oligos were double-stranded using primers that anneal to the flanking regions of CDRL3. These double-stranded CDRL3 oligos were then synthesized in 3x10 5The resulting CDRL1 and CDRL2 variants were recombined into a pre-generated library containing CDRL1 and CDRL2 variants with a diversity of 1000, and selected by one round of MACS and three rounds of FACS as described for naive discovery. In the FACS rounds, the library was examined for PSR binding, cross-reactivity, and affinity pressure, and selection was performed to obtain populations with the desired characteristics. Affinity pressure for these selections was performed as described above for CDRH1 and CDRH2 selection, with alternating antigens applied in each round to enrich for dual specificities.

[0161] Antibody production and purification - Yeast clones were grown to saturation and then induced for 48 hours at 30°C with shaking. After induction, yeast cells were pelleted and the supernatant was collected for purification. IgG was purified using a Protein A column and eluted with acetic acid at pH 2.0. Fab fragments were generated by papain digestion and purified using KappaSelect (GE Healthcare LifeSciences).

[0162] ForteBio K DMeasurements—ForteBio affinity measurements were performed on an Octet RED384 instrument as generally described previously (see, e.g., Estep et al., 2013). Briefly, ForteBio affinity measurements were performed by loading IgG online onto the AHQ sensor. The sensor was equilibrated offline in assay buffer for 30 minutes and then monitored online for 60 seconds to establish a baseline. The sensor with loaded IgG was exposed to 100 nM antigen for 3 minutes, after which it was transferred to assay buffer for 3 minutes to measure the dissociation rate. Fab was used instead of IgG for monovalent affinity evaluation. For this evaluation, non-biotinylated Fc-fusion antigen was loaded online onto the AHQ sensor. The sensor was equilibrated offline in assay buffer for 30 minutes, after which it was monitored online for 60 seconds to establish a baseline. The sensor with loaded antigen was exposed to 100 nM Fab for 3 minutes, after which it was transferred to assay buffer for 3 minutes to measure the dissociation rate. All kinetics were analyzed using a 1:1 binding model.

[0163] ForteBio Epitope Binning / Ligand Blocking - Epitope binning / ligand blocking was performed using a standard sandwich format reciprocal blocking assay. A control anti-target IgG was loaded onto the AHQ sensor, and unoccupied Fc binding sites on the sensor were blocked with an irrelevant human IgG1 antibody. The sensor was then exposed to 100 nM of target antigen, followed by a second anti-target antibody or ligand. Additional binding by the second antibody or ligand after antigen association indicates an unoccupied epitope (non-competitor), while a lack of binding indicates epitope blocking (competitor or ligand blocking).

[0164] MSD-SET K DMeasurements - Equilibrium affinity measurements of selected high-affinity antibodies were performed generally as previously described (Estep et al., 2013). Briefly, solution equilibrium titrations (SETs) were performed in PBS + 0.1% IgG-free BSA (PBSF) with antigen held constant at 50 pM and incubated with 3-5x serial dilutions of Fab starting at 20 nM. Standard-binding MSD-ECL plates were coated with antibody (20 nM in PBS) overnight at 4°C or for 30 minutes at room temperature. The plates were then blocked with BSA for 30 minutes with shaking at 700 rpm, followed by three washes with wash buffer (PBSF + 0.05% Tween 20). SET samples were applied and incubated on the plates for 150 seconds with shaking at 700 rpm, followed by one wash. Antigen captured on the plate was detected by incubating the plate with 250 ng / mL sulfo-tagged streptavidin in PBSF for 3 minutes. The plate was washed three times with wash buffer and then read on an MSD Sector Imager 2400 instrument using 1× Read Buffer T with detergent. The percent free antigen was plotted in Prism as a function of titrated antibody and fitted to a quadratic equation to obtain K D To improve throughput, a liquid-handling robot was used throughout the MSD-SET experiment, including SET sample preparation.

[0165] Cell binding analysis - Approximately 100,000 cells overexpressing the antigen were washed with wash buffer and incubated with 100 μl of 100 nM IgG for 5 minutes at room temperature. The cells were then washed twice with wash buffer and incubated with 100 μl of 1:100 Human-PE for 15 minutes on ice. The cells were then washed twice with wash buffer and analyzed on a FACS Canto II analyzer (BD Biosciences).

[0166] Antibody screening and characterization. Candidate antibodies generated from the methods presented above were tested for their ability to bind to and block PD-1 and PD-L2. 5 μg / mL of antibody was bound to CHO-PD-L1 or CHO-PD-L2 cells, followed by the addition of recombinant PD-1 (RnD Systems) labeled with Alexa 532 (ThermoFisher) for 1 hour. The maximum fluorescence intensity of PD-1 was measured. Blockade of PD-1 binding was measured by the reduction of Alexa 532 fluorescence by flow cytometry. Affinity K for human PD-L1 or PD-L2 D To generate K, BiPDL Ab was loaded onto an Anti-Human Fc Capture (AHC) biosensor at 100 nM (15 μg / mL), and the association and dissociation of human PD-L1 or PD-L2 proteins was tested in a dilution series from 30 to 0.37 nM. Analyte binding and release were recorded in real time by the Octet instrument and then used to measure K. d , K. on , and K. dis The affinity K for mouse PD-L1 or PD-L2 was calculated. Results are derived from 2:1 global fit modeling with subtraction of reference wells. D To generate K, BiPDL Ab was covalently immobilized at 100 nM (15 μg / mL) onto activated Amine Reactive 2nd Generation (AR2G) biosensors (quenched with 1 M ethanolamine at pH 8.5 after protein loading), and the association and dissociation of mouse PD-L1 and PD-L2 proteins was tested in a dilution series from 300 to 1 nM. Analyte binding and release were recorded in real time by an Octet instrument and then used to measure K. d , K. on , and K. dis was calculated. Results are derived from a 2:1 global fit modeling with subtraction of reference wells.

[0167] Antibody activity. Varying concentrations of BiPDL antibodies with a human IgG1 backbone were added to CHO cells expressing either human or mouse PD-L1 or PD-L2 (CHO-PD-L1 cells, CHO-PD-L2 cells). Binding was detected by the addition of an anti-human IgG1 secondary antibody conjugated to phycoerythrin (PE). FACS analysis was performed to detect phycoerythrin and determine fluorescent activity at various antibody concentrations. EC50 values ​​were calculated using GraphPad Prism® software.

