Human PD-L2 antibodies and methods of use thereof

Abstract

Human PD-L2 antibodies and methods of use thereof. The present disclosure relates to antibodies that bind to PD-L2 and methods of using such antibodies to treat cancer, such as those that express or overexpress PD-L2.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201980021120.0 and invention name “Human PD-L2 Antibodies and Methods of Using Them”. The parent application is the PCT international patent application PCT / US2019 / 022444 filed on March 15, 2019, which entered the Chinese national phase.

[0002] Priority claim

[0003] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 647,546, filed on March 23, 2018, the entire contents of which are hereby incorporated by reference.

[0004] Incorporation of Sequence Listing

[0005] Contained in 22.9KB (as in Microsoft The sequence listing in a file named "UTFC.P1340WO_ST25.txt" created on March 13, 2019, and is submitted herewith by electronic submission, and is incorporated herein by reference. Background of the Invention 1. Technical Field

[0006] The present disclosure relates generally to the fields of medicine, oncology and immunology. More particularly, it relates to human antibodies that bind to PD-L2 and their use in cancer treatment. 2. Background technology

[0007] Programmed death-1 (PD-1) is a cell surface molecule expressed on B cells and T cells that regulates adaptive immune responses. The PD-1 receptor on T cells is expressed after T cell activation, accumulates on the cell surface over time, and can participate in attenuating T cell responses as a mechanism of homeostatic regulation. Engagement of PD-1 by its ligands PD-L1 or PD-L2 transduces signals that inhibit T cell proliferation, cytokine production, and cytolytic function, marking an important checkpoint for cell death.

[0008] Normal cells regulate PD-L1 expression very strictly, and it is rarely expressed in normal tissues, but can be rapidly upregulated in a variety of different tissue types and by tumors in response to interferon-γ and other inflammatory mediators (Donget al., 2002). PD-L2 expression is highly restricted to antigen presenting cells, including dendritic cells and macrophages (Rozali et al., 2012). Exposure of dendritic cells and macrophages to Th2 cytokines, IFNγ and Toll-like receptor ligands both increase PD-L2 expression. It has been shown that human fibroblasts express PD-L2, resulting in T cell suppression (Pinchuk et al., 2008). In addition, not only normal fibroblasts, but also cancer-associated fibroblasts constitutively express PD-L2 (Nazareth et al., 2007).

[0009] It is well known that tumors can co-opt normal physiological checkpoints for immune regulation, resulting in an imbalance between tumor growth and host surveillance. As tumors grow, they surround themselves with stromal cells that express PD-1 ligands (i.e., PD-L1 and PD-L2). When PD-1-expressing T cells encounter PD-L1 and PD-L2 after entering the tumor microenvironment, they rapidly weaken and the tumor escapes immune control. Antibodies that block the PD-1 / PD-L1 binding interface have been in clinical trials since 2010 (Brahmer et al., 2010). Companion studies for PD-L1 antibodies have been ongoing since 2012 (Brahmer et al., 2012). However, to date, there are no clinical trials targeting PD-L2 antibodies. These interactions make the PD-L2 / PD-1 interface an attractive target for therapeutic intervention. Summary of the invention

[0010] Therefore, according to the present disclosure, antibodies or antibody fragments comprising heavy chain CDR sequences and light chain CDR sequences of clone pairs from Tables 3 and 4, respectively, are provided, which selectively bind to PD-L2. The antibodies or antibody fragments may be encoded by the variable sequences of the clone pairs shown in Table 1, may be encoded by light chain variable sequences and heavy chain variable sequences having 70%, 80% or 90% identity with the variable sequences of the clone pairs shown in Table 1, or may be encoded by light chain variable sequences and heavy chain variable sequences having 95% or greater identity with the clone pair sequences shown in Table 1. The antibodies or antibody fragments may comprise light chain variable sequences and heavy chain variable sequences according to the clone pair sequences from Table 2, may comprise light chain variable sequences and heavy chain variable sequences having 70%, 80% or 90% identity with the clone pair sequences from Table 2, or may comprise light chain variable sequences and heavy chain variable sequences having 95% or greater identity with the clone pair sequences from Table 2.

[0011] Also provided is a method of treating cancer in a subject, comprising contacting a PD-L2-positive cancer cell in the subject with an antibody as described above. The PD-L2-positive cancer cell can be a solid tumor cell, such as a lung cancer cell, a brain cancer cell, a head and neck cancer cell, a breast cancer cell, a skin cancer cell, a liver cancer cell, a pancreatic cancer cell, a stomach cancer cell, a colon cancer cell, a rectal cancer cell, a uterine cancer cell, a cervical cancer cell, an ovarian cancer cell, a testicular cancer cell, a skin cancer cell, an esophageal cancer cell, a lymphoma cell, a renal cell cancer cell, or can be a leukemia or a myeloma, such as acute myeloid leukemia, chronic myeloid leukemia, or multiple myeloma.

[0012] The method may also include contacting the PD-L2 positive cancer cells with a second anticancer agent or anticancer treatment, such as chemotherapy, radiation therapy, immunotherapy, hormone therapy, or toxin therapy. The second anticancer agent or anticancer treatment may inhibit intracellular PD-L2 function. The second anticancer agent or anticancer treatment may be administered simultaneously with the first agent, or before and / or after the first agent. The PD-L2 positive cancer cells may be metastatic cancer cells, multidrug resistant cancer cells, or recurrent cancer cells.

[0013] The antibody can be a single-chain antibody, a single-domain antibody, a chimeric antibody or a Fab fragment. The antibody can be a human antibody, a mouse antibody, an IgG, a humanized antibody or a humanized IgG. The antibody or antibody fragment can also contain a label, such as a peptide tag, an enzyme, a magnetic particle, a chromophore, a fluorescent molecule, a chemiluminescent molecule or a dye. The antibody or antibody fragment can also contain an antitumor drug connected thereto, such as an antitumor drug connected to the antibody or antibody fragment by a photolabile linker or an enzymatically cleavable linker. The antitumor drug can be a toxin, a radioisotope, a cytokine or an enzyme. The antibody or antibody fragment can be conjugated to a nanoparticle or a liposome.

[0014] In another embodiment, a method for treating cancer in a subject is provided, comprising delivering to the subject an antibody or antibody fragment having a heavy chain CDR sequence and a light chain CDR sequence of a cloned pair from Table 3 and Table 4, respectively. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab')2 fragment, or a Fv fragment. The antibody may be an IgG. The antibody may be a chimeric antibody. Delivery may include administration of the antibody or antibody fragment, or genetic delivery using an RNA or DNA sequence or vector encoding the antibody or antibody fragment.

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

[0016] Also provided are monoclonal antibodies, wherein the antibody or antibody fragment is characterized by a heavy chain CDR sequence and a light chain CDR sequence of a clone pair from Table 3 and Table 4, respectively. The antibody fragment can be a recombinant scFv (single chain variable fragment) antibody, a Fab fragment, a F(ab')2 fragment, or a Fv fragment. The antibody can be a chimeric antibody or an IgG.

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

[0018] In another embodiment, a hybridoma or modified cell encoding an antibody or antibody fragment is provided, wherein the antibody or antibody fragment is characterized by a heavy chain CDR sequence and a light chain CDR sequence of a clone pair from Table 3 and Table 4, respectively. The antibody fragment can be a recombinant scFv (single chain variable fragment) antibody, a Fab fragment, a F(ab')2 fragment, or a Fv fragment. The antibody can be a chimeric antibody or an IgG.

[0019] The antibody or antibody fragment may be encoded by a light chain variable sequence and a heavy chain variable sequence of a clone pair shown in Table 1, may be encoded by a light chain variable sequence and a heavy chain variable sequence having 95% identity to a clone pair shown in Table 1, and may be encoded by a light chain variable sequence and a heavy chain variable sequence having 70%, 80% or 90% identity to the clone pair sequence from Table 1. The antibody or antibody fragment may comprise a light chain variable sequence and a heavy chain variable sequence according to a clone pair sequence from Table 2, may comprise a light chain variable sequence and a heavy chain variable sequence having 70%, 80% or 90% identity to a clone pair sequence from Table 2, or may comprise a light chain variable sequence and a heavy chain variable sequence having 95% identity to a clone pair sequence from Table 2.

[0020] Another embodiment includes a cancer vaccine containing one or more antibodies or antibody fragments characterized by heavy chain CDR sequences and light chain CDR sequences of cloned pairs from Tables 3 and 4, respectively. At least one antibody fragment can be a recombinant scFv (single chain variable fragment) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. At least one antibody can be a chimeric antibody or an IgG. At least one antibody or antibody fragment can be encoded by a cloned pair of light chain variable sequences and heavy chain variable sequences shown in Table 1, can be encoded by a cloned pair of light chain variable sequences and heavy chain variable sequences with 95% identity as shown in Table 1, and can be encoded by a light chain variable sequence and a heavy chain variable sequence with 70%, 80% or 90% identity with the cloned pair sequences from Table 1. At least one antibody or antibody fragment may comprise a light chain variable sequence and a heavy chain variable sequence according to a clone pairing sequence from Table 2, may comprise a light chain variable sequence and a heavy chain variable sequence that are 70%, 80% or 90% identical to a clone pairing sequence from Table 2, or may comprise a light chain variable sequence and a heavy chain variable sequence that are 95% identical to a clone pairing sequence from Table 2.

[0021] In another embodiment, a method for detecting cells expressing PD-L2 in a subject is provided, comprising: contacting a sample from the subject with an antibody or antibody fragment characterized by heavy chain CDR sequences and light chain CDR sequences of clone pairs from Tables 3 and 4, respectively, and detecting cells expressing PD-L1 or PD-L2 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 cell may be a cancer cell, such as a lymphoma cell, a breast cancer cell, or a renal cell carcinoma cell. The cell may be a cell associated with immunosuppression. Cells associated with immunosuppression may be non-cancerous cells in a tumor microenvironment, such as stromal cells or endothelial cells. Detection may include ELISA, RIA, or Western blotting. The method may also include performing the method again and determining changes in orthopoxyvirus antigen levels compared to the first determination. The antibody or antibody fragment may be encoded by a light chain variable sequence and a heavy chain variable sequence of a clone pair shown in Table 1, may be encoded by a light chain variable sequence and a heavy chain variable sequence having 95% identity to a clone pair shown in Table 1, and may be encoded by a light chain variable sequence and a heavy chain variable sequence having 70%, 80% or 90% identity to the clone pair sequence from Table 1. The antibody or antibody fragment may comprise a light chain variable sequence and a heavy chain variable sequence according to a clone pair sequence from Table 2, may comprise a light chain variable sequence and a heavy chain variable sequence having 70%, 80% or 90% identity to a clone pair sequence from Table 2, or may comprise a light chain variable sequence and a heavy chain variable sequence having 95% identity to a clone pair sequence from Table 2.

[0022] Also provided is a method for treating immunosuppression in a tumor microenvironment, comprising: delivering to the subject an antibody or antibody fragment having a heavy chain CDR sequence and a light chain CDR sequence of a cloned pair from Table 3 and Table 4, respectively. The antibody or antibody fragment may be encoded by a light chain variable sequence and a heavy chain variable sequence of a cloned pair shown in Table 1, may be encoded by a light chain variable sequence and a heavy chain variable sequence of a cloned pair having 95% identity to a cloned pair shown in Table 1, and may be encoded by a light chain variable sequence and a heavy chain variable sequence having 70%, 80% or 90% identity to a cloned pair sequence from Table 1. The antibody or antibody fragment may comprise a light chain variable sequence and a heavy chain variable sequence according to a cloned pair sequence from Table 2, may comprise a light chain variable sequence and a heavy chain variable sequence having 70%, 80% or 90% identity to a cloned pair sequence from Table 2, or may comprise a light chain variable sequence and a heavy chain variable sequence having 95% identity to a cloned pair sequence from Table 2.

[0023] It is contemplated that any method or composition described herein may be implemented relative to any other method or composition described herein. Other objects, features and advantages of the present disclosure will be apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, although indicating some specific embodiments of the present disclosure, are given only by way of illustration, because, according to the detailed description, various changes and modifications within the spirit and scope of the present disclosure will be apparent to those skilled in the art.

[0024] As used herein, "substantially free" is used herein with respect to a specified component to mean that the specified component is not purposefully formulated into the composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component caused by any accidental contamination of the composition is preferably less than 0.01%. Most preferred are compositions in which the amount of the specified component cannot be detected by standard analytical methods.

[0025] As used herein in the specification and claims, a noun without a quantifier may mean one or more. As used herein in the specification and claims, when used in conjunction with the word "comprises", a noun without a quantifier may mean one or more than one. As used herein, in the specification and claims, "another" or "another" may mean at least a second or more.

[0026] As used herein in the specification and claims, the term "about" is used to indicate that a value includes the inherent variation of error for the device or method being employed to determine the value or the variation that exists between study subjects.

[0027] Through the following detailed description, other purposes, features and advantages of the present invention will be apparent. However, it should be understood that the detailed description and specific examples, although indicating some embodiments of the present invention, are given by way of illustration only, because according to the detailed description, various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following drawings constitute part of this specification and are included to further illustrate certain aspects of the present invention. The present invention may be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0029] Figures 1A to 1B: Identification of PD-L2 antibodies that block PD-L2 binding to PD-1. The antibody candidates identified as described in the examples were tested for their ability to bind to PD-L2 and block its binding to PD-1. The maximum fluorescence intensity of Alexafluor 532-labeled PD-1 was measured, where PD-L2 blocking was seen as a decrease in Alexa532 fluorescence in FACS analysis. Anti-PD-L2 clones 16501, 16425, 16510, and 16478 were evaluated against unstained cells, control antibodies, and commercially available anti-PD-L2 10C12 antibodies.

