Anti-PD-L2 antibodies

Abstract

The present invention relates to an isolated monoclonal antibody that specifically binds to Programmed Death Ligand 2 (PD-L2) and blocks activity against PD-L2 / hPD1 interaction, wherein the antibody comprises six CDR sequences in a heavy chain variable region and a light chain variable region, and comprises as heavy chain variable region CDRs, a CDRH1 region of SEQ ID NO: 13 (GYAFSNYFIE) or SEQ ID NO: 14 (GYSFSNYFIE); a CDRH2 region of SEQ ID NO: 15 (LNIPGSGGSNYAEKFKG); and a CDRH3 region of SEQ ID NO: 16 (RRLPPDWYFDV); and as light chain region CDRs, a CDRL1 region of SEQ ID NO: 17 (RSSQSLVHSGGNTYLH) or SEQ ID NO: 20 (RSSQSLVHSDGNTYLH); a CDRL2 region of SEQ ID NO: 18 (KVSNRFS); and a CDRL3 region of SEQ ID NO: 19 (SQSTHVPWT).

Description

[Technical field]

[0001] The present invention relates to isolated monoclonal antibodies that bind to Programmed Death Ligand 2 (PD-L2) and their uses as medicines.

[0002] Senescent cells have been found to accumulate in tissues and organs during the aging process, closely resembling age-related pathologies, where they play a key role in the development and progression of age-related diseases and disorders. Cellular senescence is a complex stress response in which cells irreversibly lose their proliferation capacity, and is accompanied by numerous changes in gene expression. Many potentially oncogenic insults induce the senescence response, which is recognized as a powerful tumor suppressor mechanism. Other stimuli that induce senescence include radiation, genotoxic drugs, tissue damage and remodeling, and metabolic derangements. Thus, chemotherapy and radiotherapy both induce the senescence response. Senescent cells may persist chronically, promoting local and systemic inflammation associated with undesirable secondary effects of chemotherapy and tumor recurrence.

[0003] Human PD-1 (hPD-1) was previously identified using a subtraction cloning-based approach to select proteins involved in apoptotic cell death (Yasumasa Ishida, Cells. 2020 Jun; 9(6): 1376). hPD-1 is a member of the CD28 / CTLA-4 molecular family based on its ability to bind to PD-L1. Like CTLA4, hPD-1 is rapidly induced on the surface of T cells in response to anti-CD3. However, in contrast to CTLA4, hPD-1 is also induced on the surface of B cells (in response to anti-IgM). hPD-1 is also expressed on a subset of thymocytes and myeloid cells. The hPD-1 polypeptide is an inhibitory receptor that can transmit inhibitory signals to immune cells to inhibit their effector functions or, when present, for example, in a soluble, monomeric form, can promote costimulation of immune cells (e.g., by competitive inhibition). An example of human PD-1 is shown in UniProtKB / Swiss-Prot accession number Q15116.

[0004] PD-1 has two ligands: programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2). In cancer, PD-L1 and PD-L2 are expressed on the surface of tumor cells and promote T cell exhaustion. This suggests that PD-L1 and PD-L2 play a role in tumor immune evasion. In the tumor microenvironment, T cell exhaustion begins when hPD-1 activity steadily increases with repeated exposure to tumor antigens. That is, with increased uncontrolled hPD-1 signaling, T cells begin to lose their ability to respond. Over time, exhausted T cells become progressively helpless and lose critical functions such as the ability to proliferate, fight tumor cells, and ultimately survive. Tumor-infiltrating T cells in solid tumors and hematological malignancies often show evidence of exhaustion, which includes upregulation of hPD-1 and other immune function inhibitors, reduced production of cytokines and cell signaling molecules that help induce immune responses, and a reduced ability to kill tumor cells. An exemplary human PD-L2 is shown in UniProtKB / Swiss-Prot Accession No. Q9BQ51.

[0005] Chaib, “PD-L2 mediates immune evasion of chemotherapy-treated tumors and protects from tissue injury”, PhD thesis, UNIVERSIDAD AUTOONOMA DE MADRID, 2019, discloses that PD-L2 is upregulated by various cells upon induction of senescence or injury. Furthermore, it is disclosed that in the case of cancer, this upregulation leads to immune evasion after chemotherapy, whereas in the case of tissue injury, this upregulation protects the host / organ from excessive tissue injury.

[0006] WO2021 / 197358 discloses a PD-L1 nanobody, a PD-L2 nanobody, and a bispecific antibody having both PD-L1 nanobody and PD-L2 nanobody. The bispecific antibody has good binding activity to both PD-L1 and PD-L2 molecules, and can block the interaction between PD-1 and PD-L1 and between PD-1 and PD-L2. It can interact and simultaneously block the PD-L1 / PD-1 and PD-L2 / PD-1 signaling pathways in vitro, and activate the expression of downstream reporter genes, thus having good antitumor activity. In addition, the nanobody only contains the variable region of the heavy chain, so it has no effector function. WO2020 / 036635 discloses a composition for treating cancer in a subject, comprising administering to the subject an effective amount of a nucleic acid encoding p53 and / or a nucleic acid encoding MDA-7, and at least one of a CD122 agonist and a CD132 agonist. Such a CD122 / CD132 agonist could be an IL-2 / anti-IL-2 immune complex.None of these documents address the issue of senescent cells. Summary of the Invention [Problem to be solved by the invention]

[0007] WO2019002581A1 discloses inhibitors of PD-1 or PD-L2 expression to eliminate damaged and / or senescent cells in various diseases, including cancer. It further describes that PD-L2 is highly expressed in senescent tumor cells and induces immune tolerance. However, the antitumor activity of the anti-PD-L2 agent is unsatisfactory, and there is no evidence that it can eliminate senescent cells.

[0008] An object of the present invention is to provide an agent for treating, ameliorating, or preventing a disease or condition associated with the presence of senescent cells in cancerous tissue, the agent promoting immune clearance of senescent cells in said tissue. [Means for solving the problem]

[0009] Said object is solved by an antibody according to claim 1. Further preferred embodiments are the subject of dependent claims 2 to 17.

[0010] In one aspect, the present invention relates to an isolated monoclonal antibody that specifically binds to Programmed Death Ligand 2 (PD-L2) and blocks activity against hPD-L2 / hPD1 interaction, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and comprises: a) as heavy chain variable region CDRs, a CDRH1 region of SEQ ID NO: 13 (GYAFSNYFIE) or SEQ ID NO: 14 (GYSFSNYFIE); a CDRH2 region of SEQ ID NO: 15 (LNIPGSGGSNYAEKFKG); and a CDRH3 region of SEQ ID NO: 16 (RRLPPDWYFDV); and b) As light chain region CDRs, a CDRL1 region of SEQ ID NO: 17 (RSSQSLVHSGGNTYLH) or SEQ ID NO: 20 (RSSQSLVHSDGNTYLH); a CDRL2 region of SEQ ID NO: 18 (KVSNRFS); and a CDRL3 region of SEQ ID NO: 19 (SQSTHVPWT).

[0011] The antibodies of the present invention have high affinity for human PD-L2 while also providing blocking activity for hPD1. In particular, the antibodies of the present invention have better binding activity for human PD-L2 than commercially available monoclonal PD-L2 antibodies such as MIH-18, 24F.10C12, D7U86, and TY25. Furthermore, they have been shown to not only effectively block hPD-1 / hPD-L2 interaction, but also stimulate immune-mediated clearance of senescent cells, especially senescent tumor cells, in cancer tissues. Thus, such senescent tumor cells are effectively recognized and eliminated by the immune system. Clearance of senescent cells can reduce the risk of promoting metastasis. Moreover, this allows for complete regression of associated tumors and reduces the risk of tumor recurrence.