[0168] Candidate antibodies prevent PD-1 / PD-L2 binding. The Promega PD-L1 / PD-L2 Dual Expression:PD-1 Blockade System was used to assay candidate BiPDL antibodies and FDA-approved antibodies. Various concentrations of antibodies were added to CHO-PD-L1 / L2 cells. PD-1 effector cells were Jurkat T cells, which could be stimulated by CHO-PD-L1 / L2 cells. PD-1 effector cells, which produce firefly luciferase in response to activation, were incubated with antibody and CHO cells for 6 hours and then analyzed by Bio-Glo according to the manufacturer's instructions. TM The results were read on a luminometer using an assay kit (Promega). For competition assays, biotin-rhPD-1-Fc protein was added to the cells and antibody, followed by streptavidin-APC conjugate. Blockade was evaluated as an increase in luciferase signal. Analysis was performed using GraphPad Prism® software. For competition assays, X was converted to LogX and analyzed by nonlinear regression (curve fit), dose-response inhibition, and Log(inhibitor) versus response.

[0169] BiPDL activity in mixed lymphocyte reaction. CD14+ monocytes were isolated from peripheral blood mononuclear cells using CD14 microbeads. Cells were seeded at 1 million / ml and stimulated with IL-4 and GM-CSF in 10% FCS / RPMI / P / S cell culture medium. Cells were cultured for 7 days to differentiate into immature dendritic cells (IDCs), and various concentrations of BiPDL or commercial antibodies were added. CD4+ T cells were then stimulated with IDCs at a CD4:IDC ratio of 10:1. IL-2 and IFN-γ were assayed by ELISA according to the protocol provided by R&D systems.

[0170] Antibody activity against xenograft tumors. U2940 PMBL or MDA-MB-231 triple-negative breast cancer xenograft tumors were established in immunodeficient mice. Tumors were grown in a volume of 150 mm 3 It reached 150mm 3 After reaching a tumor size of 100 mg / kg, mice were treated with the indicated antibody therapy at 10 mg / kg twice weekly for 3 weeks, with 9 mice per treatment group. Tumor volumes were calculated using caliper measurements of tumor width, length, and depth.

[0171] Survival and tumor growth in MC38-PD-L2-injected mice. Survival was measured in C57BL / 6J mice implanted with MC38-PD-L2 tumor cells. 5×10 5 MC38-PD-L2 tumor cells were implanted subcutaneously and treated with 100 μg of the indicated antibody or buffer intraperitoneally on days 3, 6, 9, 12, and 15. Tumor volume was calculated using caliper measurements of tumor width, length, and depth. Survival statistics were calculated using the Gehan-Breslow-Wilcoxon test. 1.5×10 cells were used to determine the CD8 / Treg ratio. 6 MC38-PDL2 tumor cells were implanted subcutaneously into C57BL / 6J mice in 30% Matrigel (Corning) and treated with 100 μg of the indicated antibodies by intraperitoneal injection on days 7, 10, and 13. Tumors were harvested on day 15 and FoxP3 +The ratio of infiltrating CD8 T cells to Tregs was measured by flow cytometry.

[0172] Survival of the EL4 lymphoma mouse model. EL4 T-cell lymphoma cells, which endogenously express PD-L1, were retrovirally engineered to express murine PD-L2. Expression of both PD-L1 and PD-L2 was verified by flow cytometry. Survival was measured in mice injected with EL4 cells expressing PD-L2 and luciferase. 1.5 x 10 5 EL4-PD-L2 cells were injected into the tail vein of mice to establish systemic disease in C57BL / 6J mice. Mice were treated intraperitoneally with 100 μg of the indicated antibodies on days 3, 6, 9, 12, and 15.

[0173] Epitope binning of BiPDL antibodies. The binding specificities of various BiPDL antibodies were compared using a binding competition assay performed using the ForteBio Octet® platform. Target His-tagged proteins (human PD-L1 or PD-L2) were loaded onto pre-charged nickel-NTA biosensors at 1 μg / mL. The first antibody, Ab1, was saturated at 100 nM in the target-loaded biosensors, and a reference (buffer only) well was included to determine the maximum Ab2 binding signal. The second antibody was also screened for binding signal at 100 nM, and Ab1 was included to determine the background self-blocking signal. Data Analysis HT 9.0 software was used to generate a matrix of raw signal responses for Ab2 binding, which were then converted and expressed as a percentage of the unblocked Ab2 binding signal. A response of less than 15% was considered competitive blocking.

[0174] Example 2 - Results Selection of BiPDL Antibodies Binding to Both Human PD-L1 and PD-L2. Given that PD-L1 and PD-L2 both bind to PD-1 and share approximately 40% amino acid identity, we hypothesized that it would be possible to obtain antibodies that bind to both ligands and block their binding to the T cell co-inhibitory receptor PD-1. Because we anticipated that finding high-affinity antibodies capable of bivalent binding to both ligands would be extremely rare, we discovered these antibodies through an iterative selection process from a yeast-based, fully human antibody library display system, as described above. Using recombinant ligands fused to dimerized (i.e., tightly binding) human IgG1 Fc constant regions, we first selected for antibodies that bind to both PD-L1 and PD-L2, as described in Example 1 above, and then further enriched the highest affinity hits through selection for those capable of binding to monomeric PD ligands. This initial round of screening yielded four distinct series of antibodies that could bind to both PD-L1 and PD-L2 and block their binding to PD-1 (Figures 1A-B). The antibody was named BiPDL and follows the nomenclature BiPDLX-Y, where X is the BiPDL family ( Boussiotis, 2016 ; Cheng et al., 2013 ; Latchman et al., 2001 ; Lee et al., 2016 ), Y is the clone number within that family at a given generation, and Z is the generation representing successive rounds of affinity maturation ( Boussiotis, 2016 ; Cheng et al., 2013 ; Latchman et al., 2001 ; Lee et al., 2016 ). Z Individual antibody clones are also referenced by experimental clone number. Table 5 provides both the BiPDL name and the respective clone number. Each of these antibodies exhibited moderately high avidity (K) for PD-L2 (using dimeric ligands). d ≦2×10 -9 ), but only BiPDL4 showed quantifiable avidity (K ) for human PD-L1 as measured by Octet (ForteBio). d ≦1×10 -9We further tested several antibodies for their ability to alleviate PD-1-induced inhibition of Jurkat T cells using the Promega PD-1 assay system across a wide range of antibody concentrations and found that only BiPDL4 could block PD-L1-induced T cell suppression (Figure 1C).

[0175] Affinity maturation enhances the functional ability of BiPDL to reverse PD-L1 and PD-L2 inhibition of T cell activation. Each of the first-generation leads, BiPDL1-11, BiPDL2-11, BiPDL3-11, and BiPDL4-11, was subjected to heavy chain affinity maturation to enrich for clones that bind to both PD-L1 and PD-L2 through the selection of optimized CDR1 and CDR2 sequences using the method described in Example 1 above. Various concentrations of BiPDL3 and BiPDL4 antibodies (human IgG1) were added to CHO-PD-L1 or CHO-PD-L2 cells. Binding of affinity-matured BiPDL3 and BiPDL4 was detected by the addition of a PE-conjugated anti-human IgG1 secondary antibody (Figures 2A-D). All BiPDL3 and BiPDL4 antibodies retained the ability to bind to PD-L1 (Figures 2A and 2C), whereas BiPDL3-12 showed little PD-L2 binding activity (Figure 2B).

