Human anti-PD-L2 antibodies and uses thereof

Abstract

The present invention provides antibodies or antigen-binding fragments thereof that specifically bind to human PD-L2, as well as novel complementarity determining regions CDR1, CDR2 and CDR3 for each heavy chain and light chain of the antibodies. The antibodies or antigen-binding fragments have uses in enhancing T cell function, as well as treating cancer and acute or chronic infectious diseases.

Description

Technical Field

[0001] The present invention relates to antibodies that bind to programmed death ligand 2 (PD-L2), also known as cluster of differentiation 273 (CD273). Further, the invention relates to antibodies or antibody fragments that bind to PD-L2 for use in enhancing T cell function, including treatment of cancer and acute / chronic infections. Background Art

[0002] The programmed death-1 (PD-1, CD279) receptor has two ligands: programmed death ligand 1 (PD-L1, CD274, B7-H1) and programmed death ligand 2 (PD-L2, CD273, B7-DC). The interaction between PD-1 and its ligands is a key player in the immune checkpoint pathway, inhibiting T cell activation. While the roles of PD-L1 and PD-L2 in the tumor microenvironment remain controversial, several studies have demonstrated a role for both ligands in several tumor types. Cancer cells evade immune responses by exploiting various mechanisms, including the PD-1 / PD-L1 and PD-1 / PD-L2 interactions. This inhibits T cell activation, leading to T cell immunosuppression known as anergy or T cell exhaustion, and reduces cytotoxicity, thereby favoring cancer growth by mitigating antitumor immune responses. Antibodies targeting PD-1, PD-L1, and PD-L2 compete for ligand-receptor binding and inhibit this interaction, thereby enhancing T cell activation.

[0003] Although PD-L2 has historically been considered a secondary ligand, it binds to PD-1 with an affinity two to six times higher than that of PD-L1. PD-L2 can be expressed by immune cells, stromal cells, or tumor cells. To date, most focus has been on targeting the PD-1 / PD-L1 interaction for immunotherapy, and several monoclonal antibodies specifically designed to inhibit the PD-1 / PD-L1 axis (e.g., nivolumab, pembrolizumab, atezolizumab, durvalumab, etc.) are now available for the treatment of solid and hematological malignancies.

[0004] Few antibodies targeting the PD-1 / PD-L2 interaction have been developed for cancer / tumor immunotherapy.

[0005] WO2010036959A2 describes novel composite human monoclonal antibodies that specifically bind to human PD-1, human PD-L1, and human PD-L2. While the composite human antibodies against PD-1 and PD-L1 showed better IC50 values ​​than their mouse counterparts, the composite human antibodies against PD-L2 showed lower IC50 values ​​than their mouse counterparts, reaching as high as 0.5 µg / mL.

[0006] WO2017 / 053250A1 discloses anti-human PD-L2 antibodies, providing antibodies for immunohistochemical detection of human PD-L2 expression in tissue samples. However, its use in therapy has not been shown.

[0007] US20200369772A1 discloses anti-human PD-L2 antibodies for cancer treatment. The EC50 values ​​of the provided antibodies are significantly lower than those of the standard reference antibody 24F.10C12, and their binding efficiency has been demonstrated in various in vitro assays using mammalian cancer culture cells. However, their efficacy in cancer treatment has not yet been demonstrated.

[0008] In view of the deficiencies of the prior art, the present invention provides antibodies against PD-L2 to meet medical needs.

[0009] Purpose of the Invention The main object of the present invention is to provide novel human antibodies or antigen-binding fragments against PD-L2 for enhancing T cell function and having applications in the treatment of cancer and infectious diseases (acute or chronic).

[0010] Another object of the present invention is to provide a nucleic acid encoding an antibody or an antigen-binding fragment thereof against PD-L2.

[0011] Another object of the present invention is to provide a composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof.

[0012] Another object of the present invention is to provide a composition for preventing or treating infectious diseases, which comprises the antibody or antigen-binding fragment thereof. Summary of the Invention

[0013] The present invention relates to human anti-PD-L2 antibodies or antigen-binding fragments thereof, which specifically bind to human PD-L2, thereby blocking the binding of PD-L2 to PD-1.

