Bispecific antibodies against human PD-L1 and PD-L2 and methods of using the same

Abstract

To provide bispecific antibodies which bind to both PD-L1 and PD-L2, and methods of using the antibodies to treat cancers, such as cancers expressing or overexpressing PD-L.SOLUTION: An antibody or antibody fragment comprises clone-paired heavy and light CDR sequences comprising specific sequences.SELECTED DRAWING: None

Description

Technical Field

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

[0002] Incorporation of Sequence Listing A sequence listing contained in a file named "UTFC_P1338WO_ST25" created on Mar. 14, 2019 and having a size of 49 KB (measured in Microsoft Windows®) is submitted herewith by electronic submission and is hereby incorporated by reference.

[0003] 1. Field The present disclosure relates generally to the fields of pharmaceuticals, oncology, and immunology. More specifically, the present disclosure relates to human bispecific antibodies that bind to PL-L1 and PD-L2 and their use in cancer therapy.

Background Art

[0004] 2. Description of Related Art Blocking the interaction between the T cell co-inhibitory receptor PD-1 and its ligand, PD-L1, has become a pillar of modern oncology that is available even in first-line settings for subsets of patients with melanoma and lung cancer (Boussiotis, 2016). Numerous antibodies targeting PD-1 or PD-L1 are currently FDA-approved or in clinical trials, but there are no agents targeting PD-L2, the second PD-1 ligand for which clinical research has been done. PD-L2 binds to PD-1 with an affinity approximately three-fold higher than that of 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 has generally been considered an inducible co-inhibitory molecule, and expression was thought to be limited to the tumor stroma, but improved PD-L2 detection reagents have revealed widespread PD-L2 expression both in the tumor microenvironment and on the surface of the 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] As first described in many cases of classical Hodgkin lymphoma (cHL), amplification of chromosomal region 9p24.1 leads to indirect induction via enhanced JAK2 activity in addition to direct upregulation of PD-L1 and PD-L2 (which are localized there) (Roemer et al., 2016; Shi et al., 2014; Green et al., 2010; Van Roosbroeck et al., 2016). In addition to cHL, this gene driver of high PD-L1 / PD-L2 co-expression is also found in most cases of primary mediastinal large B cell lymphoma (PMBL), T cell lymphoma, as well as 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 a number of other cancers, particularly gastric cancer, melanoma, lung, head and neck, cervical and vulvar squamous cell carcinoma, bladder cancer, and hepatocellular carcinoma (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., 2011). For many of these tumors, in addition to expression by the tumors themselves, expression of PD-L2 in stroma and endothelium has also been reported (Yearley et al., 2017). These findings suggest limitations to the therapeutic potential of PD-L1 blockade in these cancers.

[0006] Since the PD-1 co-inhibitory receptor is mainly expressed by activated T cells and NK cells, it can be the best target by an antibody that binds to the receptor and prevents the binding by the PD ligand. In contrast, PD-L1 is expressed by tumor cells and inhibitory stromal populations and can be targeted using an antibody capable of cytotoxic effector function. Although the theoretical advantages of these PD-L1 antibodies capable of antibody-dependent cell cytotoxicity (ADCC) can be demonstrated in vitro, there is no patient data demonstrating the actual effector function in patients or an improved outcome compared to purely blocking variants (Boyerinas et al., 2015).

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

[0008] Abstract Under such circumstances, in accordance with the present disclosure, an antibody or antibody fragment is provided that selectively binds to both PD-L1 and PD-L2 and has the CDR sequence pairs of the heavy and light chains of the clones from Tables 3 and 4, respectively. The antibody or antibody fragment may be encoded by the variable region sequence pairs of the clones shown in Table 1, or by the variable region sequences of the light and heavy chains having 70%, 80%, or 90% identity to the variable region sequence pairs of the clones shown in Table 1, or by the variable region sequences of the light and heavy chains having 95% or higher identity to the sequence pairs of the clones shown in Table 1. The antibody or antibody fragment may include the variable region sequences of the light and heavy chains as in the sequence pairs of the clones from Table 2, or may include the variable region sequences of the light and heavy chains having 70%, 80%, or 90% identity to the sequence pairs of the clones from Table 2, or may include the variable region sequences of the light and heavy chains having 95% or higher identity to the sequence pairs of the clones from Table 2.

[0009] A method of treating cancer in a subject, comprising contacting cancer cells that are PD-L1 or PD-L2 positive in the subject with an antibody as described above is also provided. The cancer cells that are PD-L1 or PD-L2 positive 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, renal cell carcinoma cells, or leukemia or myeloma, such as acute myeloid leukemia, chronic myeloid leukemia, or multiple myeloma.

[0010] The method may further comprise contacting PD-L1 or PD-L2 positive cancer cells with a second anti-cancer agent or anti-cancer therapy, such as chemotherapy, radiotherapy, immunotherapy, hormone therapy, or toxin therapy. The second anti-cancer agent or anti-cancer therapy may inhibit the function of intracellular PD-L1 or PD-L2. The second anti-cancer agent or anti-cancer therapy may be administered simultaneously with the first agent, or before and / or after said 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, 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, magnetic particles, a chromophore, a fluorescent molecule, a chemiluminescent molecule, or a dye). The antibody or antibody fragment may further comprise an anti-tumor drug conjugated thereto (e.g., conjugated to the antibody or antibody fragment through a photocleavable 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 nanoparticles or liposomes.

[0012] In another aspect, provided is a method of treating cancer in a subject, comprising delivering to the subject an antibody or antibody fragment having a pair of CDR sequences of the heavy and light chains of the clones from Table 3 and Table 4, respectively. The antibody fragment may be a recombinant scFv (single-chain variable region fragment) antibody, a Fab fragment, an F(ab’)2 fragment, or an Fv fragment. The antibody may be 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 variable region sequence pairs of the light and heavy chains of the clones shown in Table 1, or may be encoded by variable region sequence pairs of clones having 95% identity to those shown in Table 1, or may be encoded by variable region sequences of the light and heavy chains having 70%, 80%, or 90% identity to the sequence pairs of the clones from Table 1. The antibody or antibody fragment may contain variable region sequences of the light and heavy chains as in the sequence pairs of the clones from Table 2, or may contain variable region sequences of the light and heavy chains having 70%, 80%, or 90% identity to the sequence pairs of the clones from Table 2, or may contain variable region sequences of the light and heavy chains having 95% identity to the sequence pairs of the clones from Table 2.

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

[0015] The antibody or antibody fragment may be encoded by the variable region sequence pairs of the light and heavy chains of the clones shown in Table 1, or may be encoded by variable region sequence pairs of clones having 95% identity to those shown in Table 1, or may be encoded by variable region sequences of the light and heavy chains having 70%, 80%, or 90% identity to the sequence pairs of the clones from Table 1. The antibody or antibody fragment may contain variable region sequences of the light and heavy chains as in the sequence pairs of the clones from Table 2, or may contain variable region sequences of the light and heavy chains having 70%, 80%, or 90% identity to the sequence pairs of the clones from Table 2, or may contain variable region sequences of the light and heavy chains having 95% identity to the sequence pairs of the clones from Table 2.

[0016] In yet another aspect, provided are hybridomas or engineered cells encoding an antibody or antibody fragment, wherein the antibody or antibody fragment is characterized by the CDR sequence pairs of the heavy and light chains of the clones from Tables 3 and 4, respectively. The antibody fragment may be a recombinant scFv (single-chain variable region fragment) antibody, Fab fragment, F(ab’)2 fragment, or Fv fragment. The antibody may be a chimeric antibody or IgG.

[0017] The antibody or antibody fragment may be encoded by the variable region sequence pairs of the light and heavy chains of the clones shown in Table 1, or by the variable region sequence pairs of the light and heavy chains of clones having 95% identity to those shown in Table 1, or by the variable region sequences of the light and heavy chains having 70%, 80%, or 90% identity to the sequence pairs of the clones from Table 1. The antibody or antibody fragment may comprise the variable region sequences of the light and heavy chains as in the sequence pairs of the clones from Table 2, or may comprise the variable region sequences of the light and heavy chains having 70%, 80%, or 90% identity to the sequence pairs of the clones from Table 2, or may comprise the variable region sequences of the light and heavy chains having 95% identity to the sequence pairs of the clones from Table 2.

[0018] Further aspects include cancer vaccines comprising one or more antibodies or antibody fragments characterized by CDR sequence pairs of the heavy and light chains of the 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, an 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 the variable region sequence pairs of the light and heavy chains of the clones shown in Table 1, or by variable region sequence pairs of clones having 95% identity to those shown in Table 1, or by light and heavy chain variable region sequences having 70%, 80%, or 90% identity to the sequence pairs of the clones from Table 1. At least one antibody or antibody fragment may comprise light and heavy chain variable region sequences as in the sequence pairs of the clones from Table 2, or may comprise light and heavy chain variable region sequences having 70%, 80%, or 90% identity to the sequence pairs of the clones from Table 2, or may comprise light and heavy chain variable region sequences having 95% identity to the sequence pairs of the clones from Table 2.

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

[0020] Any method or composition described herein is intended to be practiced with respect to any other method or composition described herein. Other objects, features, and advantages of the disclosure will be apparent from the following detailed description. However, the detailed description and specific examples, while indicating particular embodiments of the disclosure, are intended for purposes of illustration only and it is understood that various changes and modifications within the spirit and scope of the disclosure will be apparent to those skilled in the art from this detailed description.

[0021] As used herein, "essentially free of" with respect to a specified component means that none of the specified components are intentionally formulated into the composition and / or are present only as contaminants or in trace amounts. The total amount of the specified component resulting from any unintended contaminants in the composition is preferably less than 0.01%. Most preferably, the composition is such that 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 combination with the word "comprising", the words "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 additional one.