[0176] The ability of fourth-generation BiPDL4 clones to restore Jurkat T cell activity was evaluated using the Promega PD-L1 / PD-L2 dual expression:PD-1 assay system (Figure 3). BiPDL4-11, -14, -24, -34, and -44 all demonstrated levels of activity similar to Keytruda (anti-PD-1) and superior to atezolizumab and avelumab. Binding of other independent family members was also evaluated (Figure 4A–F). The ability of BiPDL4-12 to reverse Jurkat T cell inhibition in the Promega system was enhanced relative to parental BiPDL4 for both PD-L1 and PD-L2, whereas BiPDL4-22 did not acquire any ability to reverse PD-L1-mediated inhibition but was able to fully reverse PD-L2-mediated suppression (Figure 4E–F). Octet affinity measurements of BiPDL3-12 demonstrated a K for PD-L1. d =2.71×10 -8 , PD-L2 K d =8.85×10 -8 (dimeric ligand) (Table 5). BiPDL4-12 expressed 6.22 × 10 -10 , 1.74 × 10 for PD-L2 -9 while BiPDL4-22 was measured to have a PD-L1 response of 8.42 × 10 -9 , 3.5 × 10 for PD-L2 -10 was measured (Table 5).

[0177] Despite improvements over the first-generation BiPDLs, none of BiPDL3-12, BiPDL4-12, or BiPDL4-22 demonstrated clinically meaningful affinity for both PD-L1 and PD-L2. For this reason, a third round of heavy chain affinity maturation was performed, focusing on BiPDL3-12 and BiPDL4-22. This process resulted in third-generation BiPDL antibodies with significantly improved affinity for both ligands. The K of Octet affinity measurements for BiPDL3-13 was 0.01. d 3.28 × 10 for PD-L1 -9 , 4.31 × 10 for PD-L2 -10(dimeric ligand). BiPDL4-13 was 6.9 × 10 for PD-L1. -10 , 4.9 × 10 for PD-L2 -10 while BiPDL4-23 was measured to have a PD-L1 response of 1.12 × 10 -9 , 3.4 × 10 for PD-L2 -10 BiPDL4-13 also inhibited monovalent PD-L1 (K d =5.04×10 -8 ) and monovalent PD-L2 (K d =8.36×10 -9 The monovalent affinity of BiPDL4-23 was K for PD-L1. d =8.4×10 -8 , PD-L2 K d =1.66×10 -9 Although all third-generation leads were able to potently reverse Jurkat T cell inhibition resulting from binding to PD-L2, the BiPDL4 antibody displayed a substantially greater ability to reverse PD-L1-induced suppression (Figure 2C).

[0178] In an attempt to improve the PD-L1 affinity of BiPDL4-13 and BiPDL4-23, a final round of light chain affinity maturation was performed as described in Example 1 above. This screening yielded a number of BiPDLs with clinically relevant affinities for both ligands, chief among them being BiPDL4-14, BiPDL4-24, BiPDL4-34, and BiPDL4-44. The affinity of these antibodies for HIS-tagged monovalent PD-L1, as measured by Octet, ranged from 2.26 to 7.9 x 10 for PD-L1. -9 , 4.98 to 9.3 × 10 for PD-L2 -10When tested for their ability to restore suppressed Jurkat T cells in the Promega system using CHO cells expressing both PD-L1 and PD-L2 as stimulator cells, these fourth-generation BiPDL4 antibodies performed nearly identically to Keytruda (Figure 2D). Furthermore, these fully matured BiPDL4s demonstrated significant superiority over both earlier-generation BiPDL4s and the FDA-approved PD-L1 antibodies atezolizumab and avelumab.

[0179] Fourth-generation BiPDL4 antibodies bind to a previously unreported epitope on PD-L1. These fourth-generation BiPDL4 antibodies bind to both human and cynomolgus monkey PD-L1 and PD-L2 with high affinity and also have significant cross-reactivity with the mouse ligands (Tables 5 and 6). All four fourth-generation BiPDL4 antibodies share the same heavy chain CDR3 but differ significantly in other heavy and light chain CDR sequences (Tables 1-4). Of note, there is no significant similarity to any of the FDA-approved PD-L1 antibodies. This lack of sequence homology suggests the potential for unique binding specificity of these PD-L1 and PD-L2 binding antibodies; therefore, they were "binned" against known PD-L1 and PD-L2 antibodies to determine comparative epitope binding.

[0180] Antibody binning showed that all four fourth-generation BiPDL4 antibodies bind to epitopes on PD-L1 that are completely distinct from avelumab and atezolizumab (Table 7). There is some overlap with the PD-L1 epitope bound by durvalumab, as evidenced by moderate levels of interference. All BiPDL4s bind to distinct epitopes on PD-L2 compared to commercially available clones 24F.10C12 and MIH18 (Table 8).

[0181] BiPDL antibodies restore effector cytokine production in primary human mixed lymphocyte reactions (MLRs). Candidate, FDA-approved, or control antibodies were evaluated in the presence of induced dendritic cells and T cells from separate donors, and IL-2 or IFN-γ production was assessed by ELISA. PD-L1 and PD-L2 expression by iDCs was confirmed by flow cytometry (Figure 5A). Both BiPDL4-14 and BiPDL4-34 demonstrated comparable abilities to restore IFN-γ secretion by primary human T cells compared with FDA-approved PD-L1 and PD-1 antibodies (Figures 5B and 5C). Despite the presence of PD-L2 in these assays, the effect of PD-L1 was found to be predominant in this setting.

[0182] BiPDL antibodies with effector function mediate effective ADCC against tumor cells expressing PD-L1 and PD-L2. U2940 is a human primary mediastinal B lymphoma (PMBL) xenograft cell line that expresses high levels of PD-L1 (approximately 50,000 molecules per cell) and low to intermediate levels of PD-L2 (approximately 7,000 molecules per cell). Murine NK cells were expanded in vitro and incubated with calcein-AM (ThermoFisher)-labeled U9240 PMBL target cells and the indicated concentrations of antibody at an effector-to-target ratio of 15:1 for 4 hours. Specific lysis (%) was calculated as the difference between experimental release and spontaneous release without antibody. All generations of BiPDL3 and BiPDL4 were able to mediate ADCC against U2940 (Figure 6). BiPDL4-12 and BiPDL4-13 were particularly effective in promoting U2940 killing.