[0014] The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to human PD-L2, comprising a light chain and a heavy chain. Furthermore, the invention provides a light chain comprising a light chain variable region having complementary determining region sequences (LCDR1, LCDR2, and LCDR3). The invention also provides a heavy chain comprising a heavy chain variable region having complementary determining region sequences (HCDR1, HCDR2, and HCDR3).

[0015] In one embodiment, the invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a light chain variable region selected from the group consisting of: Seq. ID 1, Seq. ID 2, Seq. ID 3, Seq. ID 7, Seq. ID 8, Seq. ID 9, Seq. ID 13, Seq. ID 14, Seq. ID 15, Seq. ID 19, Seq. ID 20, Seq. ID 21, Seq. ID 25, Seq. ID 26, Seq. ID 27, Seq. ID 31, Seq. ID 32, Seq. ID 33, Seq. ID37, Seq. ID 38, Seq. ID 39, Seq. ID 43, Seq. ID 44, and Seq. ID 45.

[0016] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a light chain variable region LCDR1 selected from the group consisting of: Seq. ID 1, Seq. ID 7, Seq. ID 13, Seq. ID19, Seq. ID 25, Seq. ID 31, Seq. ID 37, and Seq. ID 43.

[0017] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a light chain variable region LCDR2 selected from the group consisting of: Seq. ID 2, Seq. ID 8, Seq. ID 14, Seq. ID20, Seq. ID 26, Seq. ID 32, Seq. ID 38, and Seq. ID 44.

[0018] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a light chain variable region LCDR3 selected from the group consisting of: Seq. ID 3, Seq. ID 9, Seq. ID 15, Seq. ID21, Seq. ID 27, Seq. ID 33, Seq. ID 39, and Seq. ID 45.

[0019] In one embodiment, the present invention provides a light chain variable region of an antibody or antigen-binding fragment that specifically binds to human PD-L2, which includes LCDR1, LCDR2, and LCDR3, which are shown in Seq. ID No. 1, 2, and 3; Seq. ID No. 7, 8, and 9; Seq. ID No. 13, 14, and 15; Seq. ID No. 19, 20, and 21; Seq. ID No. 25, 26, and 27; Seq. ID No. 31, 32, and 33; Seq. ID No. 37, 38, and 39; or Seq. ID No. 43, 44, and 45, respectively.

[0020] In one embodiment, the invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a heavy chain variable region selected from the group consisting of: Seq. ID 4, Seq. ID 5, Seq. ID 6, Seq. ID 10, Seq. ID 11, Seq. ID 12, Seq. ID 16, Seq. ID 17, Seq. ID 18, Seq. ID 22, Seq. ID 23, Seq. ID 24, Seq. ID 28, Seq. ID 29, Seq. ID 30, Seq. ID 34, Seq. ID 35, Seq. ID 36, Seq. ID 40, Seq. ID 41, Seq. ID 42, Seq. ID 46, Seq. ID 47, and Seq. ID 48.

[0021] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a heavy chain variable region HCDR1 selected from the group consisting of: Seq. ID 4, Seq. ID 10, Seq. ID 16, Seq. ID22, Seq. ID 28, Seq. ID 34, Seq. ID 40, and Seq. ID 46.

[0022] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a heavy chain variable region HCDR2 selected from the group consisting of: Seq. ID 5, Seq. ID 11, Seq. ID 17, Seq. ID23, Seq. ID 29, Seq. ID 35, Seq. ID 41, and Seq. ID 47.

[0023] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a heavy chain variable region HCDR3 selected from the group consisting of: Seq. ID 6, Seq. ID 12, Seq. ID 18, Seq. ID24, Seq. ID 30, Seq. ID 36, Seq. ID 42, and Seq. ID 48.