[0012] In a preferred embodiment of the present invention, the antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region CDRs comprise the CDRH1 region of SEQ ID NO: 14 (GYSFSNYFIE), the CDRH2 region of SEQ ID NO: 15 (LNIPGSGGSNYAEKFKG), and the CDRH3 region of SEQ ID NO: 16 (RRLPPDWYFDV), and the light chain region CDRs comprise the CDRL1 region of SEQ ID NO: 17 (RSSQSLVHSGGNTYLH), the CDRL2 region of SEQ ID NO: 18 (KVSNRFS), and the CDRL3 region of SEQ ID NO: 19 (SQSTHVPWT), so that the antibody has a particularly high affinity for human PD-L2, while also providing blocking activity against hPD1. In addition, they have the great advantage of having a low risk of isomerization.

[0013] In another preferred embodiment, the isolated monoclonal antibody comprises as heavy chain variable region CDRs, a CDRH1 region of SEQ ID NO: 13 (GYAFSNYFIE); a CDRH2 region of SEQ ID NO: 15 (LNIPGSGGSNYAEKFKG); and a CDRH3 region of SEQ ID NO: 16 (RRLPPDWYFDV); and as light chain region CDRs, a CDRL1 region of SEQ ID NO: 20 (RSSQSLVHSDGNTYLH); a CDRL2 region of SEQ ID NO: 18 (KVSNRFS); and a CDRL3 region of SEQ ID NO: 19 (SQSTHVPWT), such that the antibody has a particularly high affinity for human and / or mouse PD-L2, while also providing blocking activity against hPD1. [Brief description of the drawings]

[0014] Figure 1 shows that VH0 / VK0-mIgG1 has superior binding activity to recombinant and cellular hPD-L2, and blocking activity against cellular hPD-1 / hPD-L2 interaction, compared to a panel of commercially available anti-PD-L2 antibodies. [Figure 1A]Figure 1A shows the relative binding of VH0 / VK0-mIgG1 to MIH-18 and 24F.10C12 (all commercially available antibodies against hPD-L2 known for various uses), TY-25 (a commercially available antibody known to bind mPD-L2 for various uses), and the relevant isotype negative control by ELISA using recombinant hPD-L2. [Figure 1B] Figure 1B shows the binding activity of VH0 / VK0-mIgG1 to cellular hPD-L2 expressed on CHOK-1 cells, in comparison to MIH-18, 24F.10C12, D7U86, TY-25, and the relevant isotype negative control. [Figure 1C] Figure 1C shows the blocking activity of VH0 / VK0, VH6 / VK1, VH6 / VK2, VH6 / VK3, VH7 / VK2, and VH8 / VK2, and MIH-18 against cellular hPD-L2 / hPD-1 interaction as measured by a fluorescent reporter in hPD-1-expressing Jurkat cells co-cultured with hPD-L2-expressing CHO-K1 cells. [Figure 1D] Figure 1D shows that VH0 / VK0-mIgG1 and VH6 / VK6 have superior blocking activity against cellular hPD-L2 / hPD-1 interaction compared to MIH-18, 24F.10C12, and D7U86, as measured by a fluorescent reporter in hPD-1-expressing Jurkat cells co-cultured with hPD-L2-expressing CHO-K1 cells. [Figure 1E] Figure 1E shows that VH0 / VK0-mIgG1 has superior blocking activity against recombinant hPD-1 binding to hPD-L2-expressing CHOK-1 cells compared to a panel of commercially available anti-PD-L2 antibodies. Figure 2 shows that the parent and humanized variant anti-PD-L2 antibodies have superior binding activity against recombinant and cellular mPD-L2 compared to the commercially available anti-mPD-L2 TY-25, where: [Figure 2A] FIG. 2A shows that PyMT mice treated with doxorubicin in combination with 3 mg / kg VH0 / VK0-mIgG1 for 4 weeks showed a greater reduction in final tumor volume compared to doxorubicin alone or doxorubicin in combination with TY-25. [Figure 2B] Figure 2B shows the relative binding of VH0 / VK0 and VH8 / VK2 versus TY-25 to CHO-K1 cells overexpressing mPD-L2 by flow cytometry. [Figure 2C] Figure 2C shows that VH0 / VK0-mIgG1 shows cross-reactivity with recombinant mPD-L2 by ELISA, whereas commercial MIH-18, like the isotype control mIgG1, shows no cross-reactivity with recombinant mPD-L2.Figure 3 shows that VH0 / VK0-mIgG1, in combination with doxorubicin, shows superior antitumor activity compared to TY-25 and exhibits senescence clearance activity associated with immune system activity in the mouse PyMT breast cancer model, where: [Figure 3A] Figure 3A shows that mice in the transplantable PyMT model treated with VH0 / VK0-mIgG1 in combination with doxorubicin had smaller tumor progression by day 49 compared to tumors in mice treated with TY-25 in combination with doxorubicin. Error bars represent SEM. [Figure 3B] Figure 3B shows that combined immunohistochemical double staining of senescence markers SA-β-galactosidase and PD-L2 shows that doxorubicin significantly induced double-positive cells, and treatment with VH0 / VK0-mIgG1 effectively reduced this senescent cell burden. [Figure 3C] (C) Quantification of PD-L2 and SA-βGal double positive cells in PyMT tumors shows a reduction in senescent cell burden when animals were co-treated with VH0 / VK0-mIgG1. Error bars represent SEM, and values ​​for the doxorubicin+VH0 / VK0-mIgG1 treated group are significantly lower than the vehicle or doxorubicin+mIgG1 treated groups (p<0.05 by Student's t-test). [Figure 3D]Figure 3D shows that when PyMT mice were treated with the combination of doxorubicin and VH0 / VK0-mIgG1, PyMT spontaneous tumors showed increased relative numbers of CD3+, CD8+, and CD4+ cells compared to mice treated with vehicle or doxorubicin alone, consistent with PD-L2 function as a negative immune checkpoint inhibitor for T cells. Error bars represent SEM, and values ​​for the doxorubicin+VH0 / VK0-mIgG1-treated group were significantly higher for CD3, CD8, and CD4 than the vehicle or doxorubicin+mIgG1-treated groups (p<0.05 by Student's t-test). [Figure 3E] Figure 3E shows that combined treatment with doxorubicin and VH0 / VK0-mIgG1 suppressed tumor growth more than doxorubicin and mIgG1 control treatment in the spontaneous PyMT model. Administration of depleting antibodies against CD8+ cells was able to alleviate the inhibition of doxorubicin and VH0 / VK0-mIgG1 on tumor progression, whereas depleting antibodies against CD4+ cells only slightly alleviated this inhibition, suggesting a predominant role of CD8+ in the mechanism of action of VH0 / VK0-mIgG1. Error bars represent SEM, and values ​​in the doxorubicin+VH0 / VK0-mIgG1-treated group are significantly higher than those in the doxorubicin+mIgG1 or doxorubicin+VH0 / VK0-mIgG1+anti-CD8-treated groups (p<0.05 by Student's t-test). [Figure 3F] FIG. 3F shows quantification of remaining senescent cells by SA-βGal staining in PyMT tumors after treating mice as in A. [Figure 3G] FIG. 3G is a representative image of SA-β-Gal staining in PyMT tumors after mice were treated as in 2A. [Figure 4]Figure 4 shows that FC domain effector function is required for full anti-tumor activity of anti-PD-L2 antibodies containing the VH0 / VK0 variable domain in the spontaneous PyMT model.Final tumor volumes in PyMT mice treated with doxorubicin alone at 4 mg / kg once weekly, or in combination with doxorubicin at 4 mg / kg once weekly and VH0 / VK0-mIgG2a, effector-reduced VH0 / VK0-mIgG2a / LALA, or effector-deficient VH0 / VK0-mIgG2a / LALAPG at 10 mg / Kg three times weekly for 4 weeks. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] In the context of the present invention, an antibody typically comprises at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (hereinafter V H The heavy chain constant region is composed of C H 1. C H 2, and C H Each light chain is composed of three regions: the light chain variable region (V L The light chain constant region consists of one region, C L It consists of: V H and V L The regions can be further subdivided into hypervariable regions interspersed with more conserved regions called framework regions (FWs) that are complementary determining regions (CDRs). H and V Lis composed of three CDRs and four FWs, arranged in the following order from the amino terminus to the carboxy terminus: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The variable regions of the heavy and light chains contain binding regions that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues and factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The CDR regions of Ig-derived regions can be determined as described in Kabat "Sequences of Proteins of Immunological Interest", 5th edit. NIH Publication no. 91 -3242 US Department of Health and Human Services (1991); Chothia J. Mol. Biol. 196 (1987), 901 -917 or Chothia Nature 342 (1989), 877-883.