[0183] Effector-competent BiPDLs control the growth of U2940 PBML and MDA-MB-231 tumor xenografts in SCID mice. 1 × 10 6 U2940 PMBL cells were implanted into SCID mice, and tumors grew to 150 mm 3The tumors were established until they reached a tumor volume of 10 ... 3 ) The only two mice that showed this were in the BiPDL4-13-mIgG2a group.

[0184] MDA-MB-231 triple-negative breast cancer xenograft cells (TNBC) also express both PD-L1 and PD-L2 at rates similar to U2940 (Figure 8A). 7 MDA-MB-231 TNBC cells were implanted into SCID mice, and tumors were grown to 150 mm 3 Mice were then treated twice weekly with 10 mg / kg of either the mIgG2a control antibody, Rituxan (human IgG1 control), avelumab (human IgG1), BiPDL4-14-hIgG1, or BiPDL4-14-hIgG1[S239D / I332E] engineered to enhance ADCC. In this setting, the BiPDL antibody delayed the progression of MDA-MB-231 xenografts with roughly equivalent potency to avelumab, a PD-L1 antibody capable of inducing ADCC (Figure 8B). This similarity is not surprising given the increased expression of PD-L1 relative to PD-L2 by MDA-MB-231.

[0185] BiPDL treats syngeneic MC38-PD-L2 colon cancer more effectively than any PD-L1 antibody. MC38 colon cancer cells, which endogenously express PD-L1, were retrovirally engineered to express murine PD-L2. Expression of both PD-L1 and PD-L2 was confirmed by flow cytometry (Figure 9C). 5×10 5 MC38-PD-L2 cells were implanted into C57BL / 6J mice. On days 3, 6, 9, 12, and 15, mice were injected with 100 μg of either PBS, the rat anti-mouse PD-L1 antibody 10F.9G2, the FDA-approved PD-L1 antibodies atezolizumab and avelumab, or BiPDL4-14-hIgG1[S239D / I332E]. Survival with the BiPDL4 antibody was superior to that with atezolizumab (p=0.026) and PBS (p=0.004) (Figure 9A). Tumor growth was also slowest in mice treated with BiPDL4-14-hIgG1[S239D / I332E] (Figure 9B).

[0186] BiPDL produces the most favorable intratumoral CD8 to Treg ratio in MC38-PD-L2. 1.5 × 10 cells were cultured in 30% Matrigel (Corning) to facilitate collection of infiltrating lymphocytes. 6 MC38-PD-L2 tumors were implanted into C57BL / 6J mice. PD-L1 and PD-L2 expression was confirmed by flow cytometry (Figure 10A). Mice were treated as described above, except on days 7, 10, and 13. Tumors were then harvested and infiltrating lymphocytes were analyzed by flow cytometry on day 15. In this case, BiPDL4-14-mIgG2a was used instead of the human IgG1 variant with enhanced effector function. The ratio of CD8 cytotoxic T cells to suppressive FoxP3+ regulatory T cells (Tregs) within the tumor has been established as a reliable biomarker of successful immunotherapeutic intervention. The mean CD8:Treg ratio in mice treated with BiPDL4-14-mIgG2a was 10, significantly higher than that of anti-PD-L1, anti-PD-L2, anti-PD-1, or control animals (Figure 10B).

[0187] BiPDL treats syngeneic EL4-PD-L2 T-cell lymphoma more effectively than PD-L1 or PD-L2 antibody blockade. EL4 T-cell lymphoma cells, which also endogenously express PD-L1, were retrovirally engineered to express murine PD-L2. Expression of both PD-L1 and PD-L2 was confirmed by flow cytometry (Figure 11A). 1.5 × 10 cells, which also express luciferase to facilitate bioluminescence imaging, were used. 5 Systemic disease was established in C57BL / 6J mice by transplantation of EL4-PD-L2 cells. On days 3, 6, 9, 12, and 15, mice were injected with 100 μg of either PBS, the rat anti-mouse PD-L1 antibody 10F.9G2, the rat anti-mouse PD-L2 antibody TY25, a combination of 10F.9G2 and TY25, the FDA-approved PD-L1 antibody atezolizumab, or BiPDL4-14-mIgG2a. Blockade of PD-L1 and PD-L2 was ineffective in this model, but the BiPDL4 antibody significantly prolonged survival (p=0.002 vs. no treatment; p=0.01 vs. atezolizumab) (Figure 11B). In this setting, the effector function of BiPDL antibodies appears to be particularly important for efficacy, as the combination of PD-L1 and PD-L2 blocking antibodies (10F.9G2 and TY25) had no efficacy.

[0188] Table 1: Nucleic acid sequences of antibody variable regions TIFF2026035716000004.tif211148TIFF2026035716000005.tif201144

[0189] Table 2: Protein sequences of antibody variable regions TIFF2026035716000006.tif219149TIFF2026035716000007.tif123144

[0190] Table 3: Heavy chain CDR sequences TIFF2026035716000008.tif111145

[0191] Table 4: Light chain CDR sequences TIFF2026035716000009.tif110130

[0192] (Table 5) BiPDL nomenclature TIFF2026035716000010.tif100128

[0193] Table 5. Measured affinity of antibodies binding to PD-L1 and PD-L2 TIFF2026035716000011.tif45155

[0194] Table 6. Measured affinity of antibodies binding to PD-L1 and PD-L2 TIFF2026035716000012.tif24170

[0195] Table 7. Determination of antibody overlap by binning TIFF2026035716000013.tif41152

[0196] Table 8. Determination of antibody overlap by binning TIFF2026035716000014.tif41152

[0197] Table 9. Antibody binding and competition TIFF2026035716000015.tif35156

[0198] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described in connection with preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods and steps, or to the order of the steps of the methods, described herein, without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically related agents may be substituted for the agents described herein, while still achieving the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention, as defined by the appended claims.

[0199] VIII. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2026035716000016.tif196150TIFF2026035716000017.tif230142TIFF2026035716000018.tif230130TIFF2026035716000019.tif182148