[0030] Figure 2 - Identification of PD-L2 antibody subclones that bind to PD-L2. Subclones of anti-PD-L2 antibodies (A) 16501 or (C) 16425 were tested using the PD-L2:PD-1 assay using CHO-PD-L2 cells that are able to stimulate Jurkat T cells to produce luciferase in response to activation. Antibodies were added at the indicated concentrations. Reactions were performed in ForteBio The affinity and IC of anti-PD-L2 antibodies (C) 16501 and (D) 16425 were calculated based on the curves. 50 value.

[0031] Figure 3 - Evaluation of PD-L2 antibodies compared to Keytruda. (Left) Cell surface binding and blocking of anti-PD-L2 clone 16501 and subclone 20810 were evaluated by detecting Alexafluor intensity of PD-1 cells. (Right) The ability of anti-PD-L2 clone 1650 and subclone 20810 to block PD-L2:PD-1 binding was evaluated in addition to clone 20237, Keytruda, and isotype controls in a commercial PD-L2:PD-1 binding assay. Antibodies were used at the concentrations listed.

[0032] Figures 4A to 4C -PD-L2 antibody binds to human PD-L2 with affinity. Figure 4A )Using ForteBio Detect the maximum fluorescence intensity of anti-PD-L2 subclones 16501, 20810 and 20811 binding to human PD-L2 protein. Figure 4B ) Anti-PD-L2 subclones 16425 and 20816 were tested for binding to human PD-L2 protein and the maximum fluorescence intensity was detected. Generate an affinity curve. Figure 4C ) Blockade of PD-1-FC binding to human PD-L2 by anti-PD-L2 subclones 16501 and 16510.

[0033] Figure 5 -–Equivalence to FDA-approved PD-1 / PD-L2 inhibitors. Candidate antibodies targeting PD-L2 were assayed using the Promega PD-L2:PD-1 Blockade System. Isotype control, Keytruda, Tecentriq, anti-PD-L2 20810, 20811, and 20816 were evaluated for their ability to sequester PD-L2 from PD-1. Fold induction of PD-1-luciferase response at indicated antibody concentrations is given.

[0034] Figures 6A to 6C -Candidate antibodies are active in various human mixed lymphocyte reactions. Candidate antibodies, FDA-approved antibodies, commercial antibodies, or isotype controls are evaluated in the presence of induced dendritic cells and T cells from various donors, and IL-2 or IFN-γ production is evaluated by ELISA. Fig. 6A ) Keytruda (anti-PD-1), candidate anti-PD-L2 antibodies 20811 and 20816, and isotype controls were added to IDCs prior to addition to CD4+ T cells. Figure 6B ) Anti-PD-L2 antibody 20814 or isotype control was added to IDCs prior to addition to CD4+ T cells. ( Figure 6C ) Anti-PD-L2 antibody 20814, Keytruda, PD-L1 monoclonal antibody, isotype control, or both anti-PD-L1 and 20814 were added to IDCs prior to subsequent addition to CD4+ T cells.

[0035] Figure 7 - Candidate antibodies with ADCC are highly active against human U2940 lymphoma in vivo.After establishing PBML xenograft tumors in SCID mice, mice were treated with mIgG2a control antibody, Herceptin, Rituxan, or 20810, and tumor volume was assessed on the indicated days.

[0036] Figure 8 - Candidate antibodies are active against MDA-MB-231. After establishing MDA-MB-231 xenograft tumors in SCID mice, mice were treated with mIgG2a control antibody, Rituximab, Avelumab (anti-PD-L1), or 20810. Tumor volume was assessed on the indicated days after treatment.

[0037] Fig. 9 -αPD-L2 antibody is effective in the αPD-L1-resistant EL4 lymphoma mouse model. The survival of mice injected with anti-PD-L1-resistant EL4 cells expressing PD-L2 and luciferase was measured. 5EL4 cells expressing PD-L2 and luciferase were injected into the tail vein of mice. Mice were treated intraperitoneally with 100 μg of the indicated antibodies on days 3, 6, 9, 12, and 15. Treatment with the anti-PD-L2 candidate 20810 increased survival compared with treatment with PD-L1 mAb or no treatment. DETAILED DESCRIPTION

[0038] The inventors have generated monoclonal antibodies with binding specificity to human PD-L2 protein. Since these antibodies have been shown to bind to PD-L2, they provide an opportunity to block the binding of PD-L2 to PD-1. They can also be used to deliver therapeutic payloads to cancer cells expressing PD-L2. These and other aspects of the present disclosure are described in even more detail below.

[0039] I.PD-L2

[0040] A. Structure

[0041] Programmed death-ligand 2 (PD-L2) is a protein encoded by the CD273 gene. PD-L2 is a 31 kDa protein that plays a major role in immunosuppression during multiple events such as pregnancy, tissue allografts, autoimmune diseases, cancer, and other disease states. The human PD-L2 protein is encoded by the amino acid sequence shown below:

[0042]

[0043] PD-L2 is initially produced with a signal peptide corresponding to amino acids 1 to 19 of SEQ ID NO.1, which is subsequently removed to produce the mature protein. The mature PD-L2 protein (corresponding to amino acids 20 to 273 of SEQ ID NO.1) contains an Ig-like V domain, an Ig-like C2-type domain, a transmembrane domain, and a cytoplasmic tail.

[0044] B. Function

[0045] PD-L2 is a ligand for its receptor PD-1. PD-1 can be found on activated T cells, B cells, and myeloid cells. Binding of PD-L2 to PD-1 initiates an immune cascade that impairs T cell proliferation, cytokine production, cytolytic function, and survival. PD-1 transmits inhibitory signals that reduce the proliferation of antigen-specific CD8+ T cells and CD4+ helper T cells. PD-L2 has also been shown to be an independent predictor of response to the anti-PD-1 antibody pembrolizumab in a variety of cancers (Yearley et al., 2017).

[0046] II. Monoclonal Antibodies and Their Production

[0047] A. General Methods

[0048] Antibodies to PD-L2 can be produced by standard methods known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Pat. No. 4,196,265). Methods for producing monoclonal antibodies (MAbs) generally begin along the same routes as those used to prepare polyclonal antibodies. The first step in both methods is to immunize a suitable host or identify an object that has been immunized due to a previous natural infection. As is known in the art, the immunogenicity of a given composition for immunization may vary. Therefore, it is usually necessary to strengthen the host's immune system, such as by coupling 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 may also be used as carriers. Means for conjugating polypeptides to carrier proteins are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide, and bis-biazotized benzidine. Also as is well known in the art, the immunogenicity of a particular immunogenic composition can be enhanced by the use of non-specific stimulators of the immune response known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response comprising killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant.

[0049] The amount of the immunogenic composition for producing polyclonal antibodies varies according to the properties of the immunogen and the animal used for immunization. Multiple approaches can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The generation of polyclonal antibodies can be monitored by sampling blood from the immunized animal at different points after immunization. Second, booster injections can also be given. Repetition of booster immunization and titration processes is until a suitable titer is reached. When the desired immunogenicity level is obtained, blood can be taken from the immunized animal, serum can be separated and stored, and / or the animal can be used to produce monoclonal antibodies.

[0050] After immunization, somatic cells with the potential to produce antibodies, particularly B lymphocytes (B cells) are selected for mAb production schemes. These cells can be obtained from biopsy spleen or lymph nodes or from circulating blood. Then, the B lymphocytes from the immunized animals producing antibodies are fused with cells of immortalized myeloma cells, which are usually immortalized myeloma cells of the same species as the immunized animals or human or human / mouse chimeric cells. The myeloma cell line suitable for the fusion program for producing hybridomas preferably does not produce antibodies, has high fusion efficiency and has enzyme defects, which subsequently prevents it from growing in certain selection media that only support the growth of the desired fusion cells (hybridomas).

[0051] As known to those skilled in the art, any of a variety of myeloma cells can be used (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). For example, where the immunized animal 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; for rats, R210.RCY3, Y3-Ag 1.2.3, IR983F, and 4B210 can be used; and U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6 can all be used in conjunction with human cell fusion. One particular murine myeloma cell is the NS-1 myeloma cell line (also known as P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another murine myeloma cell line that can be used is the 8-azaguanine-resistant murine myeloma SP2 / 0 non-producing cell line. Recently, additional fusion partner lines for 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 are produced using the SP2 / 0 / mIL-6 cell line, an IL-6-secreting derivative of the SP2 / 0 line.

[0052] Methods for producing hybrids of antibody-producing spleen cells or lymph node cells and myeloma cells generally include: mixing somatic cells with myeloma cells in a 2:1 ratio in the presence of one or more agents (chemical or electrical) that promote cell membrane fusion, but the ratio may vary from about 20:1 to about 1:1, respectively. Kohler and Milstein (1975; 1976) have described fusion methods using Sendai virus, and Gefter et al. (1977) have described fusion methods using polyethylene glycol (PEG), such as 37% (v / v) PEG. It is also suitable to use an electrically induced fusion method (Goding, pp. 71 to 74, 1986).

[0053] Fusion operations are usually performed at a rate of about 1×10 -6 Up to 1×10 -8 The low frequency of producing living heterozygotes. However, this does not cause problems, because by cultivating in the selection medium, the living fusion heterozygotes are distinguished from the unfused cells of the parents (particularly the unfused myeloma cells that will continue to divide indefinitely under normal circumstances). The selection medium is usually a culture medium containing a reagent that blocks the de novo synthesis of nucleotides in the tissue culture medium. Exemplary and preferred reagents are aminopterin, methotrexate and azaserine. Aminopterin and methotrexate block the de novo synthesis of both purine and pyrimidine, while azaserine only blocks purine synthesis. When using aminopterin or methotrexate, hypoxanthine and thymidine are supplemented in the culture medium as the source of nucleotides (HAT culture medium). When using azaserine, hypoxanthine is supplemented in the culture medium. If the B cell source is the human B cell line transformed by Epstein Barr virus (Epstein Barr virus, EBV), ouabain is added to remove the EBV transformed system that is not fused with myeloma.

[0054] Preferred selection medium is HAT or HAT with ouabain. Only cells that can carry out nucleotide salvage pathway can survive in HAT medium. Myeloma cells are defective in the key enzyme of salvage pathway (e.g., hypoxanthine phosphoribosyl transferase (HPRT)), so they cannot survive. B cells can carry out this pathway, but they have a limited life span in culture and usually die within about 2 weeks. Therefore, only cells that can survive in the selection medium are those hybrids formed by myeloma and B cells. When the source of the B cell used for fusion is the B cell line transformed by EBV, ouabain is also used for the drug selection of the hybrid at this time, because the B cell transformed by EBV is sensitive to drug killing, and the myeloma partner used is selected to be resistant to ouabain.

[0055] Cultivation provides a population of hybridomas from which specific hybridomas are selected. Selection of hybridomas is usually performed by culturing cells by monoclonal dilution in microtiter plates, followed by testing of individual clone supernatants for desired reactivity (after about 2 to 3 weeks). The assay should be sensitive, simple and rapid, such as radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, spot immunobinding assay, etc.

[0056] The selected hybridomas are then serially diluted or sorted by flow cytometry for single cell sorting and cloned into separate antibody-producing cell lines, which can then be infinitely propagated to provide mAbs. Cell lines can be used for MAb production in two basic ways. Hybridoma samples can be injected (usually into the peritoneal cavity) into animals (e.g., mice). Optionally, animals are primed with hydrocarbons, particularly oils (e.g., pristane (tetramethylpentadecane)) before injection. When using human hybridomas in this way, it is best to inject immunodeficient mice (e.g., SCID mice) to prevent tumor rejection. Tumors secreting specific monoclonal antibodies produced by fused cell hybrids appear in injected animals. Then, body fluids such as serum or ascites of the animal can be released to provide high concentrations of mAbs. Single cell lines can also be cultured in vitro, where mAbs are naturally secreted into the culture medium, from which high concentrations of mAbs can be easily obtained. Alternatively, human hybridoma cell lines can be used in vitro to produce immunoglobulins in cell supernatants. The cell lines can be adapted to grow in serum-free medium to optimize the ability to recover highly pure human monoclonal immunoglobulins.

[0057] If desired, the monoclonal antibodies produced in either manner can be further purified using filtration, centrifugation, and various chromatographic methods (e.g., 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 (e.g., pepsin or papain), and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, the monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

[0058] It is also contemplated that monoclonal antibodies may be generated using molecular cloning methods. To this end, RNA may be isolated from a hybridoma line, the antibody gene obtained by RT-PCR, and cloned into an immunoglobulin expression vector. Alternatively, a combinatorial immunoglobulin phagemid library may be prepared from RNA isolated from a cell line, and phagemids expressing the appropriate antibodies may be selected by panning using viral antigens. This approach has the advantage over conventional hybridoma technology that up to about 10 monoclonal antibodies may be generated and screened in a single round. 4 The antibodies are multiplied by 2.5 times, and new specificities are generated by combining H and L chains, which further improves the chances of discovering suitable antibodies.