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

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

Brief Description of the Drawings

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

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Modes for Carrying Out the Invention

[0027] Description of Exemplary Embodiments The inventors have generated monoclonal antibodies that have binding specificity for both human PD-L1 and PD-L2 proteins. Since these antibodies have been demonstrated to bind both PD-L1 and PD-L2, they provide an opportunity to block the binding of either PD-L1 or PD-L2 to PD-1. They can also be used to deliver a therapeutic payload to cancer cells that express PD-L1 or PD-L2. These and other aspects of the 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 I transmembrane protein that can play a major role in immunosuppression during various events such as pregnancy, tissue allotransplantation, autoimmune diseases, cancer, and other disease states. The human PD-L1 protein is encoded by the amino acid sequence shown below. TIFF0007713995000001.tif30149

[0029] B. Function PD-L1 is a ligand for its receptor, PD-1. PD-1 can be found on activated T cells, B cells, and myeloid cells. The binding of PD-L1 to PD-1 modulates the activation or inhibition of T cells and B cells. It transmits an inhibitory signal that reduces the proliferation of antigen-specific CD8+ T cells and CD4+ helper T cells. The 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). The upregulation of PD-L1 has been associated with the evasion of the host immune system and is thought to be a cause of increased tumor aggressiveness (Thompson et al., 2004). The role of PD-L1 in the evasion of 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 can play a major role in immunosuppression during various events such as pregnancy, tissue allotransplantation, autoimmune diseases, cancer, and other disease states. The human PD-L2 protein is encoded by the amino acid sequence shown below. TIFF0007713995000002.tif30149

[0031] PD-L2 is first produced together with a signal peptide corresponding to amino acids 1 - 19 of SEQ ID NO:2, and then the signal peptide is 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 a ligand for its receptor PD-1. PD-1 can be found on activated T cells, B cells, and myeloid cells. The 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 an inhibitory signal that reduces 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 against PD-L1 and PD-L2 can be generated by standard methods well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. 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 immunization of a suitable host or identification of an immunized subject by previous natural infection. As is well known in the art, a given composition for immunization can have varying immunogenicity. Thus, in many cases, it is necessary to enhance the host immune system, which can be achieved by conjugating 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 a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide, and bisdiazotized benzidine. Also, as is well known in the art, the immunogenicity of a particular immunogenic composition can be enhanced by use of a non-specific stimulator of the immune response known as an adjuvant. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant.

[0034] The amount of immunogenic composition used in the production of polyclonal antibodies varies depending on the nature of the immunogen and, in addition, on the animal used for immunization. Various routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous, and intraperitoneal). The production of polyclonal antibodies can be monitored by sampling the blood of the immunized animals at various times after immunization. A second booster injection may be given. The boosting and titration procedures are repeated until a suitable titer is achieved. When the desired level of immunogenicity has been obtained, the immunized animals can be bled to isolate and store the serum and / or the animals 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 the mAb production protocol. These cells can be obtained from biopsied spleen or lymph nodes, or from circulating blood. Next, antibody-producing B lymphocytes from the immunized animals are 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 the hybridoma production fusion procedure preferably have an enzyme deficiency that renders them non-antibody-producing, have a high fusion efficiency, and are unable to grow in a particular selection medium that supports the growth of only the desired fused cells (hybridomas).

[0036] As is 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 it 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 the context of 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 applying for 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 strains 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 the present disclosure were generated using the SP2 / 0 / mIL-6 cell line, which is an IL-6 secretion-inducing strain of the SP2 / 0 strain.

[0037] Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells generally involve mixing somatic cells with myeloma cells at a ratio of 2:1, although the ratio can vary from about 20:1 to about 1:1, respectively, in the presence of one or more (chemical or electrical) agents 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), such as 37% (v / v) PEG, is described by Gefter et al. (1977). The use of electrically induced fusion methods is also appropriate (Goding, pp.71-74, 1986).

[0038] The fusion procedure usually results in viable hybrids at a low frequency of about 1×10 -6 ~1×10 -8 . However, this is not a problem because viable fused hybrids differentiate from parental unfused cells (especially unfused myeloma cells, which normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally a medium containing an agent that blocks de novo synthesis of nucleotides in tissue culture medium. 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 strains that have not fused to the myeloma.

[0039] A preferred selection medium is HAT or HAT containing ouabain. Only cells capable of operating the nucleotide salvage pathway can survive in HAT medium. Myeloma cells lack the key enzyme of the salvage pathway, such as hypoxanthine phosphoribosyl transferase (HPRT), and cannot survive. B cells can operate this pathway but have a limited lifespan in culture and generally die within about two weeks. Therefore, only cells that can survive in the selection medium are hybrids formed from myeloma and B cells. When the source of B cells used for fusion is a strain of EBV-transformed B cells, as in this case, ouabain is also used for drug selection of the hybrids 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. Typically, hybridoma selection is performed by culturing cells by dilution of single clones in microtiter plates and then testing the supernatants of individual clones (after about 2 - 3 weeks) for the desired reactivity. The assay should be highly sensitive, simple, and rapid, such as radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunobinding assay, etc.

[0041] Next, the selected hybridomas can be serially diluted or single cells can be sorted by flow cytometry sorting and cloned into individual antibody-producing cell lines, and then the clones can be propagated indefinitely to provide mAbs. The cell lines can be utilized for the production of mAbs in two basic ways. A sample of the hybridomas can be injected into an animal (e.g., a mouse), often into the peritoneal cavity. Optionally, the animal can be primed with an oil such as a hydrocarbon, particularly pristane (tetramethylpentadecane), prior to injection. When human hybridomas are used in this method, it is optimal to inject into immunodeficient mice such as SCID mice to prevent tumor rejection. The injected animal develops a tumor that secretes the specific monoclonal antibody produced by the fused cell hybrid. Next, the body fluid of the animal, such as serum or ascites fluid, can be collected to provide mAbs at high concentrations. The individual cell lines can also be cultured in vitro, where the mAbs are secreted naturally into the culture medium and can be easily obtained from there at 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 high-purity human monoclonal immunoglobulins, the cell lines can be adapted to growth in serum-free media.

[0042] The monoclonal antibodies produced by any of the means can be further purified, if necessary, using filtration, centrifugation, and various chromatography 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 chemical reduction to cleave disulfide bonds. Alternatively, the monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

[0043] It is also contemplated that monoclonal antibodies may be generated using molecular cloning approaches. For this purpose, RNA can be isolated from a hybridoma strain, the antibody gene can be obtained by RT-PCR, and cloned into an immunoglobulin expression vector. Alternatively, a combinatorial immunoglobulin phagemid library can be prepared from RNA isolated from a cell line, and phagemids expressing appropriate antibodies can be selected by panning using a viral antigen. The advantages of this approach over conventional hybridoma technology are that approximately 10 4 times more antibodies can be produced and screened in a single round, and new specificities are generated by the combination of the H and L chains, which further increases the likelihood of finding appropriate antibodies.

[0044] Yeast-based antibody libraries may be rationally designed and antibodies can 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, which are hereby incorporated by reference herein and teach the production of antibodies useful in the present disclosure, include U.S. Patent No. 5,565,332, which describes the production of chimeric antibodies using combinatorial approaches; U.S. Patent No. 4,816,567, which describes recombinant immunoglobulin preparations; and U.S. Patent No. 4,867,973, which describes antibody-therapeutic agent conjugates.

[0046] B. Antibodies of the Present Disclosure Antibodies according to the present disclosure can, in a first instance, be defined by their binding specificities (i.e., binding to PD-L1 and PD-L2). One of ordinary skill in the art can determine whether such an antibody falls within the scope of the claims of this application by assessing the binding specificity / affinity of a given antibody using techniques well known to those of ordinary skill in the art. In one aspect, monoclonal antibodies are provided having the CDR pairs of the heavy and light chains of the clones shown 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, the antibodies may be defined by those variable region sequences that include additional "framework" regions. These are provided in Tables 1 and 2 that encode or represent the full length of the variable regions. Further, the antibody sequences may optionally differ from these sequences using methods discussed in more detail below. For example, the nucleic acid sequence may (a) have the variable region separated from the constant domains of the light and heavy chains, (b) the nucleic acid may differ from that shown above without affecting the residues it encodes, (c) the nucleic acid may differ from that shown above by only a given percentage of homology, such as 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, (d) the nucleic acid may differ from that shown above due to its ability to hybridize under high stringency conditions exemplified by low salt and / or high temperature conditions (e.g., conditions provided by about 0.02M to about 0.15M NaCl at a temperature of about 50°C to about 70°C), (e) the amino acids may differ from that shown above by only a given percentage of homology, such as 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or (f) the amino acids may differ from that shown above by allowing conservative substitutions (discussed below). Each of the above applies to the nucleic acid sequences shown as Table 1 and the amino acid sequences of Table 2.

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

[0049] After culturing the hybridoma, the cells can be lysed and total RNA can be extracted. After generating cDNA copies of the RNA using random hexamers with RT, PCR can be performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. After cloning the PCR products into the pGEM-T Easy vector, sequencing can be performed by automated DNA sequencing using standard vector primers. Binding and neutralization assays can be performed using the antibody recovered from the hybridoma supernatant and purified by FPLC using a Protein G column.

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

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

[0052] Antibodies, and 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. Patent No. 8,691,730, and U.S. Patent No. 9,354,228. This method of synthesizing antibodies requires that the nucleotide sequence encoding the desired or designed antibody be inserted into a vector for ectopic expression. Subsequently, the desired antibody may be expressed and purified as a full-length IgG molecule.