[0200] Sequence information SEQUENCE LISTING <110> Board of Regents, The University of Texas System <120> DUAL SPECIFICITY ANTIBODIES TO HUMAN PD-L1 AND PD-L2 AND METHODS OF USE THEREFOR <150> US 62 / 647,407 <151> 2018-03-23 <150> US 62 / 755,408 <151> 2018-11-02 <160> 101 <170> PatentIn version 3.5 <210> 1 <211> 290 <212> PRT <213> Homo sapiens <400> 1 Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60 Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser 65 70 75 80 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn 85 90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val 130 135 140 Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185 190 Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200 205 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210 215 220 Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His 225 230 235 240 Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr 245 250 255 Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys 260 265 270 Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu 275 280 285 Glu Thr 290 <210> 2 <211> 273 <212> PRT <213> Homo sapiens <400> 2 Met Ile Phe Leu Leu Leu Met Leu Ser Leu Glu Leu Gln Leu His Gln 1 5 10 15 Ile Ala Ala Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile 20 25 30 Glu His Gly Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser 35 40 45 His Val Asn Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn 50 55 60 Asp Thr Ser Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu 65 70 75 80 Pro Leu Gly Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp 85 90 95 Glu Gly Gln Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr 100 105 110 Lys Tyr Leu Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr 115 120 125 His Ile Leu Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln 130 135 140 Ala Thr Gly Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val 145 150 155 160 Pro Ala Asn Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val 165 170 175 Thr Ser Val Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys 180 185 190 Val Phe Trp Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp 195 200 205 Leu Gln Ser Gln Met Glu Pro Arg Thr His Pro Thr Trp Leu Leu His 210 215 220 Ile Phe Ile Pro Phe Cys Ile Ile Ala Phe Ile Phe Ile Ala Thr Val 225 230 235 240 Ile Ala Leu Arg Lys Gln Leu Cys Gln Lys Leu Tyr Ser Ser Lys Asp 245 250 255 Thr Thr Lys Arg Pro Val Thr Thr Thr Lys Arg Glu Val Asn Ser Ala 260 265 270 Ile <210> 3 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 3 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cgtctggatt caccttcgat gagtatggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atagggtatg atggactgaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggcggtgt actactgcgc cagagctgga 300 atagaataca gctacgccta tactgattac tggggacagg gtacattggt caccgtctcc 360 tca 363 <210> 4 <211> 327 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 4 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gttcgtttac agcagcgact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca ccaggaaaac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtctg cagttcggat ggtggcctcc taggactttc 300 ggcggaggga ccaaggtgga gatcaaa 327 <210> 5 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 5 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cgtctggatt caccttcgat gagtatggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atagggtatg atggactgaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggcggtgt actactgcgc cagagctgga 300 atagaataca gctacgccta tactgattac tggggacagg gtacattggt caccgtctcc 360 tca 363 <210> 6 <211> 327 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 6 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtaa cagtgttgtc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcagcca gcagggccaa cggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtgtt cagttcggat ggtggcctcc taggactttc 300 ggcggaggga ccaaggtgga gatcaaa 327 <210> 7 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 7 caggtgcagc tggtggagtc tgggggaggc gtgatccagc ctgggaggtc cctgagactc 60 tcctgtgcag cgtctggatt caccttcagt gcgtatctta tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagct ataggttatg atggaatgaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggcggtgt actactgcgc cagagctgga 300 atagaataca gctacgccta tactgattac tggggacagg gtacattggt caccgtctcc 360 tca 363 <210> 8 <211> 327 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 8 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gttcgtttac agcagcgact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatccg ccagggccgc cggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtatg cagttcggat ggtggcctcc taggactttc 300 ggcggaggga ccaaggttga gatcaaa 327 <210> 9 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 9 caggtgcagc tggtggagtc tgggggaggc gtgatccagc ctgggaggtc cctgagactc 60 tcctgtgcag cgtctggatt caccttcagt gcgtatctta tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagct ataggttatg atggaatgaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggcggtgt actactgcgc cagagctgga 300 atagaataca gctacgccta tactgattac tggggacagg gtacattggt caccgtctcc 360 tca 363 <210> 10 <211> 327 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 10 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca cagtgttgtc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca gcaggggaaga cggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtgtg cagttcggat ggtggcctcc taggactttc 300 ggcggaggga ccaaggtgga gatcaaa 327 <210> 11 <211> 360 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 11 caagtacaat tacaacagtg gggagctggt ttattaaagc cttcagaaac tttaagtttg 60 acctgtgctg tttacggtgg atcattatct ggttatcctt ggtcttggat tcgtcaacca 120 ccaggcaaag gattggagtg gatcggtgag acagacgtgt caggctggac tgactacaat 180 ccaagtttaa aatccagggt tactatctcc gtagacacgt ccaagaacca gttctccctg 240 aagctgagtt ctgtgaccgc cgcagacacg gcggtgtact actgcgccag agacggcaga 300 aggatgggta ccccttcatt cgacatatgg ggccagggta caatggtcac cgtctcctca 360 <210> 12 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 12 gacatccagt tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60 atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatcaagtt tgcaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240 gaagattttg caacttatta ctgtcagcag tacgtctact tccctcctac ttttggcgga 300 gggaccaagg ttgagatcaa a 321 <210> 13 <211> 357 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 13 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcagc agctggttga tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcccta tcctgggtac agcacggtac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggcggtgt actactgcgc cagagtgtac 300 agagctgctt cttggtttga tccctgggga cagggtacat tggtcaccgt ctcctca 357 <210> 14 <211> 318 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 14 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc aggcgagtca ggacattagc aactatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctacgat gcatccaatt tggaaacagg ggtcccatca 180 aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 240 gaagatattg caacatatta ctgtcagcag cccttccacc tcatcacttt tggcggaggg 300 accaaggttg agatcaaa 318 <210> 15 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 15 Gln Val Gln Leu Val Glu 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 Asp Glu Tyr 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 Gly Tyr Asp Gly Leu Asn Lys 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 Arg Ala Gly Ile Glu Tyr Ser Tyr Ala Tyr Thr Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 16 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 16 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Phe Val Tyr Ser Ser 20 25 30 Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Thr Arg Lys Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln Phe Gly Trp Trp Pro 85 90 95 Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 17 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 17 Gln Val Gln Leu Val Glu 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 Asp Glu Tyr 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 Gly Tyr Asp Gly Leu Asn Lys 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 Arg Ala Gly Ile Glu Tyr Ser Tyr Ala Tyr Thr Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 18 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 18 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Asn Ser Val Val Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ala Ser Arg Ala Asn Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Val Gln Phe Gly Trp Trp Pro 85 90 95 Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 19 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 19 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Ile Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Leu Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ala Ile Gly Tyr Asp Gly Met Asn Lys 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 Arg Ala Gly Ile Glu Tyr Ser Tyr Ala Tyr Thr Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 20 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 20 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Phe Val Tyr Ser Ser 20 25 30 Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ala Arg Ala Ala Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Met Gln Phe Gly Trp Trp Pro 85 90 95 Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 21 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 21 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Ile Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Leu Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ala Ile Gly Tyr Asp Gly Met Asn Lys 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 Arg Ala Gly Ile Glu Tyr Ser Tyr Ala Tyr Thr Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 22 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 22 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser His Ser Val Val Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Glu Asp Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Val Gln Phe Gly Trp Trp Pro 85 90 95 Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 23 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 23 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Leu Ser Gly Tyr 20 25 30 Pro Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Thr Asp Val Ser Gly Trp Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Arg Arg Met Gly Thr Pro Ser Phe Asp Ile Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 24 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 24 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser 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 Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Val Tyr Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 25 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 25 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Trp 20 25 30 Leu Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Leu Gly Thr Ala Arg 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 