[0016] As used herein, the term "monoclonal antibody" refers to an antibody that displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "humanized monoclonal antibody" refers to an antibody that displays a single binding specificity and has variable and constant regions derived from human germline or non-germline immunoglobulin sequences.

[0017] The term "epitope" as used herein refers to a portion or region of an antigen molecule (e.g., a peptide) that is specifically bound by an antigen-binding site of an antibody. An epitope usually contains at least 3, more usually at least 5 or 8-10 residues (e.g., amino acids or nucleotides). Typically, an epitope is also less than 20 residues (e.g., amino acids or nucleotides) in length, e.g., less than 15 or less than 12 residues. The term "epitope" encompasses both linear epitopes, whose consecutive amino acids are recognized by an antibody, and conformational epitopes, whose amino acids are recognized by an antibody when they are in the appropriate configuration or conformation. Thus, for some epitopes, the conformation (three-dimensional structure) is as important as the amino acid sequence (primary structure).

[0018] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to PD-L2 and is substantially free of antibodies that do not bind to PD-L2). However, an isolated antibody that specifically binds to an epitope of human PD-L2 may have cross-reactivity with other PD-L2 proteins from different species. However, in preferred embodiments, the antibody maintains high affinity and selectivity for human PD-L2. In addition, an isolated antibody is typically substantially free of other cellular material and / or chemicals.

[0019] In the context of the present invention, the term "senescent cells in cancer tissue" includes various types of senescent cells in the tumor microenvironment, i.e., normal cells and tumor cells induced to undergo senescence. Senescent tumor cells originate from tumor cells and are usually the most common type of senescent cells in the tumor microenvironment. They can be identified by known markers, such as senescence-associated beta-galactosidase.

[0020] In the context of the present invention, VHP / VKP are parental murine antibodies on a mIgG1 backbone, also called VH0 / VK0-mIgG1, and VH0 / VKP0 are chimeric antibodies on a hIgG1 backbone. The variable regions are the same in VHP / VKP and VH0 / VK0, so VHP corresponds to VH0 and VKP corresponds to VK0. Preferably, the V regions of the antibodies of the present invention have sequences that are devoid of or have reduced important T cell epitopes. Eight heavy chain (VH0 (chimeric), VH1-VH4, and VH6-VH8 (all humanized)) and four light chain (VK0 (chimeric) and VK1-VK3 (humanized)) sequences were found to provide particularly effective antibodies (Table 1).

[0021] VHO, VH1, VH2, VH3, VH4, VH6, VH7, and VH8 all contain SEQ ID NO: 13 (GYAFSNYFIE) or SEQ ID NO: 14 (GYSFSNYFIE) as CDRH1, SEQ ID NO: 15 (LNIPGSGGSNYAEKFKG) as CDRH2, and SEQ ID NO: 16 (RRLPPDWYFDV) as CDRH3.

[0022] VK0, VK1, VK2, and VK3 all contain SEQ ID NO: 17 (RSSQSLVHSGGNTYLH) or SEQ ID NO: 20 (RSSQSLVHSDGNTYLH) as CDRL1; SEQ ID NO: 18 (KVSNRFS) as CDRL2; and SEQ ID NO: 19 (SQSTHVPWT) as CDRL3. [Table 1]

[0023] Preferably, the isolated monoclonal antibody comprises: (a) a heavy chain variable region having a complementarity determining region 1 (CDRH1) comprising the amino acid sequence of SEQ ID NO: 13 (GYAFSNYFIE) or SEQ ID NO: 14 (GYSFSNYFIE); a CDRH2 comprising the amino acid sequence of SEQ ID NO: 15 (LNIPGSGGSNYAEKFKG); and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 16 (RRLPPDWYFDV), wherein the remainder of the heavy chain sequence has at least 95% identity to a heavy chain sequence selected from the group consisting of the sequences listed in Table 1; and (b) a light chain variable region having a CDRL1 comprising the amino acid sequence of SEQ ID NO: 17 (RSSQSLVHSGGNTYLH) or SEQ ID NO: 20 (RSSQSLVHSDGNTYLH); a CDRL2 comprising the amino acid sequence of SEQ ID NO: 18 (KVSNRFS); and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 19 (SQSTHVPWT), wherein the remainder of the light chain sequence has at least 95% identity to a light chain sequence selected from the group consisting of sequences listed in Table 1.

[0024] As shown in Table 2, all of the humanized variant antibodies containing VH1, VH2, VH3, VH4, VH6, VH7, and VH8 as heavy chains and VK1, VK2, and VK3 as light chains bind to the human PD-L2 antigen with high affinity. Table 2: Single cycle kinetic parameters of binding of chimeric (VH0 / VK0) and humanized variants (tested as cell culture supernatants) to human PD-L2 antigen as determined using Biacore 8K. As used herein, the term "K D " is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction, where ka is the association rate constant and kd is the dissociation rate constant. The binding affinity of the antibodies of the disclosed invention can be measured or determined by standard antibody-antigen assays. Relative K D is the humanized variant of K D The K of VH0 / VK0 assayed in the same experiment D was calculated by dividing by [Table 2]

[0025] As mentioned above, VHO / VKO correspond to the chimeric antibody (mouse Fab on a human IgG1 backbone) and VHP / VKP are the parental mouse antibodies (mouse Fab on a mouse IgG1 backbone).

[0026] Preferably, the monoclonal antibody of the invention comprises a heavy chain variable region and a light chain variable region, and (a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:1 (VH0) and the light chain variable region comprises the amino acid sequence of SEQ ID NO:9 (VK0); or (b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:6 (VH6), and the light chain variable region comprises the amino acid sequence of SEQ ID NO:10 (VK1), SEQ ID NO:11 (VK2), or SEQ ID NO:12 (VH3); or (c) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:7 (VH7), and the light chain variable region comprises the amino acid sequence of SEQ ID NO:11 (VK2); or (d) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:8 (VH8), and the light chain variable region comprises the amino acid sequence of SEQ ID NO:11 (VK2).