[0053] Antibody molecules include, for example, fragments (such as F(ab'), F(ab')2, etc.) produced by proteolytic cleavage of mAbs, or single-chain immunoglobulins that can be produced, for example, via recombinant means. Such antibody derivatives are monovalent. In one aspect, 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 that can bind to different epitopes of the same molecule.

[0054] In a related aspect, the antibody is a derivative of the disclosed antibody, for example, an antibody (such as a chimeric antibody or a CDR-grafted antibody) that contains the same CDR sequences as the CDR sequences in the disclosed antibody. Alternatively, it may be desirable to make modifications such as introducing conservative changes into the antibody molecule. In carrying out such changes, the hydrophobicity index of amino acids can be considered. The importance of the hydrophobicity amino acid index in conferring interactive biological functions to proteins is generally understood in the art (Kyte and Doolittle, 1982). The relative hydrophobicity characteristics of amino acids contribute to the secondary structure of the resulting protein, and it has been recognized that it defines the interaction of the protein with other molecules (such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).

[0055] It is also understood in the art that similar amino acid substitutions can be effectively made based on hydrophilicity. U.S. Patent No. 4,554,101, which is incorporated herein by reference, states that the maximum local average hydrophilicity of a protein governed by the hydrophilicity of adjacent amino acids is related to the biological properties of the protein. As detailed in U.S. Patent 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 non-ionic 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 non-aromatic 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 with another amino acid having similar hydrophilicity to yield a biologically or immunologically modified protein. For such changes, amino acid substitutions with hydrophilicity values within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0057] As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, such as, for example, hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions taking into account 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 different isotypes, different functions can be achieved. For example, changing to IgG1 can enhance antibody-dependent cell cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve avidity.

[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 polypeptides. Methods of recombinant expression are addressed elsewhere in this document.

[0060] D. Single-chain antibodies A single-chain variable fragment (scFv) is a fusion of the variable regions of the heavy and light chains of an immunoglobulin, linked together with a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin despite removal of the constant regions and introduction of the linker peptide. This modification typically leaves the specificity unchanged. Historically, these molecules were created to facilitate phage display, which is highly convenient for expressing the antigen-binding domain as a single peptide. Alternatively, scFvs can be made directly from subcloned heavy and light chains derived from hybridomas. Single-chain variable fragments lack the constant Fc region, which is a common binding site (e.g., protein A / G) used to purify antibodies. Since protein L interacts with the variable region of the κ light chain, these fragments can often be purified / immobilized using protein L.

[0061] Flexible linkers generally consist of helices and turn-promoting amino acid residues such as alanine, serine, and glycine. However, other residues can also function. Tang et al. (1996) used phage display to rapidly select linkers specialized for single-chain antibodies (scFvs) from a protein linker library. A random linker library was constructed in which genes for the heavy and light chain variable domains were linked by segments encoding 18 amino acid polypeptides of variable composition. The scFv repertoire (about 5×10 6 individual members) was displayed on filamentous phage and subjected to affinity selection with hapten. The selected population of variants showed a significant increase in binding activity but retained considerable sequence diversity. Screening of 1,054 individual variants subsequently yielded catalytically active scFvs that were efficiently produced in soluble form. Sequence analysis revealed conserved prolines at two residues of the linker after the C-terminus of V H and numerous arginines and prolines at other positions as the only common feature of the selected tethers.

[0062] The recombinant antibodies of the present disclosure may also be accompanied by sequences or portions that allow dimerization or multimerization of the receptor. Such sequences include those derived from IgA that allow 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 an agent that allows combination of two antibodies, such as biotin / avidin.

[0063] In another embodiment, single-chain antibodies can be created by linking the light and heavy chains of the receptor using non-peptide linkers or chemical units. Generally, the light and heavy chains are produced and purified in separate cells and then linked together in an appropriate manner (i.e., the N-terminus of the heavy chain is attached to the C-terminus of the light chain via appropriate chemical cross-linking).

[0064] Crosslinking reagents, such as stabilizers and coagulants, are used to form molecular crosslinks that link the functional groups of two different molecules. However, it is contemplated that dimers or multimers of the same analog or heteromeric complexes composed of different analogs can be produced. To link two different compounds in a step-by-step manner, a heterobifunctional crosslinking agent that removes unwanted homopolymer formation can be used.

[0065] Exemplary heterobifunctional crosslinking agents contain two reactive groups, one of which reacts with a primary amine group (e.g., N-hydroxysuccinimide), and the other of which reacts with a thiol group (e.g., pyridyldisulfide, maleimide, halogen, etc.). Through the primary amine-reactive group, the crosslinking agent may react with a lysine residue of one protein (e.g., a selected antibody or fragment), and through the thiol-reactive group, the crosslinking agent 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 preferred that a crosslinking agent having reasonable stability in the blood be used. A variety of disulfide bond-containing linkers are known that can be successful for use in conjugating targeting agents and therapeutic / prophylactic agents. Linkers containing sterically hindered disulfide bonds may confer greater stability in vivo and prevent the release of the targeting peptide before reaching the site of action. Thus, these linkers are a group of linking agents.

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

[0068] Like many other known crosslinking reagents, the SMPT crosslinking reagent confers the ability to crosslink functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of crosslinker includes hetero-bifunctional photoreactive phenyl azides containing cleavable disulfide bonds, such as sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate. The N-hydroxysuccinimidyl group reacts with primary amino groups, and the phenyl azide reacts non-selectively (by photolysis) with any amino acid residue.

[0069] In addition to crosslinkers that are subject to impairment, linkers that are not subject to impairment can also be used in accordance with the present invention. Other useful crosslinkers that are not considered to contain or generate protected disulfides include SATA, SPDP, and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such crosslinkers 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 producing conjugates of ligands with amine-containing polymers and / or proteins, particularly for forming antibody conjugates with chelators, drugs, enzymes, detectable labels, etc. U.S. Patent Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing labile bonds that can be cleaved under various mild conditions. This linker is particularly useful in that the agent of interest is directly bound to the linker such that cleavage can result in the 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 a peptide linker for use in connecting polypeptide components for preparing fusion proteins, such as single-chain antibodies. The linker is up to about 50 amino acids in length, contains the presence of at least one proline following a charged amino acid (preferably arginine or lysine), and is characterized by greater stability and reduced aggregation. U.S. Patent No. 5,880,270 discloses aminooxy-containing linkers useful in various immuno-diagnostic and separation techniques.

[0072] E. Purification In certain embodiments, the antibodies of the present disclosure may be purified. As used herein, the term "purified" is intended to refer to a composition that is isolatable from other components and that is purified to any degree compared to the state in which the protein may be obtained naturally. Thus, a purified protein also refers to a protein that has been released from the environment in which it may naturally exist. When the term "substantially purified" is used, this designation refers to a composition in which the protein or peptide forms the major component of the composition, e.g., 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. These techniques involve, at one level, a crude fractionation of the cellular environment into polypeptide and non-polypeptide fractions. Once the polypeptide has been separated from other proteins, chromatography and electrophoresis techniques can be used to further purify the polypeptide of interest to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for the preparation of pure peptides are ion-exchange chromatography, size-exclusion chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies, etc., or centrifugation after precipitation by heat denaturation; gel filtration, reverse phase, hydroxylapatite, and affinity chromatography; and combinations of such techniques and other techniques.

[0074] In the purification of the antibodies 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 cell 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 the various purification steps are performed may be changed or certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.

[0075] Generally, full antibodies are fractionated using an agent that binds to the Fc portion of the antibody (i.e., Protein A). Alternatively, an appropriate antibody may be simultaneously purified and selected using an antigen. Such methods often utilize a selection agent bound to a support such as a column, filter, or bead. The antibody is bound to the support, contaminants are removed (e.g., washed away), and the antibody is released by applying conditions (such as salt, heat).

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

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

[0078] IV. Pharmaceutical Formulations and Cancer Treatment A. Cancer Cancer results from the proliferation of a clonal population of cells from a tissue. The development of cancer, which is termed carcinogenesis, can be modeled and characterized in a number of ways. The association between cancer development and inflammation has been recognized over a long period. The inflammatory response is involved in host defense against microbial infection and also promotes tissue repair and regeneration. Considerable evidence points to a link between inflammation and cancer development risk, i.e., 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, and more specifically, any cells that overexpress either PD-L1 or PD-L2. Suitable cancer cells can be breast cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, gastric cancer, liver cancer, bone cancer, blood cancers (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, chimpanzees, or baboons), horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. The cancer may also be recurrent, metastatic, and / or multi-drug resistant, and the methods of the present disclosure may be particularly applied to such cancers to make them resectable, to prolong remission or re-induce remission, to inhibit angiogenesis, to prevent or limit metastasis, and / or to treat multi-drug resistant cancers. At the cellular level, the methods of the present disclosure can lead to the killing of cancer cells, inhibition of cancer cell proliferation, or alternatively, reversal or reduction of the malignant phenotype of tumor cells.

[0080] B. Formulations and Administration The present disclosure provides a pharmaceutical composition comprising a bispecific antibody (BiPDL) against PD-L1 and PD-L2. 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 Pharmacopeia or other generally recognized pharmacopeias. The term "carrier" refers to a diluent, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers may 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, normal saline solution, dextrose, gelatin, malt, corn, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like.

[0081] The composition can be formulated in neutral or salt form. 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 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 include classical pharmaceutical preparations. Administration of these compositions according to the present disclosure is effected via any general route as long as the target tissue is effective 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 usually administered as pharmaceutically acceptable compositions as described above. Of particular interest are direct intratumoral administration, tumor perfusion, or local or regional administration to the tumor, for example, in the local or regional vasculature or lymphatic system, or in the excised tumor bed.