[0059] Yeast-based antibody display libraries can be rationally designed, and antibodies can be selected and / or isolated from such yeast-based antibody display libraries, as disclosed in, e.g., WO2012 / 009568; WO2009 / 036379; WO2010 / 105256; WO2003 / 074679; U.S. Pat. No. 8,691,730; and U.S. Pat. No. 9,354,228. The antibodies can then be expressed from the desired cell type as full-length IgG and purified.

[0060] Other U.S. patents (each incorporated herein by reference) that teach the production of antibodies useful in the present disclosure include U.S. Patent 5,565,332, which describes the use of combinatorial methods to produce chimeric antibodies; U.S. Patent 4,816,567, which describes recombinant immunoglobulin production; and U.S. Patent 4,867,973, which describes antibody-therapeutic agent conjugates.

[0061] B. Antibodies of the Disclosure

[0062] Antibodies according to the present disclosure can be defined in the first instance by their binding specificity, i.e. binding to PD-L2. By evaluating the binding specificity / affinity of a given antibody using techniques well known to those skilled in the art, one skilled in the art can determine whether such an antibody falls within the scope of the claims of the present invention. In one aspect, monoclonal antibodies having cloned paired CDRs from heavy and light chains as shown in Tables 3 and 4, respectively, are provided. Such antibodies can be produced using the methods described herein by cloning as discussed below in the Examples section.

[0063] In a second aspect, antibodies can be defined by their variable sequences including additional "framework" regions. These are provided in Tables 1 and 2, which encode or represent complete variable regions. In addition, antibody sequences may differ from these sequences, optionally using methods discussed in more detail below. For example, nucleic acid sequences may differ from those described above in the following aspects: (a) the variable regions may be separated from the constant domains of the light and heavy chains; (b) the nucleic acids may differ from those described above without affecting the residues encoded therefrom; (c) the nucleic acids may differ from the nucleic acids described above by a given percentage, such as 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology; (d) the nucleic acids may differ from the nucleic acids described above by the ability to hybridize under high stringency conditions, as exemplified by low salt and / or high temperature conditions, for example from about 0.02 M to about 0.15 M at a temperature of about 50°C to about 70°C. NaCl provided; (e) amino acid can be different from the above amino acid with a given percentage, such as 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology; or (f) amino acid can be different from the above by allowing conservative substitutions (discussed below). The foregoing items are all applicable to the nucleotide sequences shown in Table 1 and the amino acid sequences of Table 2.

[0064] C. Modification of Antibody Sequences

[0065] In various embodiments, the sequence of the identified antibodies may be chosen to be engineered for a variety of reasons, such as improved expression, improved cross-reactivity, or reduced off-target binding. The following is a general discussion of relevant techniques for antibody engineering.

[0066] Hybridomas can be cultured, cells are then lysed, and total RNA is extracted. Random hexamers can be used together with RT to produce cDNA copies of RNA, and then PCR is performed using a multiplex mixture of PCR primers that are expected to amplify all human variable gene sequences. The PCR product can be cloned into the pGEM-T Easy vector, and then sequenced by automated DNA sequencing using standard vector primers. Antibodies collected from hybridoma supernatants and purified by FPLC using G protein columns can be used for binding and neutralization assays.

[0067] Recombinant full-length IgG antibodies were produced by subcloning the heavy and light chain Fv DNA from the cloning vector into an IgG plasmid vector, transfecting into 293 Freestyle cells or CHO cells, and collecting and purifying the antibodies from the 293 or CHO cell supernatant.

[0068] The rapid availability of antibodies produced in the same host cell and cell culture process as the final cGMP production process has the potential to reduce the duration of the process development plan. Lonza has developed a general method for rapidly producing a small amount (up to 50g) of antibodies in CHO cells using the combined transfectants cultivated in CDACF culture medium. Although slightly slower than a true transient system, its advantages include higher product concentrations and using the same host and process as the production cell line. The growth and productivity examples of the GS-CHO library expressing model antibodies in disposable bioreactors are: in the disposable bag-shaped bioreactor culture (5L working volume) carried out in fed-batch mode, the harvest antibody concentration of 2g / L was reached in 9 weeks after transfection.

[0069] Antibodies can be rationally designed and synthesized, and antibody libraries from which such antibodies can be selected and / or isolated, e.g., by Technology, such as disclosed in, for example, WO2012 / 009568; WO2009 / 036379; WO2010 / 105256; WO2003 / 074679; U.S. Pat. No. 8,691,730; and U.S. Pat. No. 9,354,228. This method of synthesizing antibodies requires inserting a nucleotide sequence encoding a desired or designed antibody into a vector for ectopic expression. The desired antibody can then be expressed as a full-chain IgG molecule and purified.

[0070] Antibody molecules will include fragments such as those produced by proteolytic cleavage of mAbs (e.g., F(ab'), F(ab')2) or single-chain immunoglobulins such as those that can be produced recombinantly. Such antibody derivatives are monovalent. In one embodiment, such fragments can be combined with each other, or with other antibody fragments or receptor ligands to form "chimeric" binding molecules. Obviously, such chimeric molecules can include substituents that can bind to different epitopes of the same molecule.

[0071] In some related embodiments, the antibody is a derivative of the disclosed antibody, for example, an antibody comprising a CDR sequence identical to the CDR sequence in the disclosed antibody (e.g., a chimeric antibody or a CDR-grafted antibody). Alternatively, it may be desirable to make modifications, such as introducing conservative changes into the antibody molecule. In making such changes, the hydropathic index of the amino acids may be considered. The importance of the amino acid hydropathic index in conferring interactive biological functions on proteins is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic nature of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).

[0072] It is also understood in the art that similar amino acid substitutions can be effectively made based on hydrophilicity. U.S. Patent 4,554,101 (incorporated herein by reference) states that the greatest local average hydrophilicity of a protein (as governed by the hydrophilicity of its neighboring amino acids) is correlated with the biological properties of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values ​​have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartic acid (+3.0±1), glutamic acid (+3.0±1), asparagine (+0.2), and glutamine (+0.2); hydrophilic nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2); The following are the hydrophobic amino acids: amide (+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).

[0073] It is understood that an amino acid can be replaced with another amino acid having a similar hydrophilicity and produce a biologically or immunologically modified protein. In such changes, replacement of amino acids whose hydrophilicity values ​​are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0074] As summarized above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account many of the foregoing 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.

[0075] 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 improve antibody-dependent cellular cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve price.

[0076] Modified antibodies can be prepared by any technique known to those skilled in the art, including expression by standard molecular biology techniques, or chemical synthesis of the polypeptide. Methods for recombinant expression are described elsewhere herein.

[0077] D. Single-chain antibody

[0078] Single chain variable fragment (scFv) is a fusion of the immunoglobulin heavy chain and light chain variable region connected together with a short (usually serine, glycine) joint. Although this chimeric molecule has removed the constant region and introduced a joint peptide, the specificity of the original immunoglobulin is retained. This modification does not usually change the specificity. Historically, these molecules were produced to facilitate phage display, in which it is very convenient to express the antigen binding domain as a single peptide. Alternatively, scFv can be directly produced by the subclone heavy chain and light chain derived from a hybridoma. Single chain variable fragments lack the constant Fc region present in the complete antibody molecule, and therefore lack the common binding sites (e.g., protein A / G) for purifying antibodies. These fragments can usually be purified / fixed using protein L because protein L interacts with the variable region of the kappa light chain.

[0079] Flexible linkers are usually composed of amino acid residues that promote helices and turns (e.g., alanine, serine, and glycine). However, other residues can also work well. Tang et al. (1996) used phage display as a way to quickly select specialized linkers for single-chain antibodies (scFv) from a protein linker library. A random linker library was constructed in which the genes for the heavy and light chain variable domains were connected by segments encoding 18-amino acid polypeptides with variable compositions. The scFv library (about 5×10 6 Different members) and affinity selection with haptens. The population of selected variants showed significantly improved binding activity, but retained considerable sequence diversity. Subsequent screening of 1054 individual variants resulted in catalytically active scFvs that were efficiently produced in soluble form. Sequence analysis revealed that the only common features of the selected tethers were: V H The 2 residues after the C-terminus are conserved prolines in linkers and there are numerous arginines and prolines at other positions.

[0080] The recombinant antibodies of the present disclosure may also involve sequences or portions that allow receptor dimerization or multimerization. Such sequences include those derived from IgA, which allow the formation of multimers in conjunction with the J chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with reagents that allow two antibodies to combine (e.g., biotin / avidin).

[0081] In an independent embodiment, single-chain antibodies can be produced by connecting receptor light and heavy chains using non-peptide linkers or chemical units. Generally, light and heavy chains are produced in different cells, purified, and then connected together in a suitable manner (i.e., the N-terminal of the heavy chain is connected to the C-terminal of the light chain by a suitable chemical bridge).

[0082] Cross-linking agents are used to form molecular bridges that tether the functional groups of two different molecules, for example, stabilizing and coagulants. However, it is contemplated that dimers or multimers of the same analogs or heteromeric complexes containing different analogs may be produced. In order to link two different compounds in a stepwise manner, hetero-bifunctional cross-linking agents may be used, which eliminate unwanted homopolymer formation.

[0083] An exemplary hetero-bifunctional cross-linker comprises two reactive groups: one that reacts with primary amine groups (e.g., N-hydroxysuccinimide) and the other that reacts with thiol groups (e.g., pyridyl disulfide, maleimide, halogen, etc.) Through the primary amine reactive group, the cross-linker can react with lysine residues of one protein (e.g., the selected antibody or fragment), and through the thiol reactive group, the cross-linker already tethered to the first protein reacts with cysteine ​​residues (free sulfhydryl groups) of another protein (e.g., the selection agent).

[0084] Preferably, a cross-linking agent with reasonable stability in blood is used. It is known that various types of disulfide-containing linkers can be successfully used to conjugate targeting agents and therapeutic / prophylactic agents. Linkers containing sterically hindered disulfide bonds can be shown to provide higher stability in vivo, thereby preventing the targeting peptide from being released before reaching the site of action. Therefore, these linkers are a group of linkers.

[0085] Another cross-linking reagent is SMPT, a bifunctional cross-linker containing a disulfide bond that is "sterically hindered" by the adjacent benzene ring and methyl groups. It is believed that the steric hindrance of the disulfide bond functions to protect the bond from attack by thiolate anions (e.g., glutathione) that may be present in tissues and blood, and thereby helps prevent the conjugate from decoupling before the attached agent is delivered to the target site.

[0086] Like many other known cross-linking reagents, the SMPT cross-linking reagent is also able to cross-link functional groups such as the SH of cysteine ​​or primary amines (e.g., the epsilon amino group of lysine). Another possible type of cross-linking agent includes hetero-bifunctional photoreactive phenylazido groups containing cleavable disulfide bonds, such as sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino groups, and the phenylazido group (after photolysis) reacts non-selectively with any amino acid residue.

[0087] In addition to hindered crosslinkers, non-hindered crosslinkers may also be used accordingly. Other useful crosslinkers, not including or generating 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 a flexible linker.

[0088] U.S. Patent No. 4,680,338 describes bifunctional linkers that can be used to produce conjugates of ligands with amine-containing polymers and / or proteins, particularly for forming antibody conjugates with chelators, drugs, enzymes, detectable labels, and the like. U.S. Patent Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing labile bonds that are cleavable under a variety of mild conditions. Such linkers are particularly useful because the agent of interest can be directly bonded to the linker and its cleavage results in the release of the active agent. Particular uses include the addition of free amino groups or free sulfhydryl groups to proteins such as antibodies or drugs.

[0089] U.S. Patent No. 5,856,456 provides peptide linkers for connecting polypeptide components to prepare fusion proteins (e.g., single-chain antibodies). The linker is up to about 50 amino acids in length; comprises a charged amino acid (preferably arginine or lysine) followed by proline, which occurs at least once; and is characterized by greater stability and reduced aggregation. U.S. Patent No. 5,880,270 discloses aminooxy-containing linkers that can be used in a variety of immunodiagnostic and separation techniques.

[0090] E. Purification

[0091] In certain embodiments, the antibodies of the present disclosure may be purified. As used herein, the term "purified" is intended to refer to compositions that are separable from other components, wherein the protein is purified to any degree relative to its naturally obtainable state. Therefore, a purified protein also refers to a protein that is freed from its naturally occurring environment. When the term "substantially purified" is used, the designation refers to a composition in which a 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.

[0092] Protein purification techniques are well known to those skilled in the art. These techniques involve, at one level, the coarse fractionation of the cellular environment into polypeptide fractions and non-polypeptide fractions. After the polypeptide is separated from other proteins, the polypeptide of interest can be further purified using chromatography and electrophoresis techniques to achieve partial purification or complete purification (or purification to homogeneity). Analytical methods particularly suitable for preparing pure peptides are ion exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric aggregation. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies, etc. or by thermal denaturation, followed by centrifugation; gel filtration, reverse phase, hydroxyapatite and affinity chromatography; and combinations of these techniques with other techniques.

[0093] In purifying 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. Affinity columns that bind to the labeled portion of the polypeptide may be used to purify the polypeptide from other cellular components. As is generally known in the art, it is contemplated that the order in which the various purification steps are performed may be altered, or that certain steps may be omitted, and still result in a method suitable for preparing a substantially purified protein or peptide.