[0027] Based on expression, iTope™-AI score and TCEM™ (a computer tool provided by Abzena for rapid screening of antibodies and other proteins with immunogenic potential), the above chimeric VH0 / VK0 antibody (SEQ ID NO:1 / SEQ ID NO:9) and the six humanized antibodies VH4 / VK2 (SEQ ID NO:5 / SEQ ID NO:11), VH6 / VK3 (SEQ ID NO:6 / SEQ ID NO:12), VH6 / VK1 (SEQ ID NO:6 / SEQ ID NO:10), VH6 / VK2 (SEQ ID NO:6 / SEQ ID NO:11), VH7 / VK2 (SEQ ID NO:7 / SEQ ID NO:11), and VH8 / VK2 (SEQ ID NO:7 / SEQ ID NO:11) are particularly preferred. The variant VH6 / VK3 (SEQ ID NO:6 / SEQ ID NO:12) has the advantage of being free of the risk of isomerization that may occur with the other five preferred humanized variants VH4 / VK2 (SEQ ID NO:5 / SEQ ID NO:11), VH6 / VK1 (SEQ ID NO:6 / SEQ ID NO:10), VH6 / VK2 (SEQ ID NO:6 / SEQ ID NO:11), VH7 / VK2 (SEQ ID NO:7 / SEQ ID NO:11) and VH8 / VK2 (SEQ ID NO:7 / SEQ ID NO:11). Table 3 shows the Ka, Kd, ​​and K D , and relative K D Indicates the value. [Table 3]

[0028] In one embodiment, the isolated antibody of the invention binds human recombinant PD-L2 at 10 -10 The dissociation constant (K D ) to join them.

[0029] In another embodiment, the isolated antibody of the invention inhibits the interaction of human programmed cell death protein 1 (PD-1) with human PD-L2 with an EC of 1.3 nM or less in a bioluminescent cell-based assay using Jurkat T cells and CHO-K1 cells. 50(half the maximal effective concentration) in vitro, where Jurkat T cells express human PD-1 and a luciferase reporter driven by the NFAT response element (NFAT-RE), and CHO-K1 cells express human PD-L2 and an engineered cell surface protein designed to activate the cognate TCR in an antigen-independent manner. Such antibodies have high clinical efficacy.

[0030] FIG. 1A shows the antibody of the present invention (EC 50 =0.219nM) to cellular PDL2, and the commercially available antibody MIH-18 (EC 50 =0.618nM), 24F.10C12(EC 50 =0.70 nM), and show better binding than TY25. The blocking activity is illustrated in Figures 1B and 1E, where recombinant hPD-1 ligand is added to CHOK-1 cells overexpressing hPD-L2 by flow cytometry. Figure 1C shows the blocking activity of VH0 / VK0 and the five humanized variants and MIH-18 against cellular hPD-L2 / hPD-1 interaction measured by fluorescent reporter, and Figure 1D shows that VH0 / VK0-mIgG1 and VH6 / VK6 have superior blocking activity against cellular hPD-L2 / hPD-1 interaction compared to the commercial antibodies MIH-18, 24F.10C12, and D7U86.

[0031] The experimental data disclosed in Figures 1A-1E clearly show that the chimeric antibody VH0 / VK0 (SEQ ID NO:1 / SEQ ID NO:9) and the five humanized antibodies VH6 / VK3 (SEQ ID NO:6 / SEQ ID NO:12), VH6 / VK1 (SEQ ID NO:6 / SEQ ID NO:10), VH6 / VK2 (SEQ ID NO:6 / SEQ ID NO:11), VH7 / VK2 (SEQ ID NO:7 / SEQ ID NO:11), and VH8 / VK2 (SEQ ID NO:7 / SEQ ID NO:11) have similar physiological properties with respect to interaction with hPD-L2. In particular, it is shown that one amino acid difference at position 3 of CDRH1 (A vs. S) and one amino acid difference at position 10 of CDRL1 (G vs. D) do not adversely affect the physiological properties. Particularly good results were obtained with VH0 / VK0 (SEQ ID NO:1 / SEQ ID NO:9), VH6 / VK3 (SEQ ID NO:6 / SEQ ID NO:12), VH6 / VK2 (SEQ ID NO:6 / SEQ ID NO:11), and VH7 / VK2 (SEQ ID NO:7 / SEQ ID NO:11).In another aspect, the isolated antibody of the invention specifically binds to both human and mouse PD-L2.

[0032] Figures 2A and 2B show that the parent antibodies and humanized variant antibodies of the invention have better binding activity to recombinant and cellular mPD-L2 than the commercially available anti-mPD-L2 TY-25. Figure 2C shows the cross-reactivity of the antibodies of the invention with recombinant mPD-L2 by ELISA, but the commercially available MIH-18 shows no cross-reactivity with recombinant mPD-L2. Particularly good results were obtained with VH0 / VK0-mIgG1.

[0033] In another embodiment, the isolated antibody of the invention has a dissociation constant (K D ) is 10 -10 M or less, and simultaneously detects the interaction between human programmed cell death protein 1 (PD-1) and human PD-L2 with an EC of 1.3 nM or less and an EC of 0.65 nM or less in a bioluminescent cell-based assay using Jurkat T cells and CHO-K1 cells. 50The study was performed in vitro, where Jurkat T cells express human PD-1 and a luciferase reporter driven by the NFAT response element (NFAT-RE), and CHO-K1 cells express human PD-L2 and an engineered cell surface protein designed to activate their cognate TCR in an antigen-independent manner.

[0034] In another embodiment, the isolated antibody of the invention has a dissociation constant (K D ) is 10 -10 It binds to human PD-L2 with an M below, and specifically binds to both human and mouse PD-L2, facilitating preclinical testing.

[0035] In another embodiment, the isolated antibody of the invention has a dissociation constant (K D ) is 10 -10 M or less, and simultaneously detects the interaction between human programmed cell death protein 1 (PD-1) and human PD-L2 with an EC of 1.3 nM or less in a bioluminescent cell-based assay using Jurkat T cells and CHO-K1 cells. 50 and specifically binds both human and mouse PD-L2, where Jurkat T cells express human PD-1 and a luciferase reporter driven by the NFAT response element (NFAT-RE), and CHO-K1 cells express human PD-L2 and an engineered cell surface protein designed to activate cognate TCRs in an antigen-independent manner.

[0036] The antibodies of the present invention have superior binding activity to cellular hPD-L2 and blocking activity against cellular hPD-1 / hPD-L2 interaction compared to a panel of commercially available anti-PD-L2 antibodies (see Figure 1a). Furthermore, the antibodies of the present invention showed higher blocking activity against cellular hPD-L2 / hPD-1 interaction measured by fluorescent reporter in hPD-1-expressing Jurkat cells co-cultured with hPD-L2-expressing CHOK-1 cells than MIH-18, 24F.10C12, D7U86, and relevant isotype negative controls (Figure 1b). In yet another embodiment, the isolated monoclonal antibody is a chimeric, humanized, or human antibody.

[0037] The term "chimeric antibody" refers to an antibody having light and heavy chain genes constructed, usually by genetic engineering, from immunoglobulin variable and / or constant region genes belonging to one or more different species. For example, the variable segments (or, for example, one or more of the complementarity determining regions (CDRs) in the variable regions) of genes from a mouse antibody (e.g., a monoclonal or polyclonal antibody) can be combined with human constant segments to generate a chimeric antibody. Thus, a therapeutic chimeric antibody of the invention is a hybrid protein composed of the variable or antigen binding region (e.g., one or more of the CDRs of a mouse antibody) and the constant or effector region of a human antibody, although other mammalian species may be used. A chimeric antibody may also include amino acid sequences obtained from protein sources other than antibodies.

[0038] The term "humanized antibody" refers to a type of chimeric antibody that contains a human framework region and one or more CDRs from a non-human (usually mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDRs is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor". Constant regions need not be present, but if present, they should be substantially identical to human immunoglobulin constant regions, i.e. at least about 85-90%, preferably about 95% or more identical. Thus, all parts of a humanized immunoglobulin (except possibly the CDRs) are substantially identical to the corresponding parts of a natural human immunoglobulin sequence. Humanized antibodies are useful as active ingredients of therapeutic agents according to the invention, due to the reduced antigenicity of the humanized antibody in the human body.