[0083] The active compound may also be administered parenterally or intraperitoneally. Solutions of the active compound as a free base or a pharmaceutically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative 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 (BiPDL) against PD-L1 and PD-L2 described herein can be used similarly in combination with interventions by chemotherapy or radiotherapy or other treatments. In particular, it may also be effective to combine bispecific antibodies against PD-L1 and PD-L2 with other therapeutic methods that target different aspects of the function of PD-L1 or PD-L2 (for example, peptides and small molecules that target the cytoplasmic domain of PD-L1 or PD-L2).

[0085] Using the methods and compositions of the present disclosure, in order to kill cells, inhibit cell proliferation, inhibit metastasis, inhibit angiogenesis, or otherwise reverse or reduce the malignant phenotype of tumor cells, generally, "target" cells are contacted with the anti-PD-L1 and anti-PD-L2 bispecific antibodies of the present disclosure and at least one other agent. These compositions are provided in a total amount effective to kill cells or inhibit cell proliferation. This method may involve contacting the cells simultaneously with the anti-PD-L1 and anti-PD-L2 bispecific antibodies of the present disclosure and other agents or factors. This may be achieved by contacting the cells with a single composition or pharmaceutical formulation containing both agents, or by contacting the cells simultaneously with two separate compositions or formulations, one composition containing the anti-PD-L1 and anti-PD-L2 bispecific antibodies of the present disclosure and the other containing the other agent.

[0086] Alternatively, anti-PD-L1 and anti-PD-L2 bispecific antibody therapy may be performed before or after treatment with other agents at intervals ranging from minutes to weeks. In embodiments where the other agent and the anti-PD-L1 and anti-PD-L2 bispecific antibody are administered to the cells separately, care is usually taken to ensure that a significant period of time does not elapse between each delivery so that the agent and the expression construct can still advantageously exert a combined effect in the cells. In such examples, it is contemplated that the cells will be contacted with both modalities within about 12 to 24 hours of each other, more preferably within about 6 to 12 hours of each other, and most preferably with a delay time of only about 12 hours. However, in some situations, it may be desirable to significantly extend the treatment period so that several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapse between each administration.

[0087] It is also contemplated that it may be desirable to administer either the anti-PD-L1 and anti-PD-L2 bispecific antibody or the other agent multiple times. Various combinations can be used, and are exemplified as follows, where the anti-PD-L1 and anti-PD-L2 bispecific antibody therapy of the present disclosure is designated "A" and the other therapy is designated "B". TIFF0007713995000003.tif17128

[0088] Administration of the therapeutic agent of the present invention to a patient follows the general protocol for administration of a particular secondary therapy, taking into account the toxicity of antibody therapy if any. Treatment cycles are expected to be repeated as necessary. In addition to the various standard therapies, surgical intervention may also be applied in combination with the described cancer treatment, as is also contemplated.

[0089] Those skilled in the art are referred to "Remington's Pharmaceutical Sciences", 15th Edition, Chapter 33, particularly pages 624 - 652. Some variation in dosage is inevitable depending on the condition of the subject being treated. In any case, the person responsible for administration determines the appropriate dosage for an individual subject. Further, for human administration, the preparation should meet the standards of sterility, pyrogenicity, general safety and purity as required by the FDA Office of Biologics standards.

[0090] 1. Chemotherapy Cancer treatment also includes various combination therapies involving both chemical - based and radiation - based treatments. Examples of combination chemotherapy include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binders, taxol, gemcitabine, navelbine, farnesyl - protein transferase inhibitors, transplatinum, 5 - fluorouracil, vincristine, vinblastine, and methotrexate, temozolomide (the 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 to treat or prevent cancer or that may be known in the art.

[0091] 2. Radiation Therapy Other factors that cause DNA damage and have been widely used include γ - rays, X - rays, and / or those generally known for tumor - cell - directed delivery of radioisotopes. Other forms of DNA - damaging factors such as microwave and UV irradiation are also contemplated. All of these factors are most likely to cause a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome construction and maintenance. The dose range of X - rays extends from a dose of 50 - 200 roentgens per day over a long period (3 - 4 weeks) to a single - dose of 2000 - 6000 roentgens. The dose range of radioisotopes varies widely and depends on the half - life of the isotope, the intensity and type of the emitted radiation, and the absorption by neoplastic cells.

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

[0093] 3. Immunotherapy Immunotherapeutic agents 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 for some marker on the surface of the tumor cell. The antibody may act alone as an effector of the therapy or may localize other cells to actually effect cell killing. The antibody may also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and act simply as a targeting agent. Alternatively, the effector may be a lymphocyte having surface molecules that interact directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells. Combinations of therapeutic modalities, namely, direct cytotoxic activity and inhibition or reduction of Fortilin, provide a therapeutic benefit in the treatment of cancer.

[0094] Immunotherapy can also be used as part of 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., that is not present on most other cells. There are many tumor markers, 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 directed against the anti-cancer effect with an immune-stimulatory effect. 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 using protein or gene delivery in combination with a tumor suppressor such as mda-7 has been shown to enhance the anti-tumor effect (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, and 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 No. 5,830,880 and U.S. Patent No. 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 is approved for use in the treatment of malignancies (Dillman, 1999). The combination therapy of cancer using Herceptin and chemotherapy has been shown to be more effective than individual therapies. Therefore, it is contemplated that one or more anti-cancer therapies may be used in combination with the tumor-associated HLA-restricted peptide therapy described herein.

[0096] In adoptive immunotherapy, the patient's circulating lymphocytes, or tumor infiltrating lymphocytes are isolated in vitro and activated by 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 antigen peptide composition as described herein. The activated lymphocytes are most preferably the patient's own cells that have been previously isolated from blood or tumor samples and activated (or "expanded") in vitro. This form of immunotherapy has caused regression of melanoma and renal carcinoma in some cases, but the proportion of responders has been small compared to non-responders.

[0097] There are many different approaches for the passive immunotherapy of cancer. It can be broadly classified as follows: injection of antibodies alone, injection of antibodies conjugated with toxins or chemotherapeutic agents, injection of antibodies conjugated with radioisotopes, injection of anti-idiotype antibodies, and finally, purging of tumor cells in the bone marrow.

[0098] Human monoclonal antibodies are used in passive immunotherapy because they cause few or no side effects in patients. However, their application has been somewhat limited by their rarity and they have so far only been administered intralesionally. Human monoclonal antibodies against ganglioside antigens have been administered intralesionally to patients with cutaneous recurrent melanoma (Irie & Morton, 1986). Regression was observed in 6 out of 10 patients after intralesional injection daily or weekly. In another study, moderate success was achieved from intralesional injection 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 also be advantageous to administer more than one monoclonal antibody targeting two different antigens, or even an antibody having multiple antigen specificities. The treatment protocol may also include administration of lymphokines or other immune enhancers as described by Bajorin et al. (1988). The development of human monoclonal antibodies is described in more detail elsewhere in this specification.

[0100] 4. Gene Therapy In yet another aspect, 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-proliferative effect on the target tissue. Alternatively, a single vector encoding both genes may be used. A variety of proteins are encompassed by the present invention, some of which are described below. A variety of genes that can be targeted by some forms of gene therapy when used in combination with the present invention are well known to those skilled in the art, and such genes can include any gene involved in cancer.

[0101] Cell growth-inducing factors. Proteins that induce cell growth are further classified into various categories according to their functions. The common feature of all these proteins is the ability to regulate cell growth. For example, one form of PDGF, the sis oncogene, is a secreted growth factor. It is rare for an oncogene to arise from a gene encoding a growth factor, and currently, sis is the only known naturally occurring oncogenic growth factor. In one aspect of the present invention, it is contemplated that antisense mRNA targeting a specific cell growth-inducing factor is used to prevent the expression of the cell growth-inducing factor.

[0102] Proteins FMS, ErbA, ErbB, and neu are growth factor receptors. Mutations in these receptors result in the loss of regulatable functions. For example, point mutations that affect the transmembrane domain of the Neu receptor protein result in the neu oncogene. The erbA oncogene is derived from the intracellular receptor for thyroid hormone. The modified oncogenic ErbA receptor is thought to compete with the endogenous thyroid hormone receptor and cause unregulated growth.

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

[0104] Cell growth inhibitors. Tumor suppressor oncogenes function to inhibit excessive cell growth. Inactivation of these genes destroys their inhibitory activity and results in unregulated growth. The most common tumor suppressors are Rb, p53, p21, and p16. Other genes that can be used according to 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 fusion, and p21 / p27 fusion.

[0105] Regulators of programmed cell death. Apoptosis, or programmed cell death, is an essential process for normal embryonic development, maintenance of homeostasis in adult tissues, and suppression of carcinogenesis (Kerr et al., 1972). Proteins of the Bcl-2 family and ICE-like proteases have been demonstrated to be important regulators and effectors of apoptosis in other systems. The Bcl-2 protein, discovered in the context of follicular lymphoma, plays a prominent role in the control of apoptosis and the promotion of 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 a death agonist or death antagonist.

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

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

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

[0109] When excising a part or all of cancer cells, cancer tissue, or a tumor, a cavity may be formed in the body. Treatment may be achieved by perfusion, direct injection, or topical application of additional anti-cancer therapy to the area. Such treatment may 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 may also be at different dosages.

[0110] V. Antibody Conjugates An antibody conjugate can be formed by linking at least one antibody with at least one agent. It is conventional to link or covalently bond or complex at least one desired molecule or moiety to enhance the effectiveness of an antibody molecule as a diagnostic or therapeutic agent. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules include molecules having a desired activity, such as immunosuppressive / anti-inflammatory. Non-limiting examples of such molecules are shown 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 an antibody 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 classes, namely those for use in in vitro diagnostics, such as in various immunoassays, and those for use in in vivo diagnostic protocols, generally known as "antibody-directed imaging". Many suitable imaging agents are known in the art, as are methods for attaching them to antibodies (see, for example, U.S. Pat. Nos. 5,021,236, 4,938,948, and 4,472,509). The imaging moieties used can be paramagnetic ions, radioisotopes, fluorescent dyes, substances detectable by NMR, 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 particularly bismuth(III).