[0094] Typically, the complete antibodies are fractionated using a reagent that binds to the Fc portion of the antibody (i.e., protein A). Alternatively, an antigen can be used to purify and select a suitable antibody simultaneously. Such methods typically use a selection agent that is bound to a support (e.g., a column, a filter, or a bead). The antibody is bound to the support, contaminants are removed (e.g., rinsed off), and the antibody is released by applying conditions (salt, heat, etc.).

[0095] In light of the present disclosure, those skilled in the art will recognize a variety of methods for quantifying the degree of purification of a protein or peptide. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptide in a 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 purity. Of course, the actual units used to express the amount of activity will depend on the particular assay technique selected for purification, and whether the expressed protein or peptide exhibits detectable activity.

[0096] It is known that the migration of polypeptides can change with different SDS / PAGE conditions, sometimes significantly (Capaldi et al., 1977). Therefore, it should be understood that the apparent molecular weight of a purified or partially purified expression product can change under different electrophoretic conditions.

[0097] III. Pharmaceutical Preparations and Cancer Treatment

[0098] A. Cancer

[0099] Cancer is caused by the overgrowth of clonal populations of cells from tissues. The occurrence of cancer (called carcinogenesis) can be modeled and characterized in many ways. The connection between cancer occurrence and inflammation has been understood for a long time. Inflammatory responses participate in host defense against microbial infections and also drive tissue repair and regeneration. A large amount of evidence points to the connection between inflammation and the risk of cancer occurrence, that is, chronic inflammation can lead to dysplasia.

[0100] Cancer cells to which the methods of the present disclosure can be applied generally include any cells expressing PD-L2 and more particularly overexpressing PD-L2. Suitable cancer cells can be cells of breast cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, gastric cancer, liver cancer, bone cancer, hematological cancer (e.g., leukemia or lymphoma), nervous tissue cancer, melanoma, ovarian cancer, testicular cancer, prostate cancer, cervical cancer, vaginal cancer, or bladder cancer. In addition, the methods of the present disclosure can be applied to a variety of species, such as humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Cancer can also be recurrent, metastatic, and / or multidrug-resistant, and the methods of the present disclosure can be particularly applied to such cancers to make them resectable, prolong or re-induce remission, inhibit angiogenesis, prevent or limit metastasis, and / or treat multidrug-resistant cancers. At the cellular level, this can translate into killing cancer cells, inhibiting cancer cell growth, or otherwise reversing or reducing the malignant phenotype of tumor cells.

[0101] B. Formulation and Administration

[0102] The present disclosure provides pharmaceutical compositions comprising anti-PD-L2 antibodies. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopeia or other recognized pharmacopeia for use in animals and more particularly in humans. The term "carrier" refers to a diluent, excipient, or vehicle administered with a therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, saline, dextrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, and the like.

[0103] The composition can be formulated as a neutral or salt form. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like; and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0104] Antibodies of the present disclosure may include classical pharmaceutical formulations. Administration of these compositions according to the present disclosure may be by any common route, as long as the target tissue can be reached by the route. This includes oral, nasal, buccal, rectal, vaginal or surface. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions will typically be administered as the above-mentioned pharmaceutically acceptable compositions. Of particular interest is direct intratumoral administration, tumor perfusion or local or regional administration to the tumor, such as administration in the local or regional vascular system or lymphatic system, or in a resected tumor bed.

[0105] The active compound can also be administered parenterally or intraperitoneally. Solutions of the active compound as free alkali or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant (e.g., hydroxypropylcellulose). Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. Under common storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.

[0106] C. Combination therapy

[0107] In the context of the present disclosure, it is also contemplated that the anti-PD-L2 antibodies described herein may similarly be used in combination with chemotherapeutic or radiotherapeutic interventions or other treatments. In particular, combining anti-PD-L2 antibodies with other treatments targeting different aspects of PD-L2 function (e.g., peptides and small molecules targeting the cytoplasmic domain of PD-L2) may also prove effective.

[0108] In order to use the methods and compositions of the present disclosure to kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis, or otherwise reverse or reduce the malignant phenotype of tumor cells, the "target" cells will generally be contacted with an anti-PD-L2 antibody according to the present disclosure and at least one other agent. These compositions will be provided in a combined amount effective to kill cells or inhibit cell proliferation. The process may include contacting cells with an anti-PD-L2 antibody according to the present disclosure and another agent or factor at the same time. This can be achieved by contacting the cells with a single composition or pharmacological preparation containing both agents, or by contacting the cells with two different compositions or preparations simultaneously, wherein one composition comprises an anti-PD-L2 antibody according to the present disclosure and the other comprises other agents.

[0109] Alternatively, anti-PD-L2 antibody treatment may be performed before or after treatment with the additional agent, with an interval of several minutes to several weeks. In some embodiments in which the additional agent and the anti-PD-L2 antibody are applied to the cell separately, it should generally be ensured that no more than a long time passes between each delivery, so that the agent and the expression construct are still able to exert a favorable combined effect on the cell. In this case, it is contemplated that the cell is contacted with both forms within about 12 to 24 hours of each other and more preferably within about 6 to 12 hours of each other, with the most preferred delay time being only about 12 hours. However, in some cases, it may be desirable to significantly extend the treatment time, with each administration separated by several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks).

[0110] It is also contemplated that it may be desirable to administer the anti-PD-L2 antibody or additional agent more than once. As shown below, a variety of combinations may be used, wherein the treatment with the anti-PD-L2 antibody according to the present disclosure is "A" and the additional treatment is "B":

[0111]

[0112] Taking into account the toxicity of the antibody treatment, if any, administration of the therapeutic agent of the present invention to the patient will follow the general scheme of administering a specific second treatment. It is expected that the treatment cycle will be repeated as needed. It is also contemplated that a variety of standard treatments as well as surgical interventions may be used in combination with the cancer treatment.

[0113] The skilled artisan is guided by "Remington's Pharmaceutical Sciences", 15th edition, Chapter 33, especially pages 624-652. Some variation in dosage will be necessary depending on the condition of the subject being treated. In any case, the person responsible for administration will determine the appropriate dosage for an individual subject. In addition, for human administration, the preparation should meet the standards of sterility, pyrogenicity, general safety, and purity required by the FDA Office of Biological Standards.

[0114] 1. Chemotherapy

[0115] Cancer treatment also includes multiple combined treatments with chemotherapy and radiation-based treatment. Combination chemotherapy includes, for example, cisplatin (CDDP), carboplatin, procarbazine, nitrogen mustard, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosoureas, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, paclitaxel, gemcitabine, navelbine, farnesyl protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, temozolomide (Temazolomide) (aqueous solution form of DTIC) or any analog or derivative variant of the aforementioned substances. The combination of chemotherapy and biological treatment is referred to as biochemical treatment. The present invention contemplates any chemotherapeutic agent that can be used in the art or is known for treating or preventing cancer.

[0116] 2. Radiation therapy

[0117] Other factors that cause DNA damage and have been widely used include commonly known gamma-rays, X-rays and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors, such as microwaves and UV irradiation, have also been considered. Most likely, all of these factors cause extensive damage to DNA, DNA precursors, replication and repair of DNA, and chromosomal assembly and maintenance. The dosage range of X-rays is a single dose of 2000 to 6000 roentgens for a period of time (3 to 4 weeks) from a daily dose of 50 to 200 roentgens. The dosage range of radioisotopes varies greatly and depends on the half-life of the isotope, the intensity and type of the radiation emitted, and the uptake of neoplastic cells.

[0118] The terms "contacting" and "exposing" as applied to cells are used herein to describe the process of delivering a therapeutic agent and a chemotherapeutic agent or radiotherapeutic agent to or placing them in direct juxtaposition with a target cell. To achieve cell killing or stasis, the two agents are delivered to the cell in a combined amount effective to kill the cell or prevent cell division.

[0119] 3. Immunotherapy

[0120] Generally speaking, immunotherapy relies on the use of immune effector cells and molecules to target and destroy cancer cells.Immune effectors can be antibodies that are, for example, specific to some markers on the surface of tumor cells.Single antibody can be used as an effector for treatment or it can recruit other cells that actually achieve cell killing.Antibodies can also be conjugated with drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chains, cholera toxin, pertussis toxin, etc.) and are only used as targeting agents.Or, the effector can be a lymphocyte carrying a surface molecule that interacts directly or indirectly with a tumor cell target.A variety of effector cells include cytotoxic T cells and NK cells.The combination of treatment modes (i.e., direct cytotoxic activity and inhibition or reduction of Fortilin) ​​will provide therapeutic benefits in treating cancer.

[0121] Immunotherapy can also be used as part of a combination therapy. The general method of combination therapy is discussed below. In one aspect of immunotherapy, tumor cells must carry some markers suitable for targeting (i.e., not present on most other cells). There are many tumor markers, and any of these 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. Another aspect of immunotherapy is the anti-cancer effect with an immunostimulatory effect. There are also immunostimulatory molecules, including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN; chemokines, such as MIP-1, MCP-1, IL-8 and growth factors, such as FLT3 ligands. Combining immunostimulatory molecules, either as proteins or using gene delivery in combination with tumor suppressors such as mda-7, has been shown to enhance anti-tumor effects (Ju et al., 2000).

[0122] As mentioned above, some examples of immunotherapy currently under investigation or in use are immune adjuvants (e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds) (U.S. Pat. No. 5,801,005; U.S. Pat. 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); al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. No. 5,830,880 and U.S. Pat. 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. Pat. No. 5,824,311). Herceptin (trastuzumab) is a chimeric (mouse-human) monoclonal antibody that blocks the HER2-neu receptor. It has anti-tumor activity and has been approved for the treatment of malignant tumors (Dillman, 1999). Combination treatment of cancer with Herceptin and chemotherapy has been shown to be more effective than single treatment. Therefore, it is contemplated that one or more anti-cancer treatments can be used in conjunction with the tumor-associated HLA-restricted peptide treatments described herein.

[0123] In adoptive immunotherapy, circulating lymphocytes or tumor-infiltrating lymphocytes of patients are separated in vitro, activated by lymphokines (e.g., IL-2) or transduced with tumor necrosis genes, and 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 as described herein incorporated into an antigenic peptide composition. The activated lymphocytes are most preferably the patient's own cells that were isolated from blood or tumor samples at an early stage and activated (or "amplified") in vitro. This form of immunotherapy has produced several cases of regression of melanoma and renal cancer, but the percentage of responders is lower than those that did not respond.

[0124] There are many different approaches for passive immunotherapy of cancer. They can be broadly categorized as follows: injection of antibodies alone; injection of antibodies coupled to toxins or chemotherapeutic agents; injection of antibodies coupled to radioisotopes; injection of anti-idiotypic antibodies; and finally, purging of tumor cells from the bone marrow.

[0125] Human monoclonal antibodies are applied to passive immunotherapy because they produce little or no side effects in patients. However, their application is limited to some extent due to its scarcity, and only intralesional administration has been performed so far. Human monoclonal antibodies for ganglioside antigens have been intralesionally administered to patients with recurrent melanoma of the skin (Irie & Morton, 1986). After intralesional injection daily or weekly, disappearance was observed in 6 of 10 patients. In another study, moderate success was achieved by 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.

[0126] It may be advantageous to administer more than one monoclonal antibody directed against two different antigens or even antibodies with multiple antigen specificities. The treatment regimen may also include administration of lymphokines or other immunopotentiators, as described by Bajorin et al. (1988). The development of human monoclonal antibodies is further described in detail in other parts of this specification.

[0127] 4. Gene therapy

[0128] In another embodiment, the second treatment is gene therapy, wherein the therapeutic polynucleotide is administered before, after or at the same time as the administration of the tumor-associated HLA restricted peptide. The delivery of a vector encoding the tumor-associated HLA restricted peptide in combination with a second vector encoding one of the following gene products will have a combined anti-hyperproliferative effect on the target tissue. Alternatively, a single vector encoding two genes can be used. A variety of proteins are encompassed within the present invention, some of which are described below. A variety of genes that can be targeted by some forms of gene therapy combined with the present invention are well known to those of ordinary skill in the art and may include any gene associated with cancer.

[0129] Inducers of cell proliferation: Proteins that induce cell proliferation are further divided into multiple categories based on their function. The commonality of all these proteins is their ability to regulate cell proliferation. For example, a form of PDGF, the sis oncogene, is a secreted growth factor. Oncogenes rarely come from genes encoding growth factors, and currently, sis is the only known naturally occurring oncogenic growth factor. In one embodiment of the present invention, it is contemplated that the expression of the cell proliferation inducer is prevented using antisense mRNA directed to a specific inducer of cell proliferation.

[0130] The proteins FMS, ErbA, ErbB, and neu are growth factor receptors. Mutations in these receptors result in loss of regulatable function. For example, point mutations affecting the transmembrane domain of the Neu receptor protein lead to the neu oncogene. The erbA oncogene is derived from the intracellular receptor for thyroid hormone. It is thought that the modified oncogenic ErbA receptor competes with the endogenous thyroid hormone receptor, resulting in uncontrolled growth.

[0131] The largest oncogene category includes signal transduction proteins (e.g., Src, Abl and Ras). Protein Src is a cytoplasmic protein tyrosine kinase, and its transformation from a proto-oncogene to an oncogene is caused by a mutation at tyrosine residue 527 in some cases. In contrast, in an example, the transformation of the GTPase protein ras from a proto-oncogene to an oncogene is caused by a mutation of the valine at the 12th amino acid in the sequence to a glycine, thereby reducing ras GTPase activity. Proteins Jun, Fos and Myc are proteins that directly exert their effects on nuclear function as transcription factors.