[0039] The term "human antibody" means that the amino acid sequences of the antibody are fully human, i.e., the variable and constant regions of the heavy and light chains are human.

[0040] In another aspect, a pharmaceutical composition is provided comprising an isolated monoclonal antibody of the invention and a pharma- ceutically acceptable excipient.

[0041] In another aspect, an isolated nucleic acid sequence encoding the amino acid sequence of the light chain variable region and / or the heavy chain variable region of an antibody of the invention is provided. A nucleic acid is "isolated" or "substantially pure" when it has been purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other methods well known in the art.

[0042] In another aspect, a recombinant expression vector comprising such a nucleic acid sequence is provided. As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop to which additional DNA segments can be ligated. Another type of vector is a viral vector to which additional DNA segments can be ligated to the viral genome. Certain vectors can replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, and are thereby replicated along with the host genome. In addition, certain vectors can induce the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors". In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably, with plasmids being the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0043] In another aspect, an isolated host cell is provided that comprises a recombinant expression vector described herein and / or expresses an antibody thereof described herein. As used herein, the term "isolated host cell" (or simply "host cell") is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in successive generations due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0044] Preferably, the antibody of the present invention is used for the treatment, amelioration, or prevention of a disease or condition associated with the presence of senescent cells in cancer tissue, preferably the senescent cells are selected from the group consisting of senescent stromal cells, immune infiltrating cells, and senescent tumor cells, most preferably senescent tumor cells.

[0045] In a preferred embodiment, the antibody of the present invention is used to treat, ameliorate, or prevent a disease or condition associated with the presence of senescent cells in cancer tissue, particularly senescent tumor cells, where the antibody is administered in combination with a chemotherapeutic agent. The combination of the antibody of the present invention with the chemotherapeutic agent results in synergistic inhibition of tumor growth. In addition, immune clearance of senescent cells is stimulated. A "chemotherapeutic agent" is a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include compounds useful in the treatment of cancer. Preferably, the chemotherapeutic agent is selected from the group consisting of alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant-derived alkaloids, topoisomerase inhibitors, hormone therapy agents, hormone antagonists, aromatase inhibitors, P-glycoprotein inhibitors, platinum complex derivatives, growth inhibitors, radioisotopes, targeted agents, particularly CDK4 / 6 inhibitors, other immunotherapeutic agents, and other anticancer agents, and combinations thereof.

[0046] More particularly, the chemotherapeutic agent is selected from the group consisting of danorubicin, etoposide, mitoxantrone, camptothecin, irinotecan, topotecan, fusulfan, temozolomide, carmustine, dacarbazine, cyclophosphamide, melphalan, mitomycin C, cisplatin, carboplatin, oxaliplatin, methotrexate, pemetrexed, gemcitabine, azacitidine, bromodeoxyuridine, 5-fluorouracil, mycophenolic acid, hydroxyurea, antinomycin D, paclitaxel, docetaxel, vincristine, vinblastine, tamoxifen, fulvestrant, androgens, most preferably doxorubicin.

[0047] Figures 3A and 3B show that PyMT mice treated with the antibody of the present invention in combination with chemotherapeutic agents, particularly doxorubicin, showed less tumor progression and smaller tumor volume compared to tumors in mice treated with TY-25 in combination with doxorubicin. Furthermore, as shown in Figures 3C and 3D, doxorubicin significantly induced double positive cells expressing both SA-beta galactosidase and PD-L2, and combination treatment with the antibody of the present invention reduced this senescent cell burden. When treated with the antibody of the present invention, the final tumor volume after 4 weeks of treatment in PyMT mice treated with 4 mg / kg doxorubicin alone once a week or with a combination of 4 mg / kg doxorubicin once a week and three times a week was significantly reduced compared to commercial TY-25 (Figure 3F). Furthermore, it was shown that the number of remaining senescent cells by SA-β-Gal staining in PyMT tumors was significantly reduced after treatment of mice with the antibody of the present invention (Figure 3G). Thus, the antibodies of the invention promote immune clearance of chemotherapy-treated cancer cells. Treatment of PyMT mice with a combination of doxorubicin and the antibodies of the invention demonstrated increased relative numbers of CD3+, CD8+, and CD4+ cells in PyMT spontaneous tumors compared to mice treated with vehicle or doxorubicin alone. This finding is consistent with PD-L2' functioning as a negative immune checkpoint inhibitor for T cells (Figure 3D), i.e., PD-L2' specifically reduces or suppresses immune signals. Furthermore, combined treatment with doxorubicin and the antibodies of the invention suppressed tumor growth more than doxorubicin and mIgG1 control treatment in the PyMT spontaneous model (Figure 3E). Administration of depleting antibodies against CD8+ cells was able to alleviate the inhibition of doxorubicin and the antibodies of the invention against tumor progression, whereas depleting antibodies against CD4+ cells did not alleviate this inhibition. This suggests that the role of CD8+ is predominant in the mechanism of action of the antibodies of the invention.

[0048] Preferably, the antibodies of the present invention provide higher antitumor activity due to the inclusion of FC domain effector functions. As shown in Figure 4, the tumor volumes of PyMT mice treated with 4 mg / kg doxorubicin alone once a week or with 4 mg / kg doxorubicin once a week in combination with either VH0 / VK0-mIgG2a three times a week indicate higher antitumor activity, but lower antitumor activity when antibodies with effector-reduced VH0 / VK0-mIgG2a / LALA or effector-deficient VH0 / VK0-mIgG2a / LALAPG were administered for 4 weeks. The targeted agent is preferably a specific CDK4 / 6 inhibitor, most preferably selected from the group consisting of imatinib, nilotinib, trametinib, vemurafenib, dasatinib, lapatinib, neratinib, afatinib, getofinib, erlotinib, sorafenib, sirolimus, rituximab, obinutuzumab, pertuzumab, trastuzumab, bevacizumab, ranibizumab, palbociclib, ademaciclib, ribociclib, olaparib, niraparib, rucaparib, and bortezomib.

[0049] Most preferably, the antibodies of the present invention are used to treat, ameliorate, or prevent a disease or condition associated with the presence of senescent cells, where the disease or condition is cancer. As used herein, the term "cancer" or "tumor" refers to the presence of cells that have properties typical of cancer-causing cells, such as, for example, uncontrolled proliferation, immortality, metastatic potential, rapid proliferation and proliferation rate, and specific morphological characteristics. Cancer cells often take the form of tumors, but such cells may exist alone in an animal or may be non-tumorigenic cancer cells, such as leukemia cells. As used herein, the term "cancer" includes pre-cancerous cancers as well as malignant cancers.