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Tyr Arg Ala Ala Ser Trp Phe Asp Pro Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 26 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 26 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Pro Phe His Leu Ile Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 27 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 27 Phe Thr Phe Asp Glu Tyr Gly Met His 1 5 <210> 28 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 28 Val Ile Gly Tyr Asp Gly Leu Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 29 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 29 Ala Arg Ala Gly Ile Glu Tyr Ser Tyr Ala Tyr Thr Asp Tyr 1 5 10 <210> 30 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 30 Phe Thr Phe Asp Glu Tyr Gly Met His 1 5 <210> 31 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 31 Val Ile Gly Tyr Asp Gly Leu Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 32 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 32 Ala Arg Ala Gly Ile Glu Tyr Ser Tyr Ala Tyr Thr Asp Tyr 1 5 10 <210> 33 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 33 Phe Thr Phe Ser Ala Tyr Leu Met His 1 5 <210> 34 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 34 Ala Ile Gly Tyr Asp Gly Met Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 35 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 35 Ala Arg Ala Gly Ile Glu Tyr Ser Tyr Ala Tyr Thr Asp Tyr 1 5 10 <210> 36 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 36 Phe Thr Phe Ser Ala Tyr Leu Met His 1 5 <210> 37 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 37 Ala Ile Gly Tyr Asp Gly Met Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 38 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 38 Ala Arg Ala Gly Ile Glu Tyr Ser Tyr Ala Tyr Thr Asp Tyr 1 5 10 <210> 39 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 39 Gly Ser Leu Ser Gly Tyr Pro Trp Ser 1 5 <210> 40 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 40 Glu Thr Asp Val Ser Gly Trp Thr Asp Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 41 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 41 Ala Arg Asp Gly Arg Arg Met Gly Thr Pro Ser Phe Asp Ile 1 5 10 <210> 42 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 42 Gly Thr Phe Ser Ser Trp Leu Ile Ser 1 5 <210> 43 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 43 Gly Ile Ile Pro Ile Leu Gly Thr Ala Arg Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 44 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 44 Ala Arg Val Tyr Arg Ala Ala Ser Trp Phe Asp Pro 1 5 10 <210> 45 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 45 Arg Ala Ser Gln Phe Val Tyr Ser Ser Asp Leu Ala 1 5 10 <210> 46 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 46 Gly Ala Ser Thr Arg Lys Thr 1 5 <210> 47 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 47 Leu Gln Phe Gly Trp Trp Pro Pro Arg Thr 1 5 10 <210> 48 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 48 Arg Ala Ser Asn Ser Val Val Ser Ser Tyr Leu Ala 1 5 10 <210> 49 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 49 Gly Ala Ala Ser Arg Ala Asn 1 5 <210> 50 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 50 Val Gln Phe Gly Trp Trp Pro Pro Arg Thr 1 5 10 <210> 51 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 51 Arg Ala Ser Gln Phe Val Tyr Ser Ser Asp Leu Ala 1 5 10 <210> 52 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 52 Gly Ala Ser Ala Arg Ala Ala 1 5 <210> 53 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 53 Met Gln Phe Gly Trp Trp Pro Pro Arg Thr 1 5 10 <210> 54 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 54 Arg Ala Ser His Ser Val Val Ser Ser Tyr Leu Ala 1 5 10 <210> 55 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 55 Gly Ala Ser Ser Arg Glu Asp 1 5 <210> 56 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 56 Val Gln Phe Gly Trp Trp Pro Pro Arg Thr 1 5 10 <210> 57 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 57 Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 <210> 58 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 58 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 59 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 59 Gln Gln Tyr Val Tyr Phe Pro Pro Thr 1 5 <210> 60 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 60 Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 61 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 61 Asp Ala Ser Asn Leu Glu Thr 1 5 <210> 62 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 62 Gln Gln Pro Phe His Leu Ile Thr 1 5 <210> 63 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 63 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Leu Ser Gly Tyr 20 25 30 Pro Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Thr Asp Val Ser Gly Trp Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Arg Arg Met Gly Thr Pro Ser Phe Asp Ile Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 64 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 64 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asn Ser Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Asn Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Lys Ala Val Tyr Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 65 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 65 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser 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 Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Lys Ala Val Tyr Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 66 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 66 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Leu Ser Gly Tyr 20 25 30 Pro Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Thr Asp Val Ser Gly Trp Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Arg Arg Met Gly Thr Pro Ser Phe Asp Ile Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 67 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 67 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Leu Ser Gly Tyr 20 25 30 Pro Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Thr Asp Val Ser Gly Trp Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Arg Arg Met Gly Thr Pro Ser Phe Asp Ile Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 68 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 68 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Lys Ser Val Tyr Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 69 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 69 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Leu Ser Gly Tyr 20 25 30 Pro Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Thr Asp Val Ser Gly Trp Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Arg Arg Met Gly Thr Pro Ser Phe Asp Ile Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 70 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 70 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Thr Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ala Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Ala Val Tyr Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 71 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 71 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Leu Ser Gly Tyr 20 25 30 Pro Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Thr Asp Val Ser Gly Trp Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Arg Arg Met Gly Thr Pro Ser Phe Asp Ile Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 72 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 72 Gly Ser Leu Ser Gly Tyr Pro Trp Ser 1 5 <210> 73 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 73 Glu Thr Asp Val Ser Gly Trp Thr Asp Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 74 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 74 Ala Arg Asp Gly Arg Arg Met Gly Thr Pro Ser Phe Asp Ile 1 5 10 <210> 75 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 75 Gly Ser Leu Ser Gly Tyr Pro Trp Ser 1 5 <210> 76 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 76 Glu Thr Asp Val Ser Gly Trp Thr Asp Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 77 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 77 Ala Arg Asp Gly Arg Arg Met Gly Thr Pro Ser Phe Asp Ile 1 5 10 <210> 78 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 78 Gly Ser Leu Ser Gly Tyr Pro Trp Ser 1 5 <210> 79 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 79 Glu Thr Asp Val Ser Gly Trp Thr Asp Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 80 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 80 Ala Arg Asp Gly Arg Arg Met Gly Thr Pro Ser Phe Asp Ile 1 5 10 <210> 81 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 81 Gly Ser Leu Ser Gly Tyr Pro Trp Ser 1 5 <210> 82 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 82 Glu Thr Asp Val Ser Gly Trp Thr Asp Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 83 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 83 Ala Arg Asp Gly Arg Arg Met Gly Thr Pro Ser Phe Asp Ile 1 5 10 <210> 84 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 84 Gly Ser Leu Ser Gly Tyr Pro Trp Ser 1 5 <210> 85 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 85 Glu Thr Asp Val Ser Gly Trp Thr Asp Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 86 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 86 Ala Arg Asp Gly Arg Arg Met Gly Thr Pro Ser Phe Asp Ile 1 5 10 <210> 87 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 87 Arg Ala Ser Gln Gly Ile Asn Ser Phe Leu Ala 1 5 10 <210> 88 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 88 Ala Ala Ser Ser Leu Asn Ser 1 5 <210> 89 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 89 Gln Lys Ala Val Tyr Phe Pro Pro Thr 1 5 <210> 90 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 90 Arg Ala Ser Gln Gly Ile Ser Asn Phe Leu Ala 1 5 10 <210> 91 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 91 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 92 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 92 Gln Lys Ala Val Tyr Phe Pro Pro Thr 1 5 <210> 93 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 93 Arg Ala Ser Gln Asp Ile Ser Ser Phe Leu Ala 1 5 10 <210> 94 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 94 Ala Ala Ser Ser Leu Gln Asp 1 5 <210> 95 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 95 Gln Lys Ser Val Tyr Phe Pro Pro Thr 1 5 <210> 96 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 96 Arg Ala Ser Gln Gly Ile Ser Thr Phe Leu Ala 1 5 10 <210> 97 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 97 Ala Ala Ser Ala Leu His Ser 1 5 <210> 98 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 98 Gln Arg Ala Val Tyr Phe Pro Pro Thr 1 5 <210> 99 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 99 Arg Ala Ser Lys Gly Ile Ser Ser Phe Leu Ala 1 5 10 <210> 100 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 100 Ala Ala Asp Ser Ile Gln Ser 1 5 <210> 101 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 101 Gln Ser Ala Val Tyr Phe Pro Pro Thr 1 5