[0024] In one embodiment, the present invention provides a heavy chain variable region of an antibody or antigen-binding fragment that specifically binds to human PD-L2, which includes HCDR1, HCDR2 and HCDR3, which are respectively as Seq. ID No. 4, 5 and 6; Seq. ID No. 10, 11 and 12; Seq. ID No. 16, 17 and 18; Seq. ID No. 22, 23 and 24; Seq. ID No. 28, 29 and 30; Seq. ID No. 34, 35 and 36; Seq. ID No. 40, 41 and 42 or Seq. ID No. 46, 47 and 48.

[0025] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human PD-L2, comprising LCDR1, LCDR2, and LCDR3 as shown in Seq. ID No. 1, 2, and 3, respectively; and HCDR1, HCDR2, and HCDR3 as shown in Seq. ID No. 4, 5, and 6, respectively.

[0026] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human PD-L2, comprising LCDR1, LCDR2, and LCDR3 as shown in Seq. ID No. 7, 8, and 9, respectively; and HCDR1, HCDR2, and HCDR3 as shown in Seq. ID No. 10, 11, and 12, respectively.

[0027] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human PD-L2, comprising LCDR1, LCDR2, and LCDR3 as shown in Seq. ID No. 13, 14, and 15, respectively; and HCDR1, HCDR2, and HCDR3 as shown in Seq. ID No. 16, 17, and 18, respectively.

[0028] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human PD-L2, comprising LCDR1, LCDR2, and LCDR3 as shown in Seq. ID No. 19, 20, and 21, respectively; and HCDR1, HCDR2, and HCDR3 as shown in Seq. ID No. 22, 23, and 24, respectively.

[0029] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human PD-L2, comprising LCDR1, LCDR2, and LCDR3 as shown in Seq. ID No. 25, 26, and 27, respectively; and HCDR1, HCDR2, and HCDR3 as shown in Seq. ID No. 28, 29, and 30, respectively.

[0030] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human PD-L2, comprising LCDR1, LCDR2, and LCDR3 as shown in Seq. ID No. 31, 32, and 33, respectively; and HCDR1, HCDR2, and HCDR3 as shown in Seq. ID No. 34, 35, and 36, respectively.

[0031] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human PD-L2, comprising LCDR1, LCDR2, and LCDR3 as shown in Seq. ID No. 37, 38, and 39, respectively; and HCDR1, HCDR2, and HCDR3 as shown in Seq. ID No. 40, 41, and 42, respectively.

[0032] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human PD-L2, comprising LCDR1, LCDR2, and LCDR3 as shown in Seq. ID No. 43, 44, and 45, respectively; and HCDR1, HCDR2, and HCDR3 as shown in Seq. ID No. 46, 47, and 48, respectively.

[0033] In one embodiment, the invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a light chain selected from the group consisting of Seq. ID 49, Seq. ID 50, Seq. ID 51, Seq. ID 52, Seq. ID 53, Seq. ID 54, Seq. ID 55, and Seq. ID 56.

[0034] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a light chain selected from the group consisting of Seq. ID 50, Seq. ID 51, Seq. ID 54, and Seq. ID 56, or a sequence with 85%–100% identity thereof; wherein the light chain variable region of the light chain sequence comprises: amino acid M or L at position 4, amino acid T or A at position 10, amino acid V or L at position 13, amino acid G or A at position 68, amino acid E or D at position 70, amino acid Q or E at position 79, amino acid K or R at position 103, and amino acid V or L at position 105.

[0035] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a light chain selected from the group consisting of: Seq. ID 49 and Seq. ID 52, or a sequence with 85%-100% identity thereof; wherein the light chain variable region of the light chain sequence comprises: amino acid T or K at position 17, amino acid V or I at position 59, amino acid S or T at position 73, amino acid G or A at position 75, amino acid S or T at position 77, amino acid S or T at position 81, and amino acid E or G at position 82.

[0036] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a light chain selected from the group consisting of Seq. ID 53 and Seq. ID 55, or a sequence with 85%–100% identity thereof; wherein the light chain variable region of the light chain sequence comprises: amino acid D or N at position 1, amino acid M or L at position 4, amino acid I or T at position 5, amino acid V or L at position 11, amino acid S or G at position 65, amino acid S or Y at position 67, amino acid D or H at position 70, and amino acid S or G at position 77.