[0050] Most preferably, the cancer is breast cancer, non-small cell lung cancer, ovarian cancer, squamous cell carcinoma of the head, squamous cell carcinoma of the neck, carcinoma, squamous cell carcinoma of the cervix, squamous cell carcinoma of the eyelid, squamous cell carcinoma of the conjunctiva, squamous cell carcinoma of the vagina, squamous cell carcinoma of the lung, squamous cell carcinoma of the oral cavity, squamous cell carcinoma of the skin, squamous cell carcinoma of the bladder, squamous cell carcinoma of the tongue, squamous cell carcinoma of the larynx, or squamous cell carcinoma of the esophagus, adenocarcinoma of the prostate, adenocarcinoma of the small intestine. , adenocarcinoma of the endometrium, adenocarcinoma of the cervix, adenocarcinoma of the colon, adenocarcinoma of the lung, adenocarcinoma of the pancreas, adenocarcinoma of the esophagus, adenocarcinoma of the rectum, adenocarcinoma of the uterus, adenocarcinoma of the stomach, adenocarcinoma of the breast, adenocarcinoma of the ovary, sarcoma in myogenic sarcoma, leukemia, neuroma, melanoma, and lymphoma, and most preferably, is selected from the group consisting of breast cancer, non-small cell lung cancer, ovarian cancer, squamous cell carcinoma of the head, and squamous cell carcinoma of the neck. EXAMPLES

[0051] Immunization of mice to generate parental monoclonal antibodies (VH0 / VK0-mIgG1) Mice were immunized subcutaneously with a 100μg dose of His-tagged anti-hPDL2 antigen (Sino Biologicals, Cat. No. 10292-H0H) together with Freund's adjuvant in a 1:1 ratio every 2 weeks for a total of 4 times over 8 weeks. The final immunization was with 80μg of antigen 96 hours before the animals were sacrificed for the fusion procedure. Spleen cells isolated from sacrificed mice were pooled between two mice and fused with P3×63 myeloma cell line and seeded in 96-well plates with HAT selection. After 10 days, the resulting hybridoma supernatants were screened for cross-reactivity with recombinant hPD-L2 and blocking activity against hPD-L2 / hPD-1 interaction, and counterscreened by ELISA for cross-reactivity to the His tag. Leads were selected from this initial screen, scaled up, purified, and further confirmed for binding to hPD-L2 and blocking of hPD-L2 / hPD-1 interaction at various antibody dilutions, and VH0 / VK0-mIgG1 was selected as the lead for humanization.

[0052] Design of humanized variants of VH0 / VK0-mIgG1 Structural modeling of the Swiss PBD first identified several residues within the variable regions of VH0 / VK0-mIgG1 as important for the binding properties of the antibody. Based on this, computational analysis selected human sequence segments with the criterion that they do not bind to MHC class II alleles. Additional independent computational analysis revealed weaknesses in the developability of the sequence, which were corrected to enhance future process development.

[0053] Based on multiple computational analyses, eight variant heavy chains (VH1-8) and three variant light chains (VK1-3), in addition to the parent murine variant heavy and light chains (VH0 / VK0), were cloned into mammalian expression vectors on the hIgG1 backbone, transiently expressed in CHO cells, and purified. Evaluation of these chimeric anti-PDL2 lead mAbs VH0 / VK0; (SEQ ID NO:1 / SEQ ID NO:9) and humanized variants VH0 / VK1 (SEQ ID NO:1 / SEQ ID NO:10); VH1 / VK0 (SEQ ID NO:2 / SEQ ID NO:9); VH1 / VK1 (SEQ ID NO:2 / SEQ ID NO:10); VH1 / VK2 (SEQ ID NO:2 / SEQ ID NO:11); VH1 / VK3 (SEQ ID NO:2 / SEQ ID NO:12); VH2 / VK1 (SEQ ID NO:3 / SEQ ID NO:10); VH2 / VK2 (SEQ ID NO:3 / SEQ ID NO:11); VH2 / VK3 (SEQ ID NO:3 / SEQ ID NO:12); VH3 / VK1 (SEQ ID NO:4 / SEQ ID NO:10); VH3 / VK2 (SEQ ID NO:4 / SEQ ID NO:11); VH3 / VK3 (SEQ ID NO:4 / SEQ ID NO: No. 12); VH4 / VK1 (SEQ ID NO:5 / SEQ ID NO:10); VH4 / VK2 (SEQ ID NO:5 / SEQ ID NO:11); VH4 / VK3 (SEQ ID NO:5 / SEQ ID NO:12); VH6 / VK1 (SEQ ID NO:6 / SEQ ID NO:10); VH6 / VK2 (SEQ ID NO:6 / SEQ ID NO:11); VH6 / VK3 (SEQ ID NO:6 / SEQ ID NO:12); VH7 / VK1 (SEQ ID NO:7 / SEQ ID NO:10); VH7 / VK2 (SEQ ID NO:7 / SEQ ID NO:11); VH7 / VK3 SEQ ID NO:7 / SEQ ID NO:12); VH8 / VK1 (SEQ ID NO:8 / SEQ ID NO:10); VH8 / VK2 (SEQ ID NO:8 / SEQ ID NO:11); VH8 / VK3 (SEQ ID NO:8 / SEQ ID NO:12) were temporarily scaled up on a large scale. To achieve this, they were transiently transfected into CHO cells using the MaxCyte STX® electroporation system equipped with an OC-400 processing assembly (MaxCyte Inc., Gaithersburg, USA). After harvesting the cells, they were cultured at 3 × 10 in CD Opti-CHO medium (ThermoFisher, Loughborough, UK) containing 8 mM L-glutamine (ThermoFisher, Loughborough, UK) and 1 × hypoxanthine-thymidine (ThermoFisher, Loughborough, UK). 6The cells were diluted to 1000 cells / mL. Two hours after resuspension, 0.5× penicillin-streptomycin solution (ThermoFisher, Loughborough, UK) was added to the cultures (5 mL / L). 24 hours after transfection, the culture temperature was reduced from 37°C to 32°C and 1 mM sodium butyrate (Sigma, Dorset, UK) was added. Cultures were fed 24 hours and 7 days after transfection with 30% and 15% (of culture volume) CHO CD Efficient Feed B (ThermoFisher, Loughborough, UK), respectively, and 3.3% and 1.65% Function Max Titre Enhancer (ThermoFisher, Loughborough, UK), respectively. CHO supernatants were harvested 14 days after transfection. Antibody concentrations were measured using a Protein A biosensor (Molecular Devices, Wokingham, Berkshire, UK) on an Octet QK 384, using an IgG1 antibody as standard.

[0054] After the cultures were harvested, the antibody supernatants were filtered using a 0.2 μM filter system (Corning, New York, US) to remove residual cell debris and supplemented with 10x PBS to neutralize the pH. Antibodies were then purified from the supernatants using a 5 mL Hitrap MabSelect PrismA column (Cytiva, Little Chalfont, UK) equilibrated with 10 CV (column volume) of 1xDPBS (pH 7.2-7.4). After loading the sample, the column was washed with 10 CV of 1xDPBS and proteins were eluted with 0.1 M sodium citrate (pH 3.0). Fractions were collected and quantified at OD280nm after adjusting the pH with 1 M Tris-HCl (pH 9.0). Fractions containing the antibody from each variant were pooled, buffer exchanged into 1x DPBS (pH 7.2-7.4), filter sterilized using a 0.2 μM syringe filter (Sartorius, Epsom, UK), and quantified at OD280nm using an extinction coefficient (Ec (0.1%)) based on the predicted amino acid sequence. PrismA purified antibodies were then analyzed by SDS-PAGE and analytical SE-HPLC.

[0055] Multi-cycle kinetic Biacore of VH0 / VK0-mIgG1 and five humanized variant leads Multi-cycle kinetic analysis was performed on purified material to assess binding of five lead humanized variants (VH6 / VK1, VH6 / VK2, VH6 / VK3; VH7 / VK2, and VH8 / VK2) to the human PD-L2 antigen. Kinetic experiments were performed at 25°C on a Biacore8K equipped with Biacore Insight Evaluation software (Cytiva, Uppsala, Sweden).

[0056] HBS-EP+ (Cytiva, Uppsala, Sweden) supplemented with 1% BSA w / v (Sigma, Dorset, UK) was used as running buffer and for dilution of ligand and analyte. Purified lead antibody was diluted to 1 μg / mL in running buffer and loaded onto all active flow cells of a Protein A sensor chip (Cytiva, Little Chalfont, UK) at the start of each cycle. Antibody was captured at a flow rate of 10 μl / min to an immobilization level (RL) of approximately 120 RU. The surface was then allowed to stabilize.