[0114] In the case of radioisotopes 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, technetium 99m and / or yttrium 90 may be mentioned. 125 I is often preferred for use in certain embodiments, and technetium 99m and / or indium 111 are also often preferred due to their suitability for low energy and long distance detection. Radioactively labeled monoclonal antibodies can be produced according to well-known methods in the art. For example, monoclonal antibodies can be iodinated by contacting them with sodium and / or potassium iodide and a chemical oxidant, such as sodium hypochlorite, or an enzymatic oxidant, such as lactoperoxidase. Monoclonal antibodies can be labeled with technetium 99m by the ligand exchange method, for example, by reducing pertechnate with a stannous solution, chelating the reduced technetium to a Sephadex column, and applying the antibody to this column. Alternatively, for example, direct labeling techniques may be used by incubating pertechnate, a reducing agent, such as SNCl2, a buffer solution, such as a sodium-potassium phthalate solution, and the antibody. Intermediate functional groups are often used to bind radioisotopes to antibodies and are present as metal ions, such as 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 contemplated is primarily intended for in vitro use, in which case 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. Pat. 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 the reaction of the antibody with a hapten-based affinity label. Essentially, the hapten-based affinity label reacts with the amino acids in the antigen-binding site, thereby destroying this site and blocking the specific antigen reaction. However, this can be disadvantageous as it results in a loss of antigen binding by the antibody conjugate.

[0118] Molecules containing an azide group 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 the nucleotide-binding domains of purified proteins (Khatoon et al, 1989; King et al, 1989; Dholakia et al, 1989) and can be used as antibody conjugates.

[0119] Several methods for attachment of an antibody to its conjugate moiety or for conjugation are known in the art. Some attachment methods involve, for example, the use of metal chelate complexes with organic chelating agents attached to the antibody, such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriamine tetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3α-6α-diphenylglycouril-3 (U.S. Patent Nos. 4,472,509 and 4,938,948). Monoclonal antibodies may 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 isothiocyanate. In U.S. Patent No. 4,938,948, imaging of breast tumors has been achieved using monoclonal antibodies, and the detectable imaging moiety is attached to the antibody using a linker such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

[0120] In another aspect, derivatization of immunoglobulins is contemplated by selectively introducing sulfhydryl groups into the Fc region of the immunoglobulin using reaction conditions that do not alter the combining site of the antibody. 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, where the reporter or effector molecule is conjugated to a hydrocarbon residue in the Fc region, has also been disclosed in the literature (O’Shannessy et al, 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies that are currently in clinical evaluation.

[0121] VI. Immunoassay In yet a further aspect, there are immunoassay methods for binding to, purifying, removing, quantifying, and otherwise generally detecting PD-L1 or PD-L2 and their related antigens. By way of non-limiting example, some immunoassay methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot. In particular, competitive assays for the detection and quantification of PD-L1 and PD-L2 antibodies are provided. The procedures for various useful immunoassays are described in scientific literature such as, for example, Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). Generally, an immunoassay method includes obtaining a sample and contacting the sample with a first antibody according to the aspects described herein under conditions effective to permit the formation of an immune complex, if desired.

[0122] Contacting a biological sample selected under effective conditions and for a sufficient period of time to enable the formation of immune complexes (primary immune complexes) with an antibody generally involves simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibody to form immune complexes, i.e., bind to the 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 antibody specifically bound within the primary immune complex 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 a number of approaches. These methods generally rely on the detection of a label or marker, e.g., a radioactive tag, fluorescent tag, biological tag, and enzyme tag. Patents related 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 is known in the art, additional advantages can be found through the use of secondary binding ligands, e.g., the construction of secondary antibodies and / or biotin / avidin ligand binding.

[0124] The antibody used for detection may itself be linked to a detectable label, in which case, by simply detecting this label, it becomes possible to determine the amount of the primary immune complex in the composition. Alternatively, the first antibody bound within the primary immune complex may be detected by a second binding ligand having 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 sometimes be referred to as a "secondary" antibody. The primary immune complex is contacted with the labeled secondary binding ligand, or antibody, under conditions effective and for a period sufficient to allow formation of the secondary immune complex. Next, the secondary immune complex is generally washed to remove any non-specifically bound labeled secondary antibody or ligand, and then the label remaining in the secondary immune complex is detected.

[0125] A further method involves detection of the primary immune complex by 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 the secondary immune complex. After washing, the secondary immune complex is contacted with a third binding ligand or antibody having binding affinity for the second antibody under conditions effective and for a period sufficient to allow formation of an immune complex (tertiary immune complex). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complex thus formed. This system can provide signal amplification if desired.

[0126] One method of immunoassay 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 that has bound to the complexed biotin. In that method, the sample being tested is first incubated in a solution containing the antibody of the first step. If the target antigen is present, a portion of the antibody binds to the antigen to form a biotinylated antibody / antigen complex. Next, the antibody / antigen complex is amplified by incubation in a sequential solution of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with additional biotin moieties being added to the antibody / antigen complex at each step. The amplification step is repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the antibody of the second step against biotin. This second-step antibody is labeled with an enzyme, for example, that can be used to detect the presence of the antibody / antigen complex by tissue enzymology using a chromogenic substrate. When suitably amplified, a macroscopically visible conjugate can be produced.

[0127] Another known method of immunoassay utilizes the methodology of immuno-PCR (polymerase chain reaction). The PCR method is similar to Cantor's method up to incubation with biotinylated DNA, but instead of using multiple rounds of incubation with streptavidin and biotinylated DNA, the DNA / biotin / streptavidin / antibody complex is washed with a low pH or high salt buffer that releases the antibody. Next, the resulting wash solution is used to perform a PCR reaction with suitable primers along with appropriate controls. At least theoretically, the very large amplification capacity and specificity of PCR can be utilized to detect a single antigen molecule.

[0128] A. ELISA In the most basic sense, an immunoassay is a binding assay. Certain preferred immunoassays are the various types of enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily understood that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analysis, etc. may also be used.

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

[0130] In another exemplary ELISA, after immobilizing a sample suspected of containing the PD-L1 and / or PD-L2 antigen on the well surface, it is contacted with an anti-PD-L1 and anti-PD-L2 bispecific antibody. After washing to remove bound and 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 complex can be detected directly. Similarly here, the immune complex may be detected using a second antibody (the second antibody is 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 commonly have certain features, such as coating, incubation and binding, washing to remove non-specifically bound species, and detection of the bound immune complex. These are described below.

[0132] For coating of the plate with either an antigen or an antibody, generally, the wells of the plate are incubated with a solution of the antigen or antibody overnight or for a specified time. Next, the wells of the plate are washed to remove incompletely adsorbed material. Next, any remaining available surface of the wells is "coated" with a non-specific protein that is neutral with respect to the antigen for the test antiserum. These include solutions of bovine serum albumin (BSA), casein or skim milk. Coating enables blocking of non-specific adsorption sites on the immobilized surface, thus reducing the background caused by non-specific binding of the antiserum to the surface.

[0133] In ELISA, it is perhaps more conventional to use secondary or tertiary detection rather than direct procedures. Thus, after binding a protein or antibody to the well, coating with non-reactive material to reduce background, and washing to remove unbound material, the biological sample being 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 a secondary binding ligand or antibody combined 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 assist in reducing non-specific background.

[0135] "Suitable" conditions also mean that the incubation is carried out at a temperature or for a period sufficient to allow effective binding. The incubation step is typically carried out at a temperature of about 1 to 2 hours to about 4 hours, preferably 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. Preferred washing procedures include washing with a solution such as PBS / Tween or borate buffer. After formation of specific immune complexes between the test sample and the material originally bound, and subsequent washing, the presence of even trace amounts of immune complexes can be determined.

[0137] To provide a detection means, the second or third antibody has an associated label for enabling detection. Preferably, this is an enzyme that generates color when incubated with an appropriate chromogenic substrate. Thus, for example, urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase conjugated antibodies are contacted or incubated with the first and second immune complexes for a period and under conditions that advantageously act on the occurrence of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).

[0138] After incubation with the labeled antibody and subsequent washing to remove unbound material, the amount of the label is quantified, for example, by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), or in the case of peroxidase as an enzyme label, with H2O2. Next, quantification is achieved by measuring the degree of color development, for example, using a visible spectrum spectrophotometer.

[0139] B. Western Blot Western blot (or protein immunoblot) is an analytical technique used to detect a specific protein in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins either by the length of the polypeptide (denaturing conditions) or by the 3D structure of the protein (native / non-denaturing conditions). Next, the protein is 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 from 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. The cells may be disrupted by one of the above mechanical methods. However, it should be noted that bacteria, viruses or environmental samples can be a source of proteins and Western blotting is not restricted to cell research only. To facilitate cell lysis and solubilize proteins, a combination of surfactants, salts, and detergents may be used. Protease and phosphatase inhibitors are often added to prevent digestion of the sample by its own enzymes. Tissue preparation is often carried out at low temperature to avoid protein denaturation.