[0037] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a heavy chain selected from the group consisting of: Seq. ID 57, Seq. ID 58, Seq. ID 59, Seq. ID 60, Seq. ID 61, Seq. ID 62, Seq. ID 63, and Seq. ID 64. Further, the present invention provides a heavy chain variable region of the heavy chain sequence, wherein the amino acid at position 1 is Q or E, the amino acid at position 5 is V or Q, the amino acid at position 74 is K or E, and the amino acid at position 114, 115, 116, or 118 is T, M, or L.

[0038] In one embodiment, the present invention provides nucleic acid sequences encoding the light chains of antibodies or antigen-binding fragments that specifically bind to human PD-L2, which are selected from the group consisting of Seq. ID 65, Seq. ID 66, Seq. ID 67, Seq. ID 68, Seq. ID 69, Seq. ID 70, Seq. ID 71, and Seq. ID 72, respectively, and the encoding light chain polypeptide sequences are Seq. ID 49, Seq. ID 50, Seq. ID 51, Seq. ID 52, Seq. ID 53, Seq. ID 54, Seq. ID55, and Seq. ID 56, respectively.

[0039] In one embodiment, the present invention provides nucleic acid sequences encoding the heavy chains of antibodies or antigen-binding fragments that specifically bind to human PD-L2, which are selected from the group consisting of: Seq. ID 73, Seq. ID 74, Seq. ID 75, Seq. ID 76, Seq. ID 77, Seq. ID 78, Seq. ID 79, and Seq. ID 80, respectively, and the light chain polypeptide sequences encoded are: Seq. ID 57, Seq. ID 58, Seq. ID 59, Seq. ID 60, Seq. ID 61, Seq. ID 62, Seq. ID 63, and Seq. ID 64, respectively.

[0040] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a light chain of Seq. ID 49 consisting of LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are selected from Seq. ID No. 1, 2, and 3, respectively; and a heavy chain of Seq. ID 57 consisting of HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are selected from Seq. ID No. 4, 5, and 6, respectively. The antibody specifically binds to human PD-L2 with an EC50 of approximately 28.38 nM.

[0041] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a light chain of Seq. ID 50 consisting of LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are selected from Seq. ID Nos. 7, 8, and 9, respectively; and a heavy chain of Seq. ID 58 consisting of HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are selected from Seq. ID Nos. 10, 11, and 12, respectively. The antibody specifically binds to human PD-L2 with an EC50 of approximately 8.159 nM.

[0042] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a light chain of Seq. ID 51 consisting of LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are selected from Seq. ID No. 13, 14, and 15, respectively; and a heavy chain of Seq. ID 59 consisting of HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are selected from Seq. ID No. 16, 17, and 18, respectively. The antibody specifically binds to human PD-L2 with an EC50 of approximately 11.64 nM.

[0043] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a light chain of Seq. ID 52 consisting of LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are selected from Seq. ID No. 19, 20, and 21, respectively; and a heavy chain of Seq. ID 60 consisting of HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are selected from Seq. ID No. 22, 23, and 24, respectively. The antibody specifically binds to human PD-L2 with an EC50 of approximately 37.66 nM.

[0044] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a light chain of Seq. ID 53 consisting of LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are selected from Seq. ID No. 25, 26, and 27, respectively; and a heavy chain of Seq. ID 61 consisting of HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are selected from Seq. ID No. 28, 29, and 30, respectively. The antibody specifically binds to human PD-L2 with an EC50 of approximately 11.18 nM.

[0045] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a light chain of Seq. ID 54 consisting of LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are selected from Seq. ID No. 31, 32, and 33, respectively; and a heavy chain of Seq. ID 62 consisting of HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are selected from Seq. ID No. 34, 35, and 36, respectively. The antibody specifically binds to human PD-L2 with an EC50 of about 15.83 nM.