[0057] Multi-cycle kinetic data were acquired using human PD-L2 antigen as the analyte, injected at a flow rate of 40 μl / min to minimize potential mass transfer effects. A seven-point two-fold dilution range of antigen from 0.37 nM to 20 nM was prepared in running buffer. For each concentration, the association phase was monitored for 240 s and the dissociation phase was measured for 1200 s. Regeneration of the sensor chip surface was performed using a single injection of 10 mM glycine-HCl (pH 1.5) between cycles. Multiple repeats of blank and antigen were programmed into the kinetic experiment to confirm the stability of both the surface and analyte throughout the kinetic cycles.

[0058] The signal from the reference flow cell (no IgG captured) was subtracted from the signal of the active flow cell for each channel used to correct for bulk effects and differences in non-specific binding to the reference surface. The signal from each IgG blank experiment (IgG captured but no antigen) was subtracted to correct for differences in surface stability. The dual-referenced sensorgrams were fitted to a Langmuir (1:1) binding model, where the goodness of fit of the data to the model is assessed using a chi-squared value that represents the deviation between the experimental and fitted (observed and predicted) curves. The fitting algorithm seeks to minimize the chi-squared value.

[0059] Assaying antibody binding to cellular human and mouse PD-L2 CHO-K1 cells overexpressing either hPDL2-GFP or mPD-L2 GFP were detached, washed with phosphate-buffered saline (PBS), and the cell concentration was adjusted to approximately 1 million cells with cytometry buffer (CB, PBS containing 1% PBS). Approximately 80,000 cells were then distributed into round-bottom 96-well plates, the plates were centrifuged at 400g for 3 min at 4 °C, and the supernatant was discarded. Antibodies at appropriate concentrations were added to the wells in a volume of 50 μL and incubated for 30 min at 6–8 °C. Plates were washed three times with PBS, and the relevant secondary antibodies (either AlexaFluor 647 goat anti-mouse IgG (H+L) (Invitrogen A21235 / lot 1915807) or AlexaFluor 647 goat anti-rat IgG (H+L) (Invitrogen A21247 / lot 1921562)) were added for 30 min at 4 °C. Cells were washed again twice with PBS, resuspended in CB, and analyzed on a Sony ID7000 Spectral Cell Analyzer (Sony) or a GALLIOS flow cytometer (Beckman-Coulter). EC50 was calculated using the sigmoidal 4PL equation in GraphPad Prism-9 software.

[0060] Assay of blocking activity of recombinant hPD-1 antibodies against cellular hPD-L2 CHO-K1 cells overexpressing hPDL2-GFP were detached, washed with phosphate-buffered saline (PBS), and adjusted to a cell concentration of approximately 1 million in CB. Approximately 80,000 cells were then distributed into round-bottom 96-well plates, the plates were centrifuged at 400g for 3 min at 4 °C, and the supernatant was discarded. A constant concentration of 15 μg / mL of the ligand, recombinant hPD-1 ligand (Sino Biological 10377-H08H / lot LC13JA2207), was incubated with the respective concentrations of antibodies and added to the cells and incubated at 6–8 °C for 30 min. The cells were washed three times with PBS, then incubated with rabbit polyclonal anti-His (Abcam Ab1187 / lot GR3369369-1) in CB, then washed three times with PBS. Cells were then incubated with labeled anti-rabbit secondary antibody (AlexaFluor 647 goat anti-rabbit IgG (H+L) (Invitrogen A21244 / lot 1910774) for 15 min at 4°C, washed twice with PBS, resuspended in CB, and analyzed on a Sony ID7000 Spectral Cell Analyzer (Sony).

[0061] ELISA for mPDL2 ELISA plates were coated with recombinant mPDL2 (Sino Biological, Cat. No. 50804-M08H-B, Lot LC10SE0706) at a concentration of 2 μg / mL for 15 h at 4°C. The coating was then washed twice with PBS and blocked with 0.5% bovine serum albumin (BSA) / PBS for 1 h at 37°C. The block was removed and appropriate concentrations of antibodies diluted in 0.5% BSA / PBS were incubated for 1 h at 37°C. The plates were then washed five times with PBS and goat anti-mouse HRP (Jackon Immuno Research, Cat. No. 115-035-071) diluted in 0.5% BSA / PBS was added and incubated for 30 min at 37°C. The plates were washed again five times with PBS and TMB substrate (Medicago Cat. No. 10-9405) was added and incubated for 30 min at 37°C in the dark. The reaction was stopped with 1N HCl and the optical density was read at 450 nM in a standard spectrophotometer plate reader.

[0062] Reporter assay for blocking activity against cellular hPD-L2 / hPD-1 interaction The cell blocking activity of the antibodies of the invention was assessed by the Promega (Southampton, Hampshire, UK) PD-1 / PD-L2 Blocking Bioassay (Promega, Cat. No. CS187131-1). This is a bioluminescent cell-based assay used to measure the potency and stability of antibodies and other biologics designed to block the hPD-1 / PD-L2 interaction. Briefly, CHO-K1 cells overexpressing hPD-L2 were thawed and seeded into 96-well plates. The following day, appropriate concentrations of antibodies were added to the wells. JurkatPD-1 effector cells expressing a luminescent NFAT reporter for T cell activation were then thawed and immediately added to the wells. The co-cultures were incubated for 6 hours and read using a SpectraMax i3x reader (Molecular Devices, Wokingham, UK). Data analysis was performed using GraphPad Prism 9.0 (GraphPad Software, La Jolla, Ca), and data were plotted using a four-parameter nonlinear regression model.

[0063] A transplantable murine PyMT mammary tumor model One million PyMT cells in PBS were injected into the right mammary fat pad number 4 with N=10 animals per treatment group. Treatment regimen was initiated when tumors reached an average of 100 cubic millimeters on day 17 post-injection. All mice were classified into study groups based on caliper estimates of tumor burden. Mice were distributed such that the mean tumor burden of all groups was within 10% of the overall mean tumor burden of the study population. The first dose of doxorubicin was administered intraperitoneally at 4 mg / kg on day 17 post-implantation, as well as all antibodies administered intraperitoneally at a dose of 10 mg / kg. Doxorubicin (MWI Animal Health, BJ0080A / NA) was administered again on day 24, and antibodies were administered on days 20, 24, 27, 31, 34, 38, and 41. The following commercially available antibodies were used in this study: anti-mouse PD-L2 (clone TY-25, Bio X Cell, lot / catalogue number 0411901). Vehicle-treated animals served as untreated controls and received only the doxorubicin vehicle.Tumor volumes were measured with standard calipers at least two to three times weekly for the duration of the study.

[0064] Spontaneous murine PyMT breast cancer model Hemizygous transgenic PyMT mice were allowed to grow (usually for approximately 9 weeks) until tumors reached an average size of approximately 200 cubic millimeters, at which point treatment was initiated, N=3-5 per treatment group. Doxorubicin (Aurovitas) was administered intraperitoneally at 4 mg / kg once weekly, and antibodies were administered every 3 days for 4 weeks at a dose of 10 mg / kg, or 3 mg / kg where indicated. Vehicle-treated animals received only the doxorubicin vehicle solvent as untreated controls. Tumors were measured weekly with standard calipers. At the end of the study, tumors were excised and further analyzed by immunohistochemistry.

[0065] In the depletion studies, the same treatment regimen as above was performed but in combination with CD4 or CD8 depleting antibodies (BioXCell) administered at a dose of 200 μg per animal on day -1, day 0, and weekly thereafter.