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

[0142] To make the 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 stacked filter papers are placed on top of that. When the entire stack is placed in a buffer solution, the buffer solution moves upward through the paper by capillary action, carrying the proteins with it. Another method of transferring proteins is called electroblotting, which uses an electric current to draw the proteins from the gel into a PVDF or nitrocellulose membrane. The proteins move 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 membranes are selected because of their non-specific protein binding properties (i.e., they bind equally to all proteins). Protein binding is based on charge interactions between the membrane and the protein, in addition to hydrophobic interactions. Nitrocellulose membranes are less expensive than PVDF membranes but are much more fragile and cannot withstand repeated probing 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, the proteins are detected using a labeled primary antibody or an indirect detection using an unlabeled primary antibody followed by a labeled protein A or secondary labeled antibody that binds to the Fc region of the primary antibody.

[0143] C. Immunohistochemistry Antibodies can also be used in combination with either fresh frozen tissue blocks and / or formalin-fixed, paraffin-embedded tissue blocks prepared for studies by immunohistochemistry (IHC). The methods for preparing tissue blocks from these particulate specimens have been used successfully in prior 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 stated, 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 freezing in isopentane at -70 °C, cutting the plastic capsule and / or removing the frozen cylindrical tissue, fixing the cylindrical tissue on a cryostat microtome chuck, and / or cutting 25 - 50 consecutive sections from the capsule. Alternatively, the entire frozen tissue sample may be used for cutting consecutive sections.

[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 for 4 hours of fixation, washing / pelleting, resuspending in warm 2.5% agar, pelleting, cooling with 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 consecutive permanent sections. Again, the entire tissue sample may be substituted.

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

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

[0148] Further suitable immunoassay reagents for use in the kits of the invention include a two-component reagent comprising a second antibody having binding affinity for a first antibody, along with a third antibody having binding affinity for the second antibody (the third antibody being linked to a detectable label). As noted above, numerous exemplary labels are known in the art and all such labels can be used in the context of the embodiments described herein.

[0149] The kit may further comprise a suitably aliquoted composition of PD-L1 antigen and PD-L2 antigen, which may be labeled or unlabeled, so as to be used for generating a standard curve for a detection assay. The kit may contain the antibody-label conjugate in a fully conjugated form, an intermediate form, or as separate moieties to be conjugated by the user of the kit. The components of the kit can be packaged either in an aqueous medium or in lyophilized form.

[0150] The container means of the kit generally includes at least one vial, test tube, flask, bottle, syringe, or other container means into which the antibody can be placed or preferably, suitably aliquoted. The kit also includes means for tightly sealing for commercial use, a container for containing the antibody, antigen, and any other reagents. Such containers can include injection-molded or blow-molded plastic containers that hold the desired vials therein.

Examples

[0151] VII. Examples The following examples are included to demonstrate preferred embodiments of the present invention. The techniques disclosed in the following examples present techniques that the inventors have discovered function well in the practice of the present invention and, accordingly, can be considered to constitute preferred modes for their practice, which should be understood by those skilled in the art. However, those skilled in the art should understand from this disclosure that many modifications can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention.

[0152] Example 1 - Materials and Methods Selection, Generation, and Production of Antibodies. Although additional details may be provided in subsequent examples, the selection, generation, and production of the disclosed antibodies were generally performed as follows.

[0153] Preparation of Antigens - 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] Naïve Discovery - Approximately 10 9Eight naive human synthetic yeast libraries, each having the diversity as described previously, were grown as described previously (see, e.g., Xu et al., 2013, WO2009036379, WO2010105256, and WO2012009568). For the first two rounds of selection, magnetic bead sorting technology using the Miltenyi MACS system was performed as described previously (see, e.g., Siegel et al., 2004). Briefly, yeast cells (about 10 10 cells / library) were incubated in wash buffer (phosphate-buffered saline (PBS) / 0.1% bovine serum albumin (BSA)) with 3 ml of 10 nM biotinylated Fc fusion-antigen 3 at 30 °C for 15 minutes. After washing once 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 this yeast and incubated at 4 °C for 15 minutes. Next, the yeast was 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. Thereafter, the column was removed from the magnetic field, the yeast was eluted with 5 mL of growth medium, and then grown overnight. Subsequent rounds of selection were performed using flow cytometry. About 2×10 7Individual yeasts were pelleted, washed three times with wash buffer, and incubated at 30 °C under equilibrium conditions with either 10 nM of the Fc-fusion antigen or, in subsequent rounds, with biotinylated antigen at decreasing concentrations (100 to 1 nM), or with 100 nM of biotinylated antigen from a different species (mouse) to obtain cross-reactivity, or with a multispecific depletion reagent (PSR) to exclude non-specific antibodies from the selection. In the case of PSR depletion, the library was incubated with a 1:10 dilution of the biotinylated PSR reagent as previously described (see, e.g., Xu et al., 2013). The yeasts were then washed twice with wash buffer and stained for 15 min at 4 °C with either LC-FITC (diluted 1:100) and SA-633 (diluted 1:500) or EAPE (diluted 1:50) secondary reagents. 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. The selection rounds were repeated until a population with all of the desired characteristics was obtained.

[0155] To generate antibodies with cross-reactivity, the above-described selections with each target antigen were performed independently and alternately round by round to enrich those with bispecificity. After the final round of sorting, the yeasts were plated, and individual colonies were picked for characterization.

[0156] Light chain batch shuffling (LCBS) - primary discovery also included a light chain batch diversification protocol from the heavy chain plasmid from naïve selection: the heavy chain plasmid from the naïve round 4 selection output was extracted from yeast and transformed into a light chain library with 5×10 6 diversity. Selection was performed by 1 round of MACS and 3 rounds of FACS using the same conditions as for naïve discovery.

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

[0158] Selection of CDRH1 and CDRH2 : 1×10 8 The CDRH3 of a single antibody was recombined into a pre-made library with CDRH1 and CDRH2 variants having a diversity of to Selection of CDRH1 and CDRH2 , and selection was performed by 1 round of MACS and 4 rounds of FACS as described in naïve discovery. In the FACS rounds, the library was examined for PSR binding, species cross-reactivity, antigen cross-reactivity, and affinity pressure, and sorting was performed to obtain a population with the desired characteristics. For these selections, affinity pressure was applied either by titrating down the biotinylated monomeric antigen or by pre-incubating the biotinylated antigen with the parental Fab for 30 minutes and then applying the pre-combined mixture to the yeast library over a period that allows the selection to reach equilibrium. Antibodies with higher affinity could then be sorted.

[0159] Selection of VH Mut : Mutations in the heavy chain variable region (VH) were induced by error-prone PCR. Next, the mutagenized VH and the heavy chain expression vector were transformed into yeast that already contained the parental light chain plasmid to create a library. Selection was performed as in the previous cycle using 3 rounds of FACS sorting. In the FACS rounds, the library was examined for cross-reactivity and affinity pressure, and sorting was performed to obtain a population with the desired characteristics.

[0160] Selection of CDRL1, CDRL2, and CDRL3 : The oligos were ordered from IDT, which included CDRL3 and were diversified by NNK diversity. The CDRL3 oligos were double-stranded using primers that anneal to the flanking regions of CDRL3. Next, these double-stranded CDRL3 oligos were to Selection of CDRL1, CDRL2, and CDRL3 with a diversity of 3×10 5Recombined into a pre-made library with CDRL1 and CDRL2 variants having diversity, and selection was performed by 1 round of MACS and 3 rounds of FACS as described in naïve discovery. In the FACS rounds, the library was examined for PSR binding, cross-reactivity, and affinity pressure, and sorting was performed to obtain a population with the desired characteristics. The affinity pressure for these selections was performed as described above for the selection of CDRH1 and CDRH2, and antigens were applied alternately for each round to enrich those with bispecificity.

[0161] Antibody production and purification - Yeast clones were grown to saturation and then induced at 30 °C for 48 hours with shaking. After induction, the 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 DMeasurement - ForteBio affinity measurements were generally performed on an Octet RED384 as previously described (see, e.g., Estep et al., 2013). Briefly, ForteBio affinity measurements were performed by loading IgG onto AHQ sensors online. The sensors were equilibrated offline in assay buffer for 30 minutes and then monitored online for 60 seconds to establish a baseline. The sensors with loaded IgG were exposed to 100 nM antigen for 3 minutes and then transferred to assay buffer for 3 minutes for measurement of the dissociation rate. Fab was used instead of IgG for monovalent affinity evaluation. For this evaluation, a non-biotinylated Fc fusion antigen was loaded onto AHQ sensors online. The sensors were equilibrated offline in assay buffer for 30 minutes and then monitored online for 60 seconds to establish a baseline. The sensors with loaded antigen were exposed to 100 nM Fab for 3 minutes and then transferred to assay buffer for 3 minutes for measurement of 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 AHQ sensors and the unoccupied Fc binding sites on the sensors were blocked with an irrelevant human IgG1 antibody. The sensors were then exposed to 100 nM target antigen, followed by a second anti-target antibody or ligand. Additional binding by the second antibody or ligand after antigen association indicates unoccupied epitopes (non-competitor), and no binding indicates epitope blocking (competitor or ligand blocking).

[0164] MSD-SET K DMeasurement - Equilibrium affinity measurements of the selected high-affinity antibodies were generally performed as previously described (Estep et al., 2013). Briefly, solution equilibrium titration (SET) was performed in PBS + 0.1% IgG-free BSA (PBSF) containing antigen maintained at 50 pM and incubated with 3-5-fold 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 while shaking at 700 rpm, followed by washing three times with wash buffer (PBSF + 0.05% Tween 20). SET samples were applied and incubated on the plate for 150 seconds while shaking at 700 rpm, followed by one wash. Antigen captured on the plate was detected by incubating on the plate for 3 minutes with 250 ng / mL of sulfo-tag-labeled streptavidin in PBSF. The plates were washed three times with wash buffer and then read on an MSD Sector Imager 2400 instrument using 1× Read Buffer T with surfactant. The percentage of free antigen (%) was plotted as a function of the titrated antibody in Prism and fit to a quadratic equation to extract K D . To improve throughput, a liquid handling robot was used throughout the MSD-SET experiment, including SET sample preparation.