[0132] Inhibitors of cell proliferation: The function of tumor suppressor oncogenes is to inhibit excessive cell proliferation. Inactivation of these genes destroys their inhibitory activity, resulting in unregulated proliferation. 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 fusions and p21 / p27 fusions.

[0133] Regulators of programmed cell death: Apoptosis or programmed cell death is an essential process for normal embryonic development, maintenance of adult tissue homeostasis, and suppression of carcinogenesis (Kerr et al., 1972). The Bcl-2 family of proteins and ICE-like proteases have been shown to be important regulators and effectors of apoptosis in other systems. Bcl-2 proteins associated with follicular lymphoma were found to play an important role in controlling apoptosis and enhancing cell survival in response to different 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 proteins are now recognized to be members of a family of related proteins that can be classified as either death agonists or death antagonists.

[0134] Following the discovery of Bcl-2, it has been shown that Bcl-2 functions to inhibit cell death triggered by a variety of stimuli. Likewise, it is now apparent that there is a family of Bcl-2 cell death regulatory proteins that share common structural and sequence homology. These different family members have been shown to have similar functions to Bcl-2 (e.g., Bcl XL , Bcl W , Bcl S , Mcl-1, A1, Bfl-1) or counteract Bcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).

[0135] 5. Surgery

[0136] About 60% of people with cancer will undergo some type of surgery, which includes preventive, diagnostic or staging, curative, and palliative surgery. Curative surgery is a cancer treatment that can be used in conjunction with other treatments (e.g., the treatment of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapy).

[0137] Curative surgery includes resection in which all or a portion of cancerous tissue is physically removed, excised and / or destroyed. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, treatment by surgery also includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). It is also contemplated that the present invention can be used in conjunction with the removal of superficial cancer, procancer, or accompanying amounts of normal tissue.

[0138] After removing a part or all of cancer cells, tissues or tumors, a cavity can be formed in vivo. Treatment can be completed by perfusion, direct injection or other anticancer treatment of the region. Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4 and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. These treatments can also have multiple doses.

[0139] IV. Antibody Conjugates

[0140] The antibody can be connected to at least one reagent to form an antibody conjugate. In order to improve the effectiveness of the antibody molecule as a diagnostic or therapeutic agent, it is conventionally connected or covalently bound or compounded with at least one desired molecule or part. Such a molecule or part can be, but is not limited to, at least one effector molecule or reporter molecule. Effector molecules include molecules with desired activities, such as immunosuppression / anti-inflammatory. Some non-limiting examples of such molecules are described above. Such molecules are optionally connected by a cleavable linker designed to allow the molecule to be released at or near the target site.

[0141] In contrast, a reporter molecule is defined as any moiety that can be detected using an assay. Some non-limiting examples of reporter molecules that have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles, or ligands, such as biotin.

[0142] It is usually preferred to use antibody conjugates as diagnostic agents. Antibody diagnostic agents generally belong to two categories: those used for in vitro diagnosis, such as for a variety of immunoassays, and those used for in vivo diagnostic protocols commonly referred to as "antibody-directed imaging". Many suitable imaging agents are known in the art, as are methods for attaching them to antibodies (see, e.g., U.S. Patents 5,021,236, 4,938,948 and 4,472,509). The imaging moieties used can be paramagnetic ions, radioisotopes, fluorescent dyes, NMR-detectable substances and X-ray imaging agents.

[0143] In the case of paramagnetic ions, the following exemplary ions may be mentioned: for example 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 that may be used in other cases (e.g. X-ray imaging) include, but are not limited to, lanthanum (III), gold (III), lead (II) and in particular bismuth (III).

[0144] Among the radioisotopes used for therapeutic and / or diagnostic applications, mention may be made of 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 In certain embodiments it is often preferred to use 125 I, and technetium is also generally preferred 99m and / or indium 111 Radiolabeled monoclonal antibodies can be produced according to methods known in the art. For example, monoclonal antibodies can be iodinated by contact with sodium iodide and / or potassium iodide and a chemical oxidant (e.g., sodium hypochlorite) or an enzymatic oxidant (e.g., lactoperoxidase). Monoclonal antibodies can be iodinated by a ligand exchange process with technetium. 99m Labeling, for example, by reducing pertechnetate with a stannous solution, chelating the reduced technetium to a Sephadex column, and applying the antibody to the column. Alternatively, direct labeling techniques may be used, such as by incubating pertechnetate, a reducing agent (e.g., SNCl2), a buffer solution (e.g., sodium potassium phthalate solution), and the antibody. Intermediate functional groups are commonly used to bind radioactive isotopes to antibodies and exist as metal ions, and are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0145] Among the fluorescent labels contemplated for use as conjugates are Alexa 350, Alexa 430, AMCA, BODIPY630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, CascadeBlue, 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.

[0146] Another type of antibody conjugate considered is those intended to be used in vitro, wherein the antibody is connected to a second binding partner and / or to an enzyme (enzyme tag) that produces a colored product after contact with a chromogenic substrate. Some examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) catalase or glucose oxidase. Preferred second binding partners are biotin and avidin and streptavidin compounds. The use of such labels is well known to those skilled in the art and is described in, for example, U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241.

[0147] Another known method for site-specific attachment of molecules to antibodies involves reacting the antibody with a hapten-based affinity tag. In essence, a hapten-based affinity tag reacts with amino acids in the antigen binding site, thereby destroying the site and blocking the specific antigen reaction. However, this can be disadvantageous because it results in a loss of antigen binding by the antibody conjugate.

[0148] Molecules containing an azide group can also be used to form covalent bonds with proteins via reactive nitrene intermediates generated by low-intensity ultraviolet light (Potter and Haley, 1983). In particular, 2-azido analogs 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-azido and 8-azido nucleotides have also been used to map nucleotide-binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and can be used as antibody binders.

[0149] Several methods for connecting or conjugating an antibody to its conjugated moiety are known in the art. Some methods of connection involve the use of metal chelate complexes using, for example, organic chelators such as diethylenetriaminepentaacetic anhydride (DTPA) attached to the antibody; ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3α-6α-diphenylglycouril-3 (tetrachloro-3α-6α-diphenylglycouril-3) (U.S. Pat. Nos. 4,472,509 and 4,938,948). Monoclonal antibodies can also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. Conjugates labeled with fluorescein are prepared in the presence of these coupling agents or by reaction with isothiocyanates. In U.S. Pat. No. 4,938,948, imaging of breast tumors was achieved using monoclonal antibodies and detectable imaging moieties were bound to the antibody using linkers such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl) propionate.

[0150] In other embodiments, it is contemplated to derivatize immunoglobulins by selectively introducing sulfhydryl groups into the Fc region of the immunoglobulin using reaction conditions that do not alter the antibody binding site. It is disclosed that antibody conjugates produced according to this method exhibit improved longevity, specificity, and sensitivity (U.S. Pat. No. 5,196,066, which is incorporated herein by reference). Site-specific attachment of effector or reporter molecules has also been disclosed in the literature, wherein the reporter or effector molecule is conjugated to a sugar residue in the Fc region (O'Shannessy et al., 1987). This method has been reported to produce promising antibodies in diagnosis and therapy that are currently under clinical evaluation.

[0151] V. Immunoassay Methods

[0152] In other embodiments, there are immunoassays for binding, purifying, removing, quantifying and otherwise generally detecting PD-L2 and its associated antigens. Some immunoassays include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays, fluorescent immunoassays, chemiluminescent assays, bioluminescent assays and Western blots, etc. In particular, competitive assays for detecting and quantifying PD-L2 antibodies are also provided. The steps of a variety of available immunoassays have been described in the scientific literature, for example, as described in Doolittle and Ben-Zeev (1999); Gulbis and Galand (1993); De Jager et al. (1993); and Nakamura et al. (1987). Generally speaking, the immunobinding method includes obtaining a sample and contacting the sample with a first antibody according to some embodiments discussed herein, as appropriate, under conditions that effectively allow the formation of immune complexes.

[0153] Contacting the selected biological sample with the antibody under effective conditions and for a time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a sufficient time to allow the antibody to form an immune complex with (i.e., bind to) PD-L2 that is present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot, or Western blot, is typically washed to remove any non-specifically bound antibody species, thereby allowing only those antibodies that are specifically bound in the primary immune complex to be detected.

[0154] Generally speaking, the detection of immune complex formation is well known in the art and can be achieved by applying a variety of methods. These methods are usually based on the detection of marks or markers (such as any of those radioactive labels, fluorescent labels, biological labels and enzyme labels). Patents related to the use of such marks include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Of course, as known in the art, by using a second binding ligand such as a second antibody and / or biotin / avidin ligand binding arrangement, additional advantages can be found.

[0155] The antibody used for detection can itself be connected to a detectable label, wherein the label will then be simply detected, thereby allowing the amount of the primary immune complex in the composition to be determined. Alternatively, the antibody bound to the primary immune complex can be detected by a second binding ligand having a binding affinity to the first antibody. In these cases, the second binding ligand can be connected to a detectable label. The second binding ligand itself is typically an antibody, which can therefore be referred to as a "second" antibody. The primary immune complex is contacted with a labeled second binding ligand or antibody under effective conditions and continued for a time sufficient to allow the formation of a secondary immune complex. The secondary immune complex is then usually washed to remove any non-specifically bound labeled second antibody or ligand, and the remaining label in the secondary immune complex is subsequently detected.

[0156] Other methods include detecting primary immune complexes by a two-step method. As described above, a second binding ligand (e.g., antibody) with binding affinity to the antibody is used to form a secondary immune complex. After washing, the secondary immune complex is again contacted with a third binding ligand or antibody with binding affinity to the second antibody under effective conditions and continues for a time sufficient to allow the formation of an immune complex (tertiary immune complex). The third ligand or antibody is connected to a detectable label, thereby allowing the detection of the tertiary immune complex thus formed. If desired, the system can provide signal amplification.

[0157] An immunodetection method uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and then a second antibody is used to detect biotin linked to the composite biotin. In this method, the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibodies bind to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then expanded by incubating in a continuous solution of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, wherein each step adds additional biotin sites to the antibody / antigen complex. The expansion step is repeated until a suitable expansion level is reached, at which point the sample is incubated in a solution containing a second step antibody for biotin. The second step antibody is labeled, such as, for example, an enzyme labeled, which can be used to detect the presence of the antibody / antigen complex by histoenzymology using a chromogenic substrate. After appropriate expansion, a macroscopically visible conjugate can be produced.

[0158] Another known immunoassay method utilizes an immuno-PCR (polymerase chain reaction) method. Before incubation with biotinylated DNA, the PCR method is similar to the Cantor method, however, as an alternative to using multiple rounds of streptavidin and biotinylated DNA incubations, the DNA / biotin / streptavidin / antibody complex is washed off with a low pH or high salt buffer, which releases the antibody. The resulting washing solution is then used to react PCR with suitable primers and suitable controls. At least in theory, the huge amplification capacity and specificity of PCR can be used to detect single antigen molecules.

[0159] A.ELISA

[0160] Immunoassays are most simply binding assays. Certain preferred immunoassays are various types of enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blots, FACS analysis, etc. may also be used.

[0161] In one exemplary ELISA, an antibody of the present disclosure is immobilized on a selected surface that exhibits protein affinity, such as a well in a polystyrene microtiter plate. A test composition suspected of containing PD-L2 is then added to the well. After binding and washing to remove non-specifically bound immune complexes, the bound antigen can be detected. Detection can be achieved by adding another anti-PD-L2 antibody linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection can also be achieved by adding a second anti-PD-L2 antibody, followed by a third antibody that has binding affinity for the second antibody, wherein the third antibody is linked to a detectable label.

[0162] In another exemplary ELISA, a sample suspected of containing PD-L2 antigen is immobilized on a well surface and subsequently contacted with an anti-PD-L2 antibody. After binding and washing to remove non-specifically bound immune complexes, the bound anti-PD-L2 antibody is detected. When the initial anti-PD-L2 antibody is linked to a detectable label, the immune complex can be detected directly. Similarly, the immune complex can be detected using a second antibody that has binding affinity for the first anti-PD-L2 antibody, wherein the second antibody is linked to a detectable label.

[0163] Regardless of the format used, ELISA has certain common features, such as coating, incubation and binding, washing to remove non-specifically bound substances, and detection of bound immune complexes. These are described below.

[0164] In coating a plate with an antigen or antibody, the wells of the plate are usually incubated with a solution of the antigen or antibody overnight or for a specified period of time. The wells of the plate are then washed to remove incompletely adsorbed material. Any remaining available surface of the wells is then "coated" with a nonspecific protein that is antigenically neutral with respect to the tested antiserum. These include bovine serum albumin (BSA), casein, or milk powder solutions. Coating allows the nonspecific adsorption sites on the fixed surface to be blocked, and thus reduces the background caused by nonspecific binding of the antiserum on the surface.

[0165] In ELISA, it may be more customary to use secondary or tertiary detection methods rather than direct manipulation. Thus, after the protein or antibody is bound to the wells, coated with a non-reactive substance to reduce background, and washed to remove unbound material, the fixed surface is contacted with the biological sample to be tested under conditions effective to allow the formation of immune complexes (antigen / antibody). Detection of the immune complex then requires a labeled second binding ligand or antibody, and the second binding ligand or antibody in combination with a labeled tertiary antibody or tertiary binding ligand.