[0066] Immunostaining of PyMT tumors For SA-beta-galactosidase / PD-L2 double staining, after excision, tumors were fixed in PBS containing 2% paraformaldehyde and 0.2% glutaraldehyde for 2 hours at room temperature. Samples were washed with PBS for 15 minutes and then stained overnight with standard procedures for SA-beta-galactosidase activity. Tumors were then washed with PBS for 15 minutes, dehydrated with 70% ethanol, embedded in paraffin, sectioned, and stained for PDL2 by standard immunohistochemistry techniques with anti-PDL2 antibody (Cell Signaling, D6L5A Cat. No. 49189). To quantify the number of SA-beta-galactosidase and PDL2 double positive cells, three random fields from two mammary glands of each animal in each treatment group were analyzed with Image J software. Staining for CD3, CD4, and CD8 was also performed with standard immunohistochemistry techniques on paraffin-embedded material and quantified according to pixel density using Image J software.

Claims

1. 1. An isolated monoclonal antibody that specifically binds to programmed death ligand 2 (PD-L2) and blocks activity against the PD-L2 / hPD1 interaction, the antibody comprising six CDR sequences in a heavy chain variable region and a light chain variable region, the antibody comprising: a) as the heavy chain variable region CDRs, CDRH1 region of SEQ ID NO: 13 (GYAFSNYFIE) or SEQ ID NO: 14 (GYSFSNYFIE); The CDRH2 region of SEQ ID NO: 15 (LNIPGSGGSNYAEKFKG); and The CDRH3 region of SEQ ID NO: 16 (RRLPPDWYFDV); and b) as the light chain region CDRs: CDRL1 region of SEQ ID NO: 17 (RSSQSLVHSGGNTYLH) or SEQ ID NO: 20 (RSSQSLVHSDGNTYLH); CDRL2 region of SEQ ID NO: 18 (KVSNRFS); and CDRL3 region of SEQ ID NO:19 (SQSTHVPWT).

2. 2. The isolated monoclonal antibody of claim 1 comprising a heavy chain variable region and a light chain variable region, the antibody comprising: a) as the heavy chain variable region CDRs, CDRH1 region of SEQ ID NO: 14 (GYSFSNYFIE); The CDRH2 region of SEQ ID NO: 15 (LNIPGSGGSNYAEKFKG); and The CDRH3 region of SEQ ID NO: 16 (RRLPPDWYFDV); and b) as the light chain region CDRs: CDRL1 region of SEQ ID NO: 17 (RSSQSLVHSGGNTYLH); CDRL2 region of SEQ ID NO: 18 (KVSNRFS); and CDRL3 region of SEQ ID NO:19 (SQSTHVPWT).

3. 2. The isolated monoclonal antibody of claim 1 comprising a heavy chain variable region and a light chain variable region, the antibody comprising: a) as heavy chain variable region CDR CDRH1 region of SEQ ID NO:13 (GYAFSNYFIE); The CDRH2 region of SEQ ID NO: 15 (LNIPGSGGSNYAEKFKG); and The CDRH3 region of SEQ ID NO: 16 (RRLPPDWYFDV); and b) As a light chain region CDR CDRL1 region of SEQ ID NO:20 (RSSQSLVHSDGNTYLH); CDRL2 region of SEQ ID NO: 18 (KVSNRFS); and CDRL3 region of SEQ ID NO:19 (SQSTHVPWT).

4. An isolated monoclonal antibody according to any one of claims 1 to 3, (a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:1 (VH0), and the light chain variable region comprises the amino acid sequence of SEQ ID NO:9 (VK0); or (b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:6 (VH6), and the light chain variable region comprises the amino acid sequence of SEQ ID NO:12 (VK3); Preferably, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 6 (VH6), and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 12 (VK3).

5. 5. The isolated antibody of any one of claims 1 to 4, comprising at least one of the following characteristics: (a) the antibody binds human PD-L2 and －１０ Dissociation constant (K Ｄ ) to bond; (b) the antibody inhibits the interaction between human programmed cell death protein 1 (PD-1) and human PD-L2 with an EC50 of 1.3 nM or less in a bioluminescent cell-based assay using Jurkat T cells and CHO-K1 cells. ５０ in vitro blocking with , where Jurkat T cells express human PD-1 and a luciferase reporter driven by the NFAT response element (NFAT-RE), and CHO-K1 cells express human PD-L2 and an engineered cell surface protein designed to activate cognate TCRs in an antigen-independent manner; (c) the antibody specifically binds to both human and mouse PD-L2.

6. The isolated antibody of any one of claims 1 to 5, wherein the antibody is a chimeric, humanized, or human antibody.

7. A composition comprising the antibody of any one of claims 1 to 6 and a pharma- ceutically acceptable excipient.

8. An isolated nucleic acid sequence encoding the amino acid sequence of the light chain variable region and / or the heavy chain variable region of the antibody according to any one of claims 1 to 5.

9. A recombinant expression vector comprising the nucleic acid of claim 8.

10. 10. An isolated host cell comprising the recombinant expression vector of claim 9.

11. The antibody according to any one of claims 1 to 5 for use in treating, ameliorating, or preventing a disease or condition associated with the presence of senescent cells in cancer tissue.

12. The antibody for use according to claim 11 , wherein the antibody is administered in combination with a chemotherapeutic agent.

13. 13. The antibody for use according to any of claims 11-12, wherein the chemotherapeutic agent is selected from the group consisting of alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant derived alkaloids, topoisomerase inhibitors, hormone therapy agents, hormone antagonists, aromatase inhibitors, P-glycoprotein inhibitors, platinum complex derivatives, growth inhibitors, radioisotopes, targeted agents, and immunotherapeutic agents, and combinations thereof.

14. The antibody for use according to any one of claims 11 to 13, wherein the chemotherapeutic agent is selected from the group consisting of doxorubicin, carboplatin, and paclitaxel.

15. 15. The antibody for use according to any of claims 11 to 14, wherein the targeted agent is a CDK4 / 6 inhibitor, preferably selected from the group consisting of: ofimatinib, nilotinib, trametinib, vemurafenib, dasatinib, lapatinib, neratinib, afatinib, getofinib, erlotinib, sorafenib, sirolimus, rituximab, obinutuzumab, pertuzumab, trastuzumab, bevacizumab, ranibizumab, palbociclib, ademaciclib, ribociclib, olaparib, niraparib, rucaparib, and bortezomib.

16. The antibody for use according to any one of claims 11 to 15, wherein the disease is cancer.

17. 17. The antibody for use according to claim 16, wherein the cancer is selected from the group consisting of breast cancer, non-small cell lung cancer, ovarian cancer, squamous cell carcinoma of the head, squamous cell carcinoma of the neck, carcinoma, squamous cell carcinoma of the cervix, squamous cell carcinoma of the eyelid, squamous cell carcinoma of the conjunctiva, squamous cell carcinoma of the vagina, squamous cell carcinoma of the lung, squamous cell carcinoma of the oral cavity, squamous cell carcinoma of the skin, squamous cell carcinoma of the bladder, squamous cell carcinoma of the tongue, squamous cell carcinoma of the larynx, or squamous cell carcinoma of the esophagus, prostate gland, or bladder. an antibody selected from the group consisting of cancer, adenocarcinoma of the small intestine, adenocarcinoma of the endometrium, adenocarcinoma of the cervix, adenocarcinoma of the large intestine, adenocarcinoma of the lung, adenocarcinoma of the pancreas, adenocarcinoma of the esophagus, adenocarcinoma of the rectum, adenocarcinoma of the uterus, adenocarcinoma of the stomach, adenocarcinoma of the breast, adenocarcinoma of the ovary, sarcoma in myogenic sarcoma, leukemia, neuroma, melanoma, and lymphoma, preferably selected from the group consisting of breast cancer, non-small cell lung cancer, ovarian cancer, squamous cell carcinoma of the head, and squamous cell carcinoma of the neck.

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