Claims

1. Antibodies or antibody fragments that specifically bind to PD-L1 and PD-L2, including: (a) heavy chain CDRH1, CDRH2, and CDRH3 having the sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and light chain CDRL1, CDRL2, and CDRL3 having the sequences set forth in SEQ ID NOs: 51, 52, and 53, respectively; or (b) heavy chains CDRH1, CDRH2, and CDRH3 having the sequences set forth in SEQ ID NOs: 72, 73, and 74, respectively, and light chains CDRL1, CDRL2, and CDRL3 having the sequences set forth in SEQ ID NOs: 87, 88, and 89, respectively.

2. The antibody or antibody fragment of claim 1, comprising heavy chains CDRH1, CDRH2 and CDRH3 having the sequences shown in SEQ ID NOs: 33, 34 and 35, respectively, and light chains CDRL1, CDRL2 and CDRL3 having the sequences shown in SEQ ID NOs: 51, 52 and 53, respectively, and encoded by light chain variable region sequences and heavy chain variable region sequences shown in SEQ ID NOs: 8 and 7, respectively.

3. The antibody or antibody fragment of claim 1, comprising heavy chains CDRH1, CDRH2 and CDRH3 having the sequences shown in SEQ ID NOs: 33, 34 and 35, respectively, and light chains CDRL1, CDRL2 and CDRL3 having the sequences shown in SEQ ID NOs: 51, 52 and 53, respectively, and encoded by light chain variable region sequences and heavy chain variable region sequences having at least 90% or 95% identity to SEQ ID NOs: 8 and 7, respectively.

4. The antibody or antibody fragment of claim 1, comprising heavy chains CDRH1, CDRH2 and CDRH3 having the sequences shown in SEQ ID NOs: 33, 34 and 35, respectively, and light chains CDRL1, CDRL2 and CDRL3 having the sequences shown in SEQ ID NOs: 51, 52 and 53, respectively, and comprising light chain variable region sequences and heavy chain variable region sequences shown in SEQ ID NOs: 20 and 19, respectively.

5. The antibody or antibody fragment of claim 1, comprising heavy chains CDRH1, CDRH2 and CDRH3 having the sequences shown in SEQ ID NOs: 33, 34 and 35, respectively, and light chains CDRL1, CDRL2 and CDRL3 having the sequences shown in SEQ ID NOs: 51, 52 and 53, respectively, and comprising light chain variable region sequences and heavy chain variable region sequences that are at least 90% or 95% identical to SEQ ID NOs: 20 and 19, respectively.

6. The antibody or antibody fragment of claim 1, comprising heavy chains CDRH1, CDRH2 and CDRH3 having the sequences shown in SEQ ID NOs: 72, 73 and 74, respectively, and light chains CDRL1, CDRL2 and CDRL3 having the sequences shown in SEQ ID NOs: 87, 88 and 89, respectively, and comprising light chain variable region sequences and heavy chain variable region sequences shown in SEQ ID NOs: 64 and 63, respectively.

7. The antibody or antibody fragment of claim 1, comprising heavy chains CDRH1, CDRH2 and CDRH3 having the sequences shown in SEQ ID NOs: 72, 73 and 74, respectively, and light chains CDRL1, CDRL2 and CDRL3 having the sequences shown in SEQ ID NOs: 87, 88 and 89, respectively, and comprising light chain variable region sequences and heavy chain variable region sequences that are at least 90% or 95% identical to SEQ ID NOs: 64 and 63, respectively.

8. The antibody or antibody fragment of any one of claims 1 to 7, wherein the antibody fragment is a recombinant scFv (single-chain variable region fragment) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment.

9. The antibody or antibody fragment of any one of claims 1 to 7, wherein the antibody is a chimeric antibody.

10. The antibody or antibody fragment of any one of claims 1 to 9, wherein the antibody is an IgG.

11. The antibody or antibody fragment of any one of claims 1 to 10, further comprising a cell-penetrating peptide and / or being an intrabody.

12. The antibody or fragment of any one of claims 1 to 11, which is a humanized antibody.

13. A pharmaceutical composition for treating cancer in a subject, comprising the antibody or antibody fragment of any one of claims 1 to 12.

14. The pharmaceutical composition of claim 13, wherein the antibody or antibody fragment comprises heavy chains CDRH1, CDRH2 and CDRH3 having the sequences shown in SEQ ID NOs: 33, 34 and 35, respectively, and light chains CDRL1, CDRL2 and CDRL3 having the sequences shown in SEQ ID NOs: 51, 52 and 53, respectively, and is encoded by the light chain variable region sequences and heavy chain variable region sequences shown in SEQ ID NOs: 8 and 7, respectively.

15. The pharmaceutical composition of claim 13, wherein the antibody or antibody fragment comprises heavy chains CDRH1, CDRH2 and CDRH3 having the sequences shown in SEQ ID NOs: 33, 34 and 35, respectively, and light chains CDRL1, CDRL2 and CDRL3 having the sequences shown in SEQ ID NOs: 51, 52 and 53, respectively, and is encoded by light chain variable region sequences and heavy chain variable region sequences having at least 90% or 95% identity to SEQ ID NOs: 8 and 7, respectively.