[0166] "Under conditions effective to allow immune complex (antigen / antibody) formation" means that the conditions preferably include diluting the antigen and / or antibody with a solution (e.g., BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS) / Tween). These added agents also tend to help reduce nonspecific background.

[0167] "Suitable" conditions also mean that the incubation is performed at a temperature or time sufficient to allow effective binding. The incubation step is generally performed at a temperature preferably about 25°C to 27°C for about 1 to 2 to 4 hours or can be performed at about 4°C overnight.

[0168] After all incubation steps in ELISA, the contacted surface is washed to remove uncomplexed material. A preferred washing process includes washing with a solution such as PBS / Tween or borate buffer. After the formation of specific immune complexes between the test sample and the initial binding material and subsequent washing, the appearance of even trace immune complexes can be determined.

[0169] In order to provide detection means, the second or third antibody has an associated label to allow detection. Preferably, this is an enzyme that produces color development after incubation with a suitable chromogenic substrate. Therefore, for example, it is desirable to contact or incubate primary and secondary immune complexes with antibodies conjugated with urease, glucose oxidase, alkaline phosphatase or catalase (for example, incubate for 2 hours at room temperature in a solution (e.g., PBS-Tween) containing PBS) at a time and under conditions conducive to further immune complex formation.

[0170] After incubation with the labeled antibody and subsequent washing to remove unbound material, the amount of label is quantified, for example by incubation with a chromogenic substrate such as urea or bromocresol purple or 2,2'-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) or H2O2 (in the case of peroxidase as enzyme label). Quantification is then achieved by measuring the degree of color produced, for example using a visible spectrum spectrophotometer.

[0171] B. Western blotting

[0172] Western blotting (or protein immunoblotting) is an analytical technique used to detect specific proteins in a given tissue homogenate or extract sample. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native / non-denaturing conditions). The proteins are then transferred to a membrane (usually nitrocellulose or PVDF) where they are probed (detected) using antibodies specific for the target protein.

[0173] The sample can be taken from the whole tissue or from cell culture. In most cases, the entity tissue is first mechanically broken using an agitator (for larger sample volumes), a homogenizer (smaller volumes) or by sonication. The cell can also be broken by one of the above-mentioned mechanical methods. However, it should be noted that bacteria, viruses or environmental samples can be the source of protein, and therefore Western blotting is not limited to cell research. A variety of detergents, salts and buffers can be used to promote cell lysis and dissolve proteins. Usually protease and phosphatase inhibitors are added to prevent the sample from being digested by its own enzymes. Tissue preparation is usually carried out at low temperatures to avoid protein denaturation.

[0174] Use gel electrophoresis to separate the protein of sample. Separation of protein can be carried out by isoelectric point (pI), molecular weight, charge or a combination of these factors. The nature of separation depends on the processing of the sample and the properties of the gel. This is a very useful method to determine protein. Two-dimensional (two-dimensional, 2-D) gels can also be used, which spread the protein from a single sample in two dimensions. In the first dimension, proteins are separated according to their isoelectric point (pH when the protein has a neutral net charge), and in the second dimension, proteins are separated according to their molecular weight.

[0175] In order to make the protein easy to carry out antibody detection, it is moved from the gel to a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF). The membrane is placed on the top of the gel, and a stack of filter paper is placed on top of it. The whole stack is placed in a buffer solution, which moves to the paper by capillary action, so that the protein moves therewith. Another method for transferring proteins is called electroblotting, and an electric current is used to pull the protein from the gel to the PVDF or nitrocellulose membrane. The protein moves from the gel to the membrane while maintaining the organization it has in the gel. As a result of the blotting process, the protein is exposed to a thin surface layer for detection (see below). These two membranes are selected because of their non-specific protein binding properties (that is, all proteins are equally well combined). Protein binding is based on hydrophobic interactions and charged interactions between membrane and protein. Nitrocellulose membrane is cheaper than PVDF, but it is more brittle and cannot withstand repeated detection 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 (Ponceau S) dyes. Once transferred, the protein is detected using either a labeled primary antibody or an unlabeled primary antibody followed by indirect detection using labeled protein A or a secondary labeled antibody that binds to the Fc region of the primary antibody.

[0176] C. Immunohistochemistry

[0177] Antibodies can also be used in combination with both fresh frozen and / or formalin fixed paraffin embedded tissue blocks prepared for study by immunohistochemistry (IHC). Methods for preparing tissue blocks from these pelleted samples have been successfully used in previous IHC studies of a variety of prognostic factors and are well known to those skilled in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990).

[0178] Briefly, cryosections can be prepared by rehydrating 50 ng of frozen "pulverized" tissue in phosphate-buffered saline (PBS) at room temperature in a small plastic capsule; pelleting the pellet by centrifugation; resuspending it in a viscous embedding medium (OCT); inverting the capsule and / or pelleting it again by centrifugation; snap freezing in -70°C isopentane; cutting the plastic capsule and / or removing the frozen tissue cylinder; mounting the tissue cylinder on a cryostat chuck; and / or cutting 25 to 50 serial sections from the capsule. Alternatively, the entire frozen tissue sample can be used for serial section cutting.

[0179] Permanent sections can be prepared by a similar method, which includes rehydrating 50 mg of sample in a plastic microcentrifuge tube; sedimentation; resuspending in 10% formalin for 4 hours; washing / sedimentation; resuspending in warm 2.5% agar; sedimentation; cooling in ice water to harden agar; removing the tissue / agar block from the tube; immersing and / or embedding the block in paraffin; and / or cutting up to 50 consecutive permanent sections. Likewise, the entire tissue sample can be replaced.

[0180] D. Immunoassay kit

[0181] In other embodiments, there is an immunoassay kit for use with the above-described immunoassay method. The immunoassay kit thus comprises a first antibody that binds to the PD-L2 antigen and optionally an immunoassay reagent in a suitable container means.

[0182] In certain embodiments, the PD-L2 antibody may be pre-bound to a solid support, such as a column matrix and / or a well of a microtiter plate. The immunodetection reagents of the kit may take any of a variety of forms, including those detectable labels associated or linked to a given antibody. Detectable labels associated or linked to a second binding ligand are also contemplated. Exemplary second ligands are those second antibodies that have binding affinity for the first antibody.

[0183] Other suitable immunodetection reagents for use in kits of the invention include two-component reagents comprising a second antibody having binding affinity to the first antibody, and a third antibody having binding affinity to the second antibody, the third antibody being linked to a detectable label. As described above, many exemplary labels are known in the art, and all such labels may be used in conjunction with the embodiments discussed herein.

[0184] The kit may also contain appropriate aliquots of a PD-L2 antigen composition, whether labeled or unlabeled, such as may be used to prepare a standard curve for a detection assay. The kit may contain the antibody-label conjugate in fully conjugated form, in intermediate form, or as a separate part to be conjugated by the user of the kit. The components of the kit may be packaged in an aqueous medium or in lyophilized form.

[0185] The container means of the test kit generally include at least one vial, test tube, flask, bottle, syringe or other container means in which the antibody can be placed, or preferably the antibody is appropriately aliquoted. The test kit also includes means for enclosing and restricting the antibody, antigen and any other reagent containers for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the desired vials are held.

[0186] VI. Examples

[0187] The following examples are included to demonstrate some preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention, and therefore can be considered to constitute preferred modes for its practice. However, it will be appreciated by those skilled in the art that, according to this disclosure, many changes may be made to the disclosed specific embodiments and still the same or similar results may be obtained without departing from the spirit and scope of the present invention.

[0188] Example 1 - Materials and Methods

[0189] Antibody Selection, Generation, and Production: Although additional details may be provided in the examples that follow, selection, generation, and production of the disclosed antibodies are generally performed as follows.

[0190] Materials and Methods - Antigens were biotinylated using the EZ-Link Sulfo-NHS-biotinylation kit from Pierce. Goat F(ab')2 anti-human κ-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 SouthernBiotech.

[0191] Initial Discovery - As previously described, eight initial human synthetic yeast libraries (approximately 10 9 For the first two rounds of selection, magnetic bead sorting using the Miltenyi MACS system was performed as previously described (see, e.g., Siegel et al., 2004). Briefly, yeast cells (approximately 10 10Cells / library) were incubated with 3 ml of 10 nM biotinylated Fc fusion antigen in wash buffer (phosphate buffered saline (PBS) / 0.1% bovine serum albumin (BSA)) 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, streptavidin microbeads (500 μl) were added to the 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 3 times with 3 ml of wash buffer. The column was then removed from the magnetic field, and the yeast was eluted with 5 mL of growth medium and then grown overnight. Subsequent rounds of selection were performed using flow cytometry. Approximately 2×10 7 Yeast, washed three times with wash buffer, and incubated at 30°C with 10 nM Fc fusion antigen or in later rounds with decreasing concentrations of biotinylated antigen (100 to 1 nM) under equilibrium conditions, with 100 nM biotinylated antigen of a different species (mouse) to obtain species cross-reactivity, or with poly-specificity depletion reagent (PSR) to remove non-specific antibodies from the selection. For PSR depletion, the library was incubated with a 1:10 dilution of biotinylated PSR reagent as previously described (see, e.g., Xu et al., 2013). Yeast were then washed twice with wash buffer and stained with LC-FITC (1:100 dilution) with SA-633 (1:500 dilution) or EAPE (1:50 dilution) secondary reagent for 15 minutes at 4°C. After washing twice with washing buffer, the cell pellet is resuspended in 0.3mL washing buffer and transferred to the sorting tube covered with a filter. Sorting is performed using a FACSARIA sorter (BD Biosciences), and a sorting gate is determined to select antibodies with desired characteristics. Rounds of selection are repeated until a colony with all desired characteristics is obtained. After the last round of sorting, yeast plates are inoculated and single colonies are selected for characterization.

[0192] Light chain batch shuffle (LCBS) - Key findings also include a light chain batch diversification protocol from heavy chain plasmids from initial selection: heavy chain plasmids from the initial fourth round of selection output were extracted from yeast and transformed to a diversity of 5×10 6 The selection was performed using the same conditions as the initial discovery, with one round of MACS and three rounds of FACS.

[0193] Antibody Optimization - Antibodies were optimized by introducing diversity into the heavy and light chain variable regions as described below. A combination of some of these approaches was used for each antibody.

[0194] CDRH1 and CDRH2 selection: CDRH3 of a single antibody was recombined to have a diversity of 1 × 10 8 Premade libraries of CDRH1 and CDRH2 variants of 1000 bp were prepared and selected with one round of MACS and four rounds of FACS as described in the initial discovery. In the FACS rounds, the libraries were investigated for PSR binding, species cross-reactivity, antigen cross-reactivity, and affinity pressure, and sorted to obtain populations with the desired characteristics. For these selections, affinity pressure was applied by titrating biotinylated monomeric antigen or pre-incubating the biotinylated antigen with the parent Fab for 30 minutes and then applying the pre-complexed mixture to the yeast library for the length of time that the selection was allowed to reach equilibrium. Then, antibodies with higher affinity could be sorted.

[0195] VH Mut selection: The heavy chain variable region (VH) was mutagenized by error-prone PCR. The library was then created by transforming the mutagenized VH and heavy chain expression vectors into yeast already containing the parental light chain plasmid. The selection was performed similarly to the previous cycle using three rounds of FACS sorting. In the FACS rounds, the library was studied for cross-reactivity and affinity pressure and sorted to obtain a population with the desired characteristics.

[0196] CDRL1, CDRL2, and CDRL3 selection: Oligos were ordered from IDT that contained CDRL3 and were diversified by NNK diversity. CDRL3 oligos were duplexed using primers that annealed to the flanking regions of CDRL3. These duplexed CDRL3 oligos were then recombined to have a diversity of 3 × 10 5 Pre-made libraries of CDRL1 and CDRL2 variants of 1000 bp were prepared and selected with one round of MACS and three rounds of FACS as described in the initial discovery. In the FACS rounds, the libraries were investigated for PSR binding, cross-reactivity, and affinity pressure, and sorted to obtain populations with the desired characteristics. Affinity pressure for these selections was performed as described above in the CDRH1 and CDRH2 selections.

[0197] Antibody production and purification - Yeast clones were grown to saturation and then induced with shaking at 30°C for 48 hours. After induction, yeast cells were precipitated and supernatants were harvested for purification. IgG was purified using a protein A column and eluted with acetic acid pH 2.0. Fab fragments were produced by papain digestion and purified by KappaSelect (GE Healthcare Life Sciences).

[0198] ForteBio K D Measurement - ForteBio affinity measurements were performed on Octet RED384 as described previously (see, e.g., Estep et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs 5(2), 270-278(2013)). In brief, ForteBio affinity measurements were performed by loading IgG online onto an AHQ sensor. The sensor was balanced offline in assay buffer for 30 minutes, and then monitored online for 60 seconds to establish a baseline. The sensor loaded with IgG was exposed to 100 nM antigen for 3 minutes, and then transferred to assay buffer for 3 minutes for off-rate measurement. For monovalent affinity evaluation, Fab was used instead of IgG. For this evaluation, non-biotinylated Fc fusion antigen was loaded online onto an AHQ sensor. The sensor was balanced offline in assay buffer for 30 minutes, and then monitored online for 60 seconds to establish a baseline. Antigen loaded sensors were exposed to 100 nM Fab for 3 min and then transferred to assay buffer for 3 min for off-rate measurements. All kinetics were analyzed using a 1:1 binding model.