[0165] Cell binding assay - 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 above-presented methods were tested for their ability to bind to PD-1 and PD-L2 and block their binding to PD-1. Antibodies at 5 μg / mL were bound to CHO-PD-L1 cells or CHO-PD-L2 cells, and then recombinant PD-1 (RnD Systems) labeled with Alexa 532 (ThermoFisher) was added for 1 hour. The maximum fluorescence intensity of PD-1 was measured. Blockade of PD-1 binding was measured by reduction of Alexa 532 fluorescence by flow cytometry. The affinity K D To generate, 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 protein were tested in a dilution series from 30 to 0.37 nM. The binding and release of the analyte were recorded in real time by an Octet instrument and then used to calculate K d , K on , and K dis . The results are derived from 2:1 global fit modeling with subtraction of the reference well. The affinity K D To generate for mouse PD-L1 or PD-L2, BiPDL Ab was covalently immobilized at 100 nM (15 μg / mL) on an activated Amine Reactive 2nd Generation (AR2G) biosensor (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 were tested in a dilution series from 300 to 1 nM. The binding and release of the analyte were recorded in real time by an Octet instrument and then used to calculate K d , K on , and K dis . The results are derived from 2:1 global fit modeling with subtraction of the reference well.

[0167] Antibody activity. BiPDL antibodies with a human IgG1 backbone at various concentrations were added to CHO cells (CHO-PD-L1 cells, CHO-PD-L2 cells) expressing either human or mouse PD-L1 or PD-L2. 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 the fluorescence activity at various antibody concentrations. The EC50 was calculated using GraphPad Prism® software.

[0168] The candidate antibody prevents the binding of PD-1 / PD-L2. The candidate BiPDL antibody and an FDA-approved antibody were assayed using the Promega PD-L1 / PD-L2 dual-expression: PD-1 blockade system. Antibodies at various concentrations were added to CHO-PD-L1 / L2 cells. The PD-1 effector cells were Jurkat T cells, which can be stimulated by CHO-PD-L1 / L2 cells. The PD-1 effector cells that produce firefly luciferase in response to activation were incubated with the antibody and CHO cells for 6 hours, and then the results were read on a luminometer using the Bio-Glo TM assay kit (Promega) according to the manufacturer's instructions. For the competitive assay, biotin-rhPD-1-Fc protein was added to the cells and the antibody, followed by the addition of streptavidin-APC conjugate. Blockade was evaluated as an increase in the luciferase signal. The analysis was performed using GraphPad Prism® software. For the competitive assay, X was transformed to LogX and analyzed by non-linear regression (curve fit), dose-response inhibition, and Log(inhibitor) vs. response.

[0169] BiPDL activity in the 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 a commercial antibody were added. Next, CD4+ T cells were stimulated using 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. In immunodeficient mice, U2940 PMBL or MDA-MB-231 triple-negative breast cancer xenograft tumors were established. Tumors were allowed to reach a volume of 150 mm 3 Once the tumors reached 150 mm 3 After reaching 150 mm

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

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

[0173] Epitope binding of BiPDL antibodies. Binding competition assays performed using the ForteBio Octet® platform were used to compare the binding specificities of various BiPDL antibodies. Target His-tagged protein (human PD-L1 or PD-L2) was loaded onto nickel NTA biosensors pre-charged at 1 μg / mL. The first antibody, Ab1, was saturated at 100 nM on the biosensor loaded with the target, and reference (buffer only) wells were 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. A matrix of raw signal responses of Ab2 binding was generated using Data Analysis HT 9.0 software and then converted to represent the percentage of non-blocked Ab2 binding signal. A response of less than 15% was considered competitive blocking.

[0174] Example 2 - Results Selection of BiPDL antibodies that bind to both human PD-L1 and PD-L2. Considering that both PD-L1 and PD-L2 bind to PD-1 and share approximately 40% amino acid identity, a hypothesis was established that it would be possible to obtain antibodies that bind to both ligands and prevent their binding to the T cell co-inhibitory receptor PD-1. Since the discovery of high-affinity antibodies capable of bivalent binding to both ligands was expected to be extremely rare, these antibodies were discovered through an iterative selection process from a yeast-based fully human antibody library presentation system as described above. Using a recombinant ligand fused to a dimerized (i.e., tightly binding) human IgG1 Fc constant region, antibodies that bind to both PD-L1 and PD-L2 were first selected as described in Example 1 above, and then the highest affinity hits were further enriched through selection of those that could bind to the monomeric PD ligand. This initial round of screening yielded four distinct series of antibodies that bind to both PD-L1 and PD-L2 and can block their binding to PD-1 (Figures 1A - B). The antibodies were named BiPDL, with X representing the BiPDL family (Boussiotis, 2016; Cheng et al., 2013; Latchman et al., 2001; Lee et al., 2016), Y representing the clone number within that family in a given generation, and Z representing the generation (Boussiotis, 2016; Cheng et al., 2013; Latchman et al., 2001; Lee et al., 2016) of a series of rounds of affinity maturation, using the nomenclature BiPDL X-Y Z was used. Individual antibody clones were also referenced by their experimental clone numbers. Table 5 provides both the BiPDL name and each respective clone number. Each of these antibodies showed moderately high avidity (K d ≤ 2×10 -9 ) for PD-L2 (using the dimeric ligand), but only BiPDL4 had a quantifiable avidity (K d ≤ 1×10 -9) and several antibodies were further tested for their ability to relieve the inhibition of Jurkat T cells by PD-1 using the Promega PD-1 assay system over a wide range of antibody concentrations. It was found that only BiPDL4 was able to block PD-L1-induced T cell suppression (Figure 1C).

[0175] Affinity maturation enhances the functional ability of BiPDL to reverse the inhibition of T cell activation by PD-L1 and PD-L2. Each of the first-generation leads, BiPDL1-11, BiPDL2-11, BiPDL3-11, and BiPDL4-11, was subjected to heavy chain affinity maturation to enrich 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. The 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 of the BiPDL3 and BiPDL4 antibodies retained the ability to bind to PD-L1 (Figures 2A and 2C), but BiPDL3-12 showed little PD-L2 binding activity (Figure 2B).

[0176] Promega PD-L1 / PD-L2 Dual Expression: Using the PD-1 assay system, the ability of the fourth-generation BiPDL4 clones to restore Jurkat T cell activity was evaluated (Figure 3). BiPDL4-11, -14, -24, -34, -44 all showed activity levels comparable to those of Keytruda (anti-PD1) and exceeded those of atezolizumab and avelumab. The binding of other independent families was also evaluated (Figures 4A - F). The ability of BiPDL4-12 to reverse the inhibition of Jurkat T cells in the Promega system was enhanced for both PD-L1 and PD-L2 compared to the parental BiPDL4, while BiPDL4-22 did not acquire any ability to reverse PD-L1-mediated inhibition but was able to completely reverse PD-L2-mediated suppression (Figures 4E - F). The Octet affinity measurements of BiPDL3-12 were K d = 2.71×10 -8 for PD-L1 and K d = 8.85×10 -8 (dimeric ligand) for PD-L2 (Table 5). BiPDL4-12 was measured to be 6.22×10 -10 for PD-L1 and 1.74×10 -9 for PD-L2, while BiPDL4-22 was measured to be 8.42×10 -9 for PD-L1 and 3.5×10 -10 for PD-L2 (Table 5).

[0177] Despite the improvements from the first-generation BiPDL, neither BiPDL3-12, BiPDL4-12, nor BiPDL4-22 showed clinically significant affinity for both PD-L1 and PD-L2. For this reason, the third round of heavy-chain affinity maturation was focused on BiPDL3-12 and BiPDL4-22. This process resulted in third-generation BiPDL antibodies with significantly enhanced affinity for both ligands. The K d of the Octet affinity measurement for BiPDL3-13 was 3.28×10 -9 for PD-L1 and 4.31×10 -10(The dimer ligand). BiPDL4-13 was 6.9×10 for PD-L1 -10 and 4.9×10 for PD-L2 -10 while BiPDL4-23 was 1.12×10 for PD-L1 -9 and 3.4×10 for PD-L2 -10 as measured. BiPDL4-13 also showed measurable affinity for 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 d =8.4×10 -8 for PD-L1 and K d =1.66×10 -9 for PD-L2. All third-generation leads were able to potently reverse the Jurkat T cell inhibition resulting from binding to PD-L2, while the BiPDL4 antibodies showed a substantially higher ability to reverse the inhibition induced by PD-L1 (Figure 2C).

[0178] In an attempt to improve the PD-L1 affinity of BiPDL4-13 and BiPDL4-23, the final round of light chain affinity maturation was performed as described in Example 1 above. This screening yielded a number of BiPDLs with clinically significant affinity for both ligands, among which the major ones were BiPDL4-14, BiPDL4-24, BiPDL4-34, and BiPDL4-44. The affinity of these antibodies for HIS-tagged monovalent PD-L1 measured by Octet was 2.26 - 7.9×10 for PD-L1 -9 and 4.98 - 9.3×10 for PD-L2 -10reached. When 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, the performance of these fourth-generation BiPDL4 antibodies was almost equivalent to Keytruda (Figure 2D). Furthermore, these fully mature BiPDL4 showed significant superiority over both earlier-generation BiPDL4 and the FDA-approved PD-L1 antibodies atezolizumab and avelumab.