16. The pharmaceutical composition of claim 13, wherein the antibody or antibody fragment comprises heavy chains CDRH1, CDRH2 and CDRH3 having the sequences shown in SEQ ID NOs: 33, 34 and 35, respectively, and light chains CDRL1, CDRL2 and CDRL3 having the sequences shown in SEQ ID NOs: 51, 52 and 53, respectively, and comprises light chain variable region sequences and heavy chain variable region sequences shown in SEQ ID NOs: 20 and 19, respectively.

17. The pharmaceutical composition of claim 13, wherein the antibody or antibody fragment comprises heavy chains CDRH1, CDRH2 and CDRH3 having the sequences shown in SEQ ID NOs: 33, 34 and 35, respectively, and light chains CDRL1, CDRL2 and CDRL3 having the sequences shown in SEQ ID NOs: 51, 52 and 53, respectively, and comprises light chain variable region sequences and heavy chain variable region sequences having at least 90% or 95% identity to SEQ ID NOs: 20 and 19, respectively.

18. The pharmaceutical composition of claim 13, wherein the antibody or antibody fragment comprises heavy chains CDRH1, CDRH2 and CDRH3 having the sequences shown in SEQ ID NOs: 72, 73 and 74, respectively, and light chains CDRL1, CDRL2 and CDRL3 having the sequences shown in SEQ ID NOs: 87, 88 and 89, respectively, and comprises light chain variable region sequences and heavy chain variable region sequences shown in SEQ ID NOs: 64 and 63, respectively.

19. The pharmaceutical composition of claim 13, wherein the antibody or antibody fragment comprises heavy chains CDRH1, CDRH2 and CDRH3 having the sequences shown in SEQ ID NOs: 72, 73 and 74, respectively, and light chains CDRL1, CDRL2 and CDRL3 having the sequences shown in SEQ ID NOs: 87, 88 and 89, respectively, and comprises light chain variable region sequences and heavy chain variable region sequences having at least 90% or 95% identity to SEQ ID NOs: 64 and 63, respectively.

20. 1. An engineered cell encoding an antibody or antibody fragment, wherein the antibody or antibody fragment specifically binds to PD-L1 and PD-L2, and characterized by: (a) heavy chain CDRH1, CDRH2, and CDRH3 having the sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and light chain CDRL1, CDRL2, and CDRL3 having the sequences set forth in SEQ ID NOs: 51, 52, and 53, respectively; or (b) heavy chains CDRH1, CDRH2, and CDRH3 having the sequences set forth in SEQ ID NOs: 72, 73, and 74, respectively, and light chains CDRL1, CDRL2, and CDRL3 having the sequences set forth in SEQ ID NOs: 87, 88, and 89, respectively.

21. 1. A pharmaceutical formulation comprising one or more antibodies or antibody fragments, wherein the antibodies or antibody fragments specifically bind to PD-L1 and PD-L2, and characterized by: (a) heavy chain CDRH1, CDRH2, and CDRH3 having the sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and light chain CDRL1, CDRL2, and CDRL3 having the sequences set forth in SEQ ID NOs: 51, 52, and 53, respectively; or (b) heavy chains CDRH1, CDRH2, and CDRH3 having the sequences set forth in SEQ ID NOs: 72, 73, and 74, respectively, and light chains CDRL1, CDRL2, and CDRL3 having the sequences set forth in SEQ ID NOs: 87, 88, and 89, respectively.

22. The pharmaceutical formulation of claim 21, wherein at least one antibody or antibody fragment comprises heavy chains CDRH1, CDRH2 and CDRH3 having the sequences shown in SEQ ID NOs: 33, 34 and 35, respectively, and light chains CDRL1, CDRL2 and CDRL3 having the sequences shown in SEQ ID NOs: 51, 52 and 53, respectively, and is encoded by the light chain variable region sequence and heavy chain variable region sequence shown in SEQ ID NOs: 8 and 7, respectively.

23. 22. The pharmaceutical formulation of claim 21, wherein at least one antibody or antibody fragment comprises heavy chains CDRH1, CDRH2 and CDRH3 having the sequences shown in SEQ ID NOs: 33, 34 and 35, respectively, and light chains CDRL1, CDRL2 and CDRL3 having the sequences shown in SEQ ID NOs: 51, 52 and 53, respectively, and is encoded by light chain variable region sequences and heavy chain variable region sequences having at least 90% or 95% identity to SEQ ID NOs: 8 and 7, respectively.

24. The pharmaceutical formulation of claim 21, wherein at least one antibody or antibody fragment comprises heavy chains CDRH1, CDRH2 and CDRH3 having the sequences shown in SEQ ID NOs: 33, 34 and 35, respectively, and light chains CDRL1, CDRL2 and CDRL3 having the sequences shown in SEQ ID NOs: 51, 52 and 53, respectively, and comprises light chain variable region sequences and heavy chain variable region sequences shown in SEQ ID NOs: 20 and 19, respectively.

25. 22. The pharmaceutical formulation of claim 21, wherein at least one antibody or antibody fragment comprises heavy chains CDRH1, CDRH2, and CDRH3 having the sequences shown in SEQ ID NOs: 33, 34, and 35, respectively, and light chains CDRL1, CDRL2, and CDRL3 having the sequences shown in SEQ ID NOs: 51, 52, and 53, respectively, and comprises light chain variable region sequences and heavy chain variable region sequences having at least 90% or 95% identity to SEQ ID NOs: 20 and 19, respectively.

26. The pharmaceutical formulation of claim 21, wherein at least one antibody or antibody fragment comprises heavy chains CDRH1, CDRH2 and CDRH3 having the sequences shown in SEQ ID NOs: 72, 73 and 74, respectively, and light chains CDRL1, CDRL2 and CDRL3 having the sequences shown in SEQ ID NOs: 87, 88 and 89, respectively, and comprises light chain variable region sequences and heavy chain variable region sequences shown in SEQ ID NOs: 64 and 63, respectively.

27. 22. The pharmaceutical formulation of claim 21, wherein at least one antibody or antibody fragment comprises heavy chains CDRH1, CDRH2 and CDRH3 having the sequences shown in SEQ ID NOs: 72, 73 and 74, respectively, and light chains CDRL1, CDRL2 and CDRL3 having the sequences shown in SEQ ID NOs: 87, 88 and 89, respectively, and comprises light chain variable region sequences and heavy chain variable region sequences having at least 90% or 95% identity to SEQ ID NOs: 64 and 63, respectively.

28. 1. A method for detecting cells expressing PD-L1 or PD-L2 in a sample from a subject, comprising: (a) contacting the sample with an antibody or antibody fragment according to any one of claims 1 to 12; and (b) detecting cells in the sample that express PD-L1 or PD-L2 by binding of the antibody or antibody fragment to cells in the sample. A method comprising:

29. A pharmaceutical composition for treating immunosuppression in the tumor microenvironment, comprising an antibody or antibody fragment of any one of claims 1 to 12.

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