[0199] ForteBio Epitope Binning / Ligand Blocking - Epitope Binning / Ligand Blocking was performed using a standard sandwich format cross-blocking assay. A control anti-target IgG was loaded onto the AHQ sensor, and unoccupied Fc binding sites on the sensor were blocked with an irrelevant human IgG1 antibody. The sensor was then exposed to 100 nM target antigen, followed by exposure to a second anti-target antibody or ligand. Additional binding of the second antibody or ligand after antigen association indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blocking (competitor or ligand blocking).

[0200] MSD-SET K DMeasurement - Equilibrium affinity measurements of selected high affinity antibodies were generally performed as previously described (Estep et al., Mabs, Vol. 5 (2), pp. 270-278 (2013)). Briefly, solution equilibrium titration (SET) was performed in PBS + 0.1% IgG-free BSA (PBSF) with the antigen kept constant at 50 pM, and incubated with Fab serially diluted 3 to 5 times starting from 20 nM. Antibodies (20 nM in PBS) were coated onto standard binding MSD-ECL plates at 4 ° C overnight or at room temperature for 30 minutes. The plates were then blocked by BSA for 30 minutes at 700 rpm shaking, followed by washing 3 times with wash buffer (PBSF + 0.05% Tween 20). SET samples were applied and incubated on the plates for 150 seconds at 700 rpm shaking, followed by washing once. Antigen captured on the plate was detected with 250 ng / mL sulfo-tagged streptavidin in PBSF by incubating the plate for 3 minutes. The plate was washed 3 times with wash buffer and then read on an MSD Sector Imager 2400 instrument using 1x read buffer T with surfactant. The percentage of free antigen was plotted in Prism as a function of titrated antibody and a quadratic equation was fitted to extract K D To improve the workload, a liquid handling robot was used throughout the MSD-SET experiments, including SET sample preparation.

[0201] Cell binding assay - Approximately 100,000 cells overexpressing 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 on ice for 15 minutes. The cells were then washed twice with wash buffer and analyzed on a FACS Canto II analyzer (BD Biosciences).

[0202] Antibody screening and characterization: The antibody candidates generated by the above-mentioned display method were tested for their ability to bind to PD-L2 and block its binding to PD-1. 5 μg / mL of antibody was bound to CHO-PD-L2 cells, and recombinant PD-1 (RnD Systems) labeled with Alexa 532 (ThermoFisher) was subsequently added for 1 hour. The maximum fluorescence intensity of PD-1 was measured. The blocking of PD-1 binding was measured by flow cytometry by the reduction of Alexa 532 fluorescence. In order to generate an affinity KD for human PD-L2, the anti-PD-L2 Ab was loaded onto an anti-human Fc capture (AHC) biosensor at 100 nM (15 μg / mL) and tested for human PD-L2 protein association and dissociation in a dilution series from 30 to 0.37 nM. The binding and release of the analyte (PD-L2) were recorded in real time by the Octet instrument and subsequently used to calculate KD, K on and K dis ;The results are derived from 2: 1 global fit modeling minus the reference well. In order to generate the affinity KD for mouse PD-L2, the anti-PD-L2 Ab was covalently fixed to the activated amine-reactive second generation (AR2G) biosensor (quenched with 1M ethanolamine pH 8.5 after protein loading) at 100nM (15μg / mL), and the mouse PD-L2 protein association and dissociation were tested in a dilution series of 300 to 1nM. The binding and release of the analyte (PD-L2) were recorded in real time by the Octet instrument and then used to calculate KD, K on and K dis ; Results are derived from 2:1 global fit modeling minus the reference well.

[0203] Antibody activity: Different concentrations of PD-L2 antibodies with a human IgG1 backbone were added to CHO cells expressing human or mouse PD-L2 (CHO-PD-L2 cells). Binding was detected by adding a secondary anti-human IgG1 antibody conjugated to phycoerythrin (PE). FACS analysis was performed to detect phycoerythrin to determine the fluorescence activity at different antibody concentrations. GraphPad The software calculates EC50.

[0204] Candidate antibodies block PD-1 / PD-L2 binding: Candidate PD-L2 monoclonal antibodies and FDA-approved antibodies were assayed using the Promega PD-L2:PD-1 blocking system. Antibodies were added to CHO-PD-L2 cells at varying concentrations. PD-1 effector cells are Jurkat T cells that can be stimulated by CHO-PD-L2 cells. PD-1 effector cells, which produce firefly luciferase in response to activation, were incubated with antibodies and CHO cells for 6 hours, and the results were then read on a luminometer using the Bio-glo assay kit (Promega) according to the manufacturer's instructions. For competition assays, biotin-rhPD-1-Fc protein was added to the cells and antibodies, followed by the addition of streptavidin-APC conjugate. Blocking was assessed as an increase in luciferase signal. GraphPad For competition assays, × was converted to LogX and analyzed by nonlinear regression (curve fitting), dose-response inhibition, and Log(inhibitor) vs response.

[0205] PD-L2 activity in mixed lymphocyte reaction: CD14+ monocytes were isolated from peripheral blood mononuclear cells using CD14 microbeads. The cells were seeded at 1 million / ml in 10% FCS / RPMI / P / S cell culture medium and stimulated with IL-4 and GM-CSF. The cells were cultured for 7 days to differentiate into immature dendritic cells (IDCs) and different concentrations of PD-L2 or commercially available antibodies were added. CD4+ T cells were then stimulated with IDCs at a ratio of 10:1CD4:IDC. IL-2 and IFN-γ were determined by ELISA according to the protocol provided by R&D systems.

[0206] Antibody activity against xenograft tumors: U2940PMBL or MDA-MB-231 triple-negative breast cancer xenograft tumors were established in immunodeficient mice. Tumors were allowed to reach 150 mm 3 volume. When reaching 150mm 3 Thereafter, mice were treated twice weekly with the indicated antibody therapeutics, 9 mice per treatment group, at 10 mg / kg for 3 weeks. Caliper measurements of tumor width, length, and depth were used to calculate tumor volume.

[0207] Survival of the αPD-L1-resistant EL4 lymphoma mouse model: The survival of mice injected with αPD-L1-resistant EL4 cells expressing PD-L2 and luciferase was measured. 5EL4 cells expressing PD-L2 and luciferase were injected into the tail vein of mice. On days 3, 6, 9, 12, and 15, mice were treated intraperitoneally with 100 μg of the indicated antibodies.

[0208] Epitope binning of PD-L2 antibodies: using ForteBio The binding competition assay performed by the platform compared the binding specificity of various PD-L2 antibodies. The HIS-tagged target protein (human PD-L2) was loaded onto a pre-charged nickel NTA biosensor at 1 μg / mL. The first antibody Ab1 was saturated at 100nM on the biosensor loaded with the target, and a reference (buffer only) well was included to determine the maximum Ab2 binding signal. The binding signal of the second Ab2 was also screened at 100nM, and Ab1 was included to determine the background self-blocking signal. Data Analysis HT 9.0 software was used to generate a matrix of the raw signal response of Ab2 binding, which was then converted to a percentage of the unblocked Ab2 binding signal. Less than 15% response is considered competitive blocking.

[0209] Example 2 - Results

[0210] Identification of PD-L2 antibodies that block PD-L2 binding to PD-1: The antibody candidates identified as described above in Example 1 were tested for their ability to bind to PD-L2 and block its binding to PD-1. The maximum fluorescence intensity of PD-1 labeled with Alexafluor 532 was measured, where PD-L2 blocking was seen as a decrease in Alexa532 fluorescence in FACS analysis. Anti-PD-L2 clones 16501, 16425, 16510, and 16478 were evaluated against unstained cells, control Ig and antibodies, and the commercially available antibody 24F. It was found that the binding of PD-L2 to PD-1 was significantly reduced using clones 16501, 16425, and 16478. The subclones were tested using a PD-L2: PD-1 assay using CHO-PD-L2 cells that can stimulate Jurkat T cells that produce luciferase in response to activation ( Figure 2 IC was generated from the PD-L2:PD-1 assay curve. 50 The clones were also evaluated for their ability to block PD-L2:PD-1 binding ( Figure 3 ). It was found that each clone evaluated (16501, 20810, 20237) blocked PD-L2 binding to PD-1 better than the FDA-approved antibody therapeutic Keytruda ( Figure 3 ).

[0211] PD-L2 Antibodies Affine Binding Human PD-L2: At ForteBio Affinity curves for subclones 16425, 16501, 20810, 20811, and 20816 were generated on the ELISA kit. The antibody subclones were immobilized on an anti-human Fc capture (AHC) biosensor at 100 nM (15 μg / mL) and tested for human PD-L2 protein association and dissociation in a dilution series from 30 to 0.37 nM ( Figures 4A to 4B ). Based on the data, the affinities of subclones 20810, 20811, and 20816 were calculated (Table 5). As the antibody concentration increased, it was observed that the addition of anti-PD-L2 blocked the binding of PD-1-FC to human PD-L2 ( Figure 4C ).

[0212] Equivalence to FDA-approved PD-1 / PD-L2: Candidate antibodies targeting PD-L2 were tested using the Promega PD-L2:PD-1 Blockade System. It was found that 20810, 20811, and 20816 each caused significant sequestration of PD-2 from PD-1, thereby inducing the production of firefly luciferase, compared to isotype controls ( Figure 5 The candidate antibodies induced luciferase production at least equivalent to that of FDA-approved immunotherapeutics: Keytruda and Tecentriq ( Figure 5 ).

[0213] The candidate antibodies were active in various human mixed lymphocyte reactions: The candidate antibodies were evaluated in the presence of induced dendritic cells and T cells from different donors. It was found that 20811, 20814 and 20816 induced more IL-2 production compared to the isotype control ( Figures 6A to 6B ). Treatment with candidate antibody 20814 induced IFN-γ production that exceeded isotype control at treatments above 10 nM. Combination anti-PD-L1 + 20814 treatment significantly induced IFN-γ production, better than either Keytruda or PD-L1 mAb alone ( Figure 6C ).

[0214] Candidate antibodies with ADCC are highly active against human U2940 lymphoma in vivo: After establishing PBML xenograft tumors in SCID mice, mice were treated with mIgG2a control antibody, Herceptin, Rituxan, or candidate antibody 20810. When compared to Herceptin, 20810 showed significant reduction in tumor growth during the treatment course, which was similar to Rituxan ( Figure 7 Similarly, 20810 also reduced MDA-MB-231 xenograft tumor growth as well as or better than avelumab or rituximab ( Figure 8 ).

[0215] Survival of the αPD-L1-resistant EL4 lymphoma mouse model: The survival of mice injected with αPD-L1-resistant EL4 cells expressing PD-L2 and luciferase was measured. 5 EL4 cells expressing PD-L2 and luciferase were injected into the tail vein of mice. Mice were treated intraperitoneally with 100 μg of the indicated antibodies on days 3, 6, 9, 12, and 15. Treatment with the anti-PD-L2 candidate 20810 increased survival compared to treatment with PD-L1 mAb or no treatment ( Fig. 9 ).

[0216] Candidate Antibody Binding Overlap: Using ForteBio The percent overlap of candidate antibodies was evaluated in a tandem competition assay. Based on the competition studies, candidate antibodies 20810 and 16510 bind to very similar epitopes on PD-L2 (Table 6).

[0217] Table 1 - Nucleotide sequences of antibody variable regions

[0218]

[0219]

[0220] Table 2 – Protein sequences of antibody variable regions

[0221]

[0222] Table 3 - CDR heavy chain sequences

[0223]

[0224] Table 4 - CDR light chain sequences

[0225]

[0226] Table 5 – Affinity measurements of antibodies binding to PD-L2

[0227]

[0228] Table 6 - Antibody overlap

[0229]

[0230] ***

[0231] According to the present disclosure, all methods disclosed and claimed herein can be carried out and implemented without excessive experimentation. Although the compositions and methods of the present invention have been described according to some preferred embodiments, it is obvious to those skilled in the art that the steps or the order of steps of the methods described herein and the methods can be changed without departing from the concept, spirit and scope of the present invention. More specifically, it is obvious that certain reagents related in both chemistry and physiology can be substituted for the reagents described herein while achieving the same or similar results. All these similar substitutions and modifications obvious 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.

[0232] VII. References

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[0321] The present invention also relates to the following embodiments (which correspond to the claims of the original application):

[0322] 1. An antibody or antibody fragment comprising a heavy chain CDR sequence and a light chain CDR sequence of a clone pair from Table 3 and Table 4, respectively.

[0323] 2. The antibody or antibody fragment of embodiment 1, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence according to the clone pairing sequence from Table 1.

[0324] 3. The antibody or antibody fragment of embodiment 1, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence that are at least 70%, 80% or 90% identical to the cloned mate sequence from Table 1.

[0325] 4. The antibody or antibody fragment of embodiment 1, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence that are at least 95% identical to the cloned pairing sequence from Table 1.

[0326] 5. The antibody or antibody fragment of embodiment 1, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence according to the clone pairing sequence from Table 2.

[0327] 6. The antibody or antibody fragment of embodiment 1, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence that are 70%, 80% or 90% identical to the cloned mate sequence from Table 2.

[0328] 7. The antibody or antibody fragment of embodiment 1, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence that are 95% identical to the cloned paired sequence from Table 2.

[0329] 8. The antibody or antibody fragment of embodiments 1 to 7, wherein the antibody fragment is a recombinant scFv (single-chain variable fragment) antibody, a Fab fragment, a F(ab')2 fragment or a Fv fragment.