[0179] Fourth-generation BiPDL4 binds to an epitope on PD-L1 that has not been previously reported. These fourth-generation BiPDL4 antibodies bind with high affinity to both human and cynomolgus monkey PD-L1 and PD-L2 and also have significant cross-reactivity with mouse ligands (Tables 5 and 6). All four fourth-generation BiPDL4 antibodies share the same heavy-chain CDR3 but are significantly different in other heavy-chain and light-chain CDR sequences (Tables 1-4). As a note, there is no significant similarity to any of the FDA-approved PD-L1 antibodies. This lack of sequence homology suggests the possibility of unique binding specificities for these PD-L1 and PD-L2 binding antibodies, and thus, they were "binned" against known PD-L1 and PD-L2 antibodies to determine epitope binding by comparison.

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

[0181] The BiPDL antibody restores effector cytokine production in the primary human mixed lymphocyte reaction (MLR). Candidate antibodies, FDA-approved antibodies, or control antibodies were evaluated in the presence of dendritic cells and T cells derived from separate donors, and evaluated for IL-2 or IFN-γ production by ELISA. Expression of PD-L1 and PD-L2 by iDCs was confirmed by flow cytometry (Figure 5A). Both BiPDL4-14 and BiPDL4-34 showed an ability to restore IFN-γ secretion by human primary T cells that was approximately equivalent compared to 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 dominant in this context.

[0182] The BiPDL antibody with effector function mediates 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 medium levels of PD-L2 (approximately 7,000 molecules per cell). Mouse NK cells were expanded in vitro and incubated for 4 hours at an effector-to-target ratio of 15:1 with calcein-AM (ThermoFisher)-labeled U9240 PMBL target cells and the indicated concentrations of antibody. Specific lysis rate (%) 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 the killing of U2940.

[0183] Effector-capable BiPDL controls the growth of U2940 PBML and MDA-MB-231 tumor xenografts in SCID mice. 1×10 6 U2940 PMBL cells were transplanted into SCID mice, and the tumors reached 150 mm 3It was established until it reached. Cells were evaluated for the expression of PD-L1 and PD-L2 (Figure 7A). Next, mice were treated twice a week with either 10 mg / kg of mIgG2a control antibody, Herceptin, Rituxan (U2940 is CD20+), BiPDL4-12-mIgG2a, or BiPDL4-22-mIgG2a. Both BiPDL4 antibodies significantly delayed tumor growth compared to the control mAb and exceeded Rituxan (Figure 7B). When the same experiment was repeated using BiPDL3-13-mIgG2a and BiPDL4-13-mIgG2a, a significant therapeutic benefit of both BiPDL antibodies compared to the control was demonstrated again (Figure 7C). In this case, neither antibody significantly exceeded Rituxan, but only two mice with no tumors remaining (tumor volume ≤ 20 mm 3 ) at the end of the study 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 a similar rate to U2940 (Figure 8A). 1×10 7 MDA-MB-231 TNBC cells were transplanted into SCID mice and established until the tumors reached 150 mm 3 . Next, mice were treated twice a week with either 10 mg / kg of 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 situation, the BiPDL antibody delayed the progression of MDA-MB-231 xenografts with an ability almost equivalent to that of Avelumab, a PD-L1 antibody capable of inducing ADCC (Figure 8B). This similarity is not surprising considering that the expression of PD-L1 by MDA-MB-231 is higher than that of PD-L2.

[0185] BiPDL treats syngeneic MC38-PD-L2 colon cancer more effectively than any PD-L1 antibody. MC38 colon cancer cells that endogenously express PD-L1 were engineered with a retrovirus to express murine PD-L2. Expression of both PD-L1 and PD-L2 was confirmed by flow cytometry (Figure 9C). 5×10 5 cells of MC38-PD-L2 were transplanted into C57BL / 6J mice. On days 3, 6, 9, 12, and 15, 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] was injected into the mice. Survival when using the BiPDL4 antibody was superior to that of 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 yields the most favorable intratumoral CD8 to Treg ratio in MC38-PD-L2. To facilitate the recovery of infiltrating lymphocytes, 1.5×10 6 cells of MC38-PD-L2 were transplanted into C57BL / 6J mice in 30% Matrigel (Corning). Expression of PD-L1 and PD-L2 was confirmed by flow cytometry (Figure 10A). Mice were treated as above except on days 7, 10, and 13, and subsequently tumors were 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 mean CD8:Treg ratio of mice treated with BiPDL4-14-mIgG2a was 10, significantly higher than that of any of the 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. Similarly, EL4 T cell lymphoma cells that 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 5 cells of EL4-PD-L2 that also express luciferase to facilitate bioluminescence imaging were transplanted to establish systemic disease in C57BL / 6J mice. On days 3, 6, 9, 12, and 15, mice were injected with either PBS, rat anti-mouse PD-L1 antibody 10F.9G2, rat anti-mouse PD-L2 antibody TY25, a combination of 10F.9G2 and TY25, FDA-approved PD-L1 antibody atezolizumab, or 100 μg of BiPDL4-14-mIgG2a. Blockade of PD-L1 and PD-L2 was not effective in this model, but the BiPDL4 antibody significantly prolonged survival (p = 0.002 vs untreated; p = 0.01 vs. atezolizumab) (Figure 11B). In this context, the combination of a PD-L1 blocking antibody and a PD-L2 blocking antibody (10F.9G2 and TY25) was not effective, so the effector function of the BiPDL antibody appears to be particularly important for efficacy.

[0188] (Table 1) Nucleic acid sequences of antibody variable regions TIFF0007713995000004.tif211148TIFF0007713995000005.tif201144

[0189] (Table 2) Protein sequences of antibody variable regions TIFF0007713995000006.tif219149TIFF0007713995000007.tif123144

[0190] (Table 3) Heavy chain CDR sequences TIFF0007713995000008.tif111145

[0191] (Table 4) Light chain CDR sequences TIFF0007713995000009.tif110130

[0192] (Table 5) BiPDL Nomenclature TIFF0007713995000010.tif100128

[0193] (Table 5) Affinity Measurement Values of Antibodies that Bind to PD-L1 and PD-L2 TIFF0007713995000011.tif45155

[0194] (Table 6) Affinity Measurement Values of Antibodies that Bind to PD-L1 and PD-L2 TIFF0007713995000012.tif24170

[0195] (Table 7) Determination of Antibody Duplication by Binning TIFF0007713995000013.tif41152

[0196] (Table 8) Determination of Antibody Duplication by Binning TIFF0007713995000014.tif41152

[0197] (Table 9) Antibody Binding and Competition TIFF0007713995000015.tif35156

[0198] All of the methods disclosed and claimed in this specification can be constructed and implemented without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in relation to preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods and processes described herein, or to the order of the process steps, without departing from the concepts, spirit, and scope of the present invention. More specifically, certain chemically and physiologically related agents may be used in place of the agents described herein, and it will be apparent that they may achieve the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

[0199] VIII. References The following references are specifically incorporated herein by reference to the extent that they provide exemplary procedures or other detailed supplements to those described herein. TIFF0007713995000016.tif196150TIFF0007713995000017.tif230142TIFF0007713995000018.tif230130TIFF0007713995000019.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 gcagggaaga 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. Use of an antibody or antibody fragment in the manufacture of a medicament for the treatment of cancer cells that are solid tumor cells and overexpress PD-L1 and / or PD-L2, wherein the antibody or antibody fragment is (a) a heavy chain CDR1 sequence of SEQ ID NO: 33, a heavy chain CDR2 sequence of SEQ ID NO: 34, and a heavy chain CDR3 sequence of SEQ ID NO: 35, and a light chain CDR1 sequence of SEQ ID NO: 51, a light chain CDR2 sequence of SEQ ID NO: 52, and a light chain CDR3 sequence of SEQ ID NO: 53, or (b) a heavy chain CDR1 sequence of SEQ ID NO: 72, a heavy chain CDR2 sequence of SEQ ID NO: 73, and a heavy chain CDR3 sequence of SEQ ID NO: 74, and a light chain CDR1 sequence of SEQ ID NO: 87, a light chain CDR2 sequence of SEQ ID NO: 88, and a light chain CDR3 sequence of SEQ ID NO: 89 and specifically binds to PD-L1 and PD-L2 Use.

2. The use according to claim 1, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence shown in SEQ ID NO: 8 and 7, respectively.

3. The use according to claim 1, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence having at least 90% identity to SEQ ID NO: 8 and 7, respectively.

4. The use according to claim 1, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence having at least 95% identity to SEQ ID NO: 8 and 7, respectively.

5. The use according to claim 1, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence shown in SEQ ID NO: 20 and 19, respectively.

6. The use according to claim 1, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence having 90% identity to SEQ ID NO: 20 and 19, respectively.

7. The use according to claim 1, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence having 95% identity to SEQ ID NO: 20 and 19, respectively.

8. The use according to claim 1, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence shown in SEQ ID NO: 64 and SEQ ID NO: 63, respectively.

9. The use according to claim 1, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence having 90% identity to each of SEQ ID NO: 64 and SEQ ID NO:

63.

10. The use according to claim 1, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence having 95% identity to each of SEQ ID NO: 64 and SEQ ID NO:

63.

11. The antibody fragment is a recombinant scFv (single-chain variable region fragment) antibody, Fab fragment, F(ab’) 2 fragment, or Fv fragment, and the use according to any one of claims 1 to 10

12. The use according to any one of claims 1 to 10, wherein the antibody is a chimeric antibody.

13. The use according to any one of claims 1 to 10, wherein the antibody is IgG.

14. The use according to any one of claims 1 to 10, wherein the antibody or antibody fragment further comprises a cell-penetrating peptide and / or is an intrabody.

15. The use according to any one of claims 1 to 10, wherein the antibody or antibody fragment is a humanized antibody.

Images