[0046] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a light chain of Seq. ID 55 consisting of LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are selected from Seq. ID No. 37, 38, and 39, respectively; and a heavy chain of Seq. ID 63 consisting of HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are selected from Seq. ID No. 40, 41, and 42, respectively. The antibody specifically binds to human PD-L2 with an EC50 of approximately 28.32 nM.

[0047] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human PD-L2, comprising a light chain of Seq. ID 56 consisting of LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are selected from Seq. ID No. 43, 44, and 45, respectively; and a heavy chain of Seq. ID 64 consisting of HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are selected from Seq. ID No. 46, 47, and 48, respectively. The antibody specifically binds to human PD-L2 with an EC50 of approximately 32.41 nM. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1A -D depicts a graphical representation of the FACS blocking results of 36 candidate antibodies on huPD-L2-HEK293 cells compared to the negative control antibody R15-F7 (LGG1) and the reference antibody or positive control 24F.10C12; Figure 2Depicts a graphical representation of the results of luciferase assays on the PD-1-NF-AT luciferase-Jurkat reporter system for seven candidate molecules, A57, A96, A115, A117, A125, A131, A133, and A154, compared to the negative control antibody R15-F7 (LGG1) and the reference antibody or positive control 24F.10C12; Figure 3 Depicted are graphical representations of tumor growth / volume in mice up to 30 days following tumor cell inoculation and administration of three candidate antibodies (A96, A125, and A131) at the two doses listed in Table 6, either 2 mpk or 5 mpk, compared to the negative control antibody R15-F7 (IgG1) and the reference antibody or positive control 24F.10C12; Figure 4 depicts a graphical representation of mouse body weight 25-30 days following tumor cell inoculation and administration of three candidate antibodies (A96, A125, and A131) at the 5 mpk dose listed in Table 6, compared to the negative control antibody R15-F7 (LGG1) and the reference antibody or positive control 24F.10C12; and Figure 5 Depicted are graphical representations of tumor weights in mice on day 30 following tumor cell inoculation and administration of A96, A125, A131, 24F.10C12, and R15-F7 (IgG1) using a 5 mpk dose. Detailed Description of the Invention

[0049] definition

[0050] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, or polyclonal antibody that binds to a specific antigen. A natural intact antibody comprises two heavy chains (H) and two light chains (L). Each heavy chain consists of a variable region (V H ) and the first, second and third constant regions (C H ) and each light chain consists of a variable region (V L ) and constant region (C L ). This antibody has a "Y" shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together by disulfide bonds. Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions.

[0051] As used herein, the term "CDR or complementarity determining region" includes the variable region portions of both antibody chains. These are typically three highly variable loops in each light chain and each heavy chain. The light (L) chain CDRs include LCDR1, LCDR2, and LCDR3, and the heavy (H) chain CDRs include HCDR1, HCDR2, and HCDR3.

[0052] As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed by a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain the entire native antibody structure. An antigen-binding fragment is capable of binding to the same antigen as the parent antibody.

[0053] As used herein, the term "fully human antibody" or "human antibody" refers to an antibody or antigen-binding fragment that has or consists of an amino acid sequence corresponding to an antibody produced by a human or human immune cell, or is derived from a non-human source (such as a transgenic non-human animal utilizing a human antibody library or other human antibody encoding sequence). In certain embodiments, a fully human antibody does not comprise amino acid residues (particularly antigen-binding residues) derived from a non-human antibody.

[0054] The term "humanized antibody" as used herein with reference to antibodies or antigen-binding fragments refers to antibodies derived from non-human species whose amino acid sequences have been modified to increase their similarity to naturally occurring antibody variants in humans.

[0055] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homologous antibody group, i.e., except for a small amount of possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), the individual antibodies comprising the group are identical. Monoclonal antibodies are highly specific, being directed to a single antigenic site. In contrast to polyclonal antibody preparation (which typically includes directed to different antibodies for different determinants (epitopes)), every kind of monoclonal antibody is directed to a single determinant on the antigen.

[0056] As used herein, the term "PD-L2" refers to programmed cell death ligand 2, also known as CD273, B7-DC, PDCD1L2, PDCD1LG2, or PDL2. PD-L2 is a transmembrane protein expressed on various tumor cells, stromal cells, or both, and binds to PD-1.