[0330] 9. The antibody or antibody fragment of embodiments 1 to 7, wherein the antibody is a chimeric antibody.

[0331] 10. The antibody or antibody fragment of embodiments 1 to 9, wherein the antibody is IgG.

[0332] 11. The antibody or antibody fragment of embodiments 1 to 10, wherein the antibody or antibody fragment further comprises a cell penetrating peptide and / or is an intracellular antibody.

[0333] 12. The antibody or fragment of embodiments 1 to 11, wherein the antibody or antibody fragment is a human antibody.

[0334] 13. The antibody or fragment of embodiments 1 to 11, wherein the antibody or antibody fragment is a humanized antibody.

[0335] 14. A method of treating a subject having cancer, comprising delivering to the subject an antibody or antibody fragment having a heavy chain CDR sequence and a light chain CDR sequence of a clonal pairing from Table 3 and Table 4, respectively.

[0336] 15. The method of embodiment 14, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence of the clone pair shown in Table 1.

[0337] 16. The method of embodiments 14 to 15, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence that are paired with a clone having 95% identity as shown in Table 1.

[0338] 17. The method of embodiments 14 to 15, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence that are 70%, 80% or 90% identical to the cloned pairing sequence from Table 1.

[0339] 18. The method of embodiment 14, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence according to the clone pairing sequence from Table 2.

[0340] 19. The method of embodiment 14, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence that are 70%, 80% or 90% identical to the cloned matching sequence from Table 2.

[0341] 20. The method of embodiment 14, encoded by a light chain variable sequence and a heavy chain variable sequence that have 95% identity with the cloned matching sequence from Table 2.

[0342] 21. The method of embodiments 14 to 20, wherein the antibody fragment is a recombinant scFv (single-chain variable fragment) antibody, a Fab fragment, a F(ab')2 fragment or a Fv fragment.

[0343] 22. The method of embodiments 14 to 21, wherein the antibody is IgG.

[0344] 23. The method of embodiments 14 to 20, wherein the antibody is a chimeric antibody.

[0345] 24. The method of embodiments 14 to 23, wherein the delivery comprises administration of an antibody or antibody fragment, or gene delivery using an RNA or DNA sequence or vector encoding the antibody or antibody fragment.

[0346] 25. A hybridoma or modified cell encoding an antibody or antibody fragment, wherein the antibody or antibody fragment is characterized by a heavy chain CDR sequence and a light chain CDR sequence of a clone pair from Table 3 and Table 4, respectively.

[0347] 26. The hybridoma or modified cell of embodiment 25, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence according to the clone pairing sequence from Table 1.

[0348] 27. The hybridoma or modified cell of embodiment 25, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence that are at least 70%, 80% or 90% identical to the variable sequence paired with a clone from Table 1.

[0349] 28. The hybridoma or modified cell of embodiment 25, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence that are 95% identical to the variable sequence paired with a clone from Table 1.

[0350] 29. The hybridoma or modified cell of embodiment 25, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence according to the clone pairing sequence from Table 2.

[0351] 30. The hybridoma or modified cell of embodiment 25, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence that are at least 70%, 80% or 90% identical to the variable sequence paired with a clone from Table 2.

[0352] 31. The hybridoma or modified cell of embodiment 25, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence that are 95% identical to the cloned matching sequence from Table 2.

[0353] 32. The hybridoma or engineered cell of embodiments 25 to 31, wherein the antibody fragment is a recombinant scFv (single-chain variable fragment) antibody, a Fab fragment, a F(ab')2 fragment, or a Fv fragment.

[0354] 33. The hybridoma or engineered cell of embodiments 25 to 32, wherein the antibody is a chimeric antibody.

[0355] 34. The hybridoma or engineered cell of embodiments 25 to 32, wherein the antibody is IgG.

[0356] 35. The hybridoma or engineered cell of embodiments 25 to 34, wherein the antibody or antibody fragment further comprises a cell penetrating peptide and / or is an intracellular antibody.

[0357] 36. A vaccine formulation comprising one or more antibodies or antibody fragments characterized by heavy chain CDR sequences and light chain CDR sequences of clone pairs from Table 3 and Table 4, respectively.

[0358] 37. The vaccine formulation of embodiment 36, wherein at least one antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence according to the clone pairing sequences from Table 1.

[0359] 38. The vaccine formulation of embodiment 36, wherein at least one antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence that are at least 70%, 80% or 90% identical to the clone pairing sequences from Table 1.

[0360] 39. The vaccine formulation of embodiment 36, wherein at least one antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence that are at least 95% identical to the clone pairing sequences from Table 1.

[0361] 40. The vaccine formulation of embodiment 36, wherein at least one antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence according to the clone pairing sequences from Table 2.

[0362] 41. The vaccine formulation of embodiment 36, wherein at least one antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence that are 95% identical to the cloned pairing sequences from Table 2.

[0363] 42. The vaccine formulation of embodiments 36 to 41, wherein at least one antibody fragment is a recombinant scFv (single-chain variable fragment) antibody, a Fab fragment, a F(ab')2 fragment, or a Fv fragment.

[0364] 43. The vaccine formulation of embodiments 36 to 41, wherein at least one antibody is a chimeric antibody.

[0365] 44. The vaccine formulation of embodiments 36 to 43, wherein at least one antibody is IgG.

[0366] 45. The vaccine formulation of embodiments 36 to 44, wherein at least one antibody or antibody fragment further comprises a cell penetrating peptide and / or is an intracellular antibody.

[0367] 46. ​​A method for detecting cells expressing PD-L2 in a subject, comprising:

[0368] (a) contacting a sample from the subject with an antibody or antibody fragment having heavy chain CDR sequences and light chain CDR sequences of clone pairs from Tables 3 and 4, respectively; and

[0369] (b) detecting cells expressing PD-L2 in the sample by allowing the antibody or antibody fragment to bind to the cells in the sample.

[0370] 47. The method of embodiment 46, wherein the sample is a body fluid.

[0371] 48. The method of embodiments 46 to 47, wherein the sample is a tissue sample.

[0372] 49. The method of embodiments 46 to 47, wherein detection comprises ELISA, RIA or Western blot.

[0373] 50. The method of embodiments 46 to 49, further comprising performing steps (a) and (b) again and determining the change in the level of orthopoxvirus antigen compared to the first determination.

[0374] 51. The method of embodiments 46 to 50, wherein the antibody or antibody fragment is encoded by a variable sequence of a clone pair shown in Table 1.

[0375] 52. The method of embodiments 46 to 50, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence that are 70%, 80% or 90% identical to the variable sequences of the clone pairing shown in Table 1.

[0376] 53. The method described in embodiments 46 to 50, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence that have 95% identity with the clone pairing sequences shown in Table 1.

[0377] 54. The method of embodiments 46 to 50, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence according to the clone pairing sequence from Table 2.

[0378] 55. The method of embodiments 46 to 50, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence that are 70%, 80% or 90% identical to the cloned matching sequence from Table 2.

[0379] 56. The method of embodiments 46 to 50, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence that are 95% identical to the cloned matching sequence from Table 2.

[0380] 57. The method described in embodiments 46 to 56, wherein the antibody fragment is a recombinant ScFv (single-chain variable fragment) antibody, a Fab fragment, a F(ab')2 fragment or a Fv fragment.

[0381] 58. The method of embodiments 46 to 57, wherein the cell is a cancer cell.

[0382] 59. The method of embodiment 58, wherein the cancer cell is a lymphoma cell, a breast cancer cell, or a renal cell carcinoma cell.

[0383] 60. The method of embodiments 46 to 57, wherein the cell is a cell associated with immunosuppression.

[0384] 61. The method of embodiment 60, wherein the cells associated with immunosuppression are non-cancerous cells in the tumor microenvironment.

[0385] 62. The method described in embodiment 61, wherein the non-cancerous cells in the tumor microenvironment are stromal cells or endothelial cells.

[0386] 63. A method for treating immunosuppression in a tumor microenvironment, comprising: delivering to the subject an antibody or antibody fragment having a heavy chain CDR sequence and a light chain CDR sequence of a clone pair from Table 3 and Table 4, respectively.

Claims

1. An antibody or antibody fragment comprising heavy and light chain CDR 1-3 sequences, wherein the heavy and light chain CDR 1-3 sequences comprise SEQ ID NOs: 22, 23 and 24, and SEQ ID NOs: 37, 38 and 39, respectively, SEQ ID NOs: 25, 26 and 27, and SEQ ID NOs: 40, 41 and 42, respectively, SEQ ID NOs: 31, 32 and 33, and SEQ ID NOs: 46, 47 and 48, or SEQ ID NOs: 34, 35 and 36, and SEQ ID NOs: 49, 50 and 51, respectively.

2. The antibody or antibody fragment of claim 1, comprising heavy chain and light chain CDR 1-3 sequences, wherein the heavy chain and light chain CDR 1-3 sequences comprise SEQ ID NOs: 34, 35 and 36, and SEQ ID NOs: 49, 50 and 51, respectively.

3. The antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence comprising SEQ ID NOs: 3 and 2, SEQ ID NOs: 5 and 4, SEQ ID NOs: 9 and 8, and SEQ ID NOs: 11 and 10, respectively.

4. The antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence that are at least 70%, 80%, 90% or 95% identical to SEQ ID NOs: 3 and 2, SEQ ID NOs: 5 and 4, SEQ ID NOs: 9 and 8, and SEQ ID NOs: 11 and 10, respectively.

5. The antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence, and the light chain variable sequence and the heavy chain variable sequence comprise SEQ ID NOs: 13 and 12, SEQ ID NOs: 15 and 14, SEQ ID NOs: 19 and 18, and SEQ ID NOs: 21 and 20, respectively.

6. The antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence that are 70%, 80%, 90% or 95% identical to SEQ ID NOs: 13 and 12, SEQ ID NOs: 15 and 14, SEQ ID NOs: 19 and 18, and SEQ ID NOs: 21 and 20, respectively.

7. The antibody or antibody fragment of claim 1, wherein the antibody fragment is a recombinant scFv (single-chain variable fragment) antibody, a Fab fragment, a F(ab')2 fragment or a Fv fragment.

8. The antibody or antibody fragment of claim 1, wherein the antibody is IgG.

9. The antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment further comprises a cell penetrating peptide and / or is an intrabody.

10. The antibody or fragment of claim 1, wherein the antibody or antibody fragment is a human antibody.

11. Use of the antibody or antibody fragment of claim 1 in the preparation of a medicament for treating a subject suffering from cancer, wherein the medicament is formulated for delivery of the antibody or antibody fragment to the subject, wherein the cancer cells and / or immune cells of the subject express PD-L2.

12. The use of claim 11, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence that are 70%, 80%, 90% or 95% identical to SEQ ID NOs: 3 and 2, SEQ ID NOs: 5 and 4, SEQ ID NOs: 9 and 8, and SEQ ID NOs: 11 and 10, respectively.

13. The use of claim 11, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence, and the light chain variable sequence and the heavy chain variable sequence comprise SEQ ID NOs: 13 and 12, SEQ ID NOs: 15 and 14, SEQ ID NOs: 19 and 18, and SEQ ID NOs: 21 and 20, respectively.

14. The use of claim 11, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence that are 70%, 80%, 90% or 95% identical to SEQ ID NOs: 13 and 12, SEQ ID NOs: 15 and 14, SEQ ID NOs: 19 and 18, and SEQ ID NOs: 21 and 20, respectively.

15. A hybridoma or engineered cell encoding the antibody or antibody fragment according to claim 1.

16. A pharmaceutical formulation comprising one, two, three or all four antibodies or antibody fragments according to claim 1.

17. The pharmaceutical formulation of claim 16, wherein at least one antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence that are at least 70%, 80%, 90% or 95% identical to SEQ ID NOs: 3 and 2, SEQ ID NOs: 5 and 4, SEQ ID NOs: 9 and 8, and SEQ ID NOs: 11 and 10, respectively.

18. The pharmaceutical formulation of claim 16, wherein at least one antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence that are 70%, 80%, 90% or 95% identical to SEQ ID NOs: 13 and 12, SEQ ID NOs: 15 and 14, SEQ ID NOs: 19 and 18, and SEQ ID NOs: 21 and 20, respectively.

19. Use of the antibody or antibody fragment of claim 1 in the preparation of a kit for detecting cells expressing PD-L2 in a subject, wherein the kit is formulated for: (a) contacting a sample from the subject with the antibody or antibody fragment; and (b) detecting cells expressing PD-L2 in the sample by allowing the antibody or antibody fragment to bind to the cells in the sample.

20. Use of the antibody or antibody fragment of claim 1 in the preparation of a medicament for treating immunosuppression in a tumor microenvironment comprising PD-L2 in a subject, wherein the medicament is formulated for delivery of the antibody or antibody fragment to the subject.

21. The pharmaceutical preparation of claim 16, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence, and the light chain variable sequence and the heavy chain variable sequence comprise SEQ ID NOs: 13 and 12, SEQ ID NOs: 15 and 14, SEQ ID NOs: 19 and 18, and SEQ ID NOs: 21 and 20, respectively.

22. The use of claim 11, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence comprising SEQ ID NOs: 3 and 2, SEQ ID NOs: 5 and 4, SEQ ID NOs: 9 and 8, and SEQ ID NOs: 11 and 10, respectively.

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