[0057] As used herein, the term "PD-1" refers to programmed cell death-1 (also known as CD279), a key immune checkpoint receptor known to be expressed by activated T cells that mediate immunosuppression. Inhibiting the interaction between PD-1 and PD-L1 can enhance T cell responses and thereby mediate anti-cancer activity.

[0058] As used herein, the term "anti-PD-L2 antibody" refers to an antibody that specifically binds to PD-L2 (e.g., human PD-L2) with an affinity sufficient to provide diagnostic and / or therapeutic use.

[0059] As used herein, the ability to "block binding" refers to the ability of an antibody or antigen-binding fragment to inhibit, to any detectable extent, the binding interaction between two molecules (e.g., human PD-L2 and an anti-PD-L2 antibody).

[0060] The term "EC50" as used herein refers to the antibody concentration required to block PD-1 / PD-L2 binding to 50% of the maximum binding amount.

[0061] As used herein, the term "vector" refers to a polynucleotide that can be operably inserted into a protein encoding protein to achieve a vehicle for expressing the protein. A vector can be used to transform, transduce, or transfect a host cell so that the genetic elements it carries are expressed in the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived from artificial chromosomes (PAC)), bacteriophages (such as lambda phage or M13 phage), and animal viruses. The vector may include various elements for regulating expression, including promoter sequences, transcription initiation sequences, enhancer sequences, screening elements, and reporter genes. In addition, the vector may include an origin of replication. The vector may also include materials that assist its entry into cells, including but not limited to viral particles, liposomes, or protein coatings.

[0062] The present invention will now be described with reference to the following detailed description, which shows some, but not all, embodiments of the invention. Indeed, the invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. The invention is fully described herein by way of non-limiting examples and illustrative experimental descriptions.

[0063] In the primary embodiment of the present invention, the present invention provides human antibodies or antigen-binding fragments that specifically bind to human PD-L2. Furthermore, the present invention provides the CDRs of the light and heavy chains of each human antibody or antigen-binding fragment that specifically binds to human PD-L2. The amino acid sequences of exemplary antibodies and CDRs are shown in Table 1.

[0064] Table 1: List of polypeptide sequences of anti-PD-L2 antibody light chain, heavy chain, and light chain and heavy chain CDRs

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071] In another embodiment of the present invention, the present invention provides nucleic acid sequences encoding human antibodies or antigen-binding fragments that specifically bind to human PD-L2. Further, the present invention provides nucleic acid sequences encoding the light and heavy chains of each human antibody or antigen-binding fragment that specifically binds to human PD-L2. The nucleic acid sequences encoding the light and heavy chains of exemplary antibodies are shown in Table 2.

[0072] Table 2: List of nucleic acid sequences encoding light and heavy chains of anti-PD-L2 antibodies

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] In one embodiment, the present invention provides a human anti-PD-L2 antibody comprising a light chain consisting of variable regions LCDR1, LCDR2, and LCDR3, and a heavy chain consisting of variable regions HCDR1, HCDR2, and HCDR3.

[0082] In one embodiment, the invention provides the LCDR1 of Seq. ID 7 or an amino acid sequence having at least 89% identity to Seq. ID 7. LCDR1 is a nine amino acid sequence long polypeptide. The LCDR1 consists of the amino acid Asn (N) or Tyr (Y) at position 6. Additionally, LCDR1 is Seq. ID 43. LCDR2 is Seq. ID 8 or an amino acid sequence having 85% or more identity to Seq. ID 8. LCDR2 comprises a thirteen amino acid sequence having at least 85% identity to Seq. ID 8, the sequence consisting of the amino acid Gly (G) or Asp (D) at position 7 and the amino acid Thr (T) or Asn (N) at position 10. Additionally, LCDR2 is Seq. ID 14. LCDR3 is Seq. ID 9 or has at least 50% or more identity to Seq. ID 9. LCDR3 comprises a fourteen amino acid sequence that is at least 50% or more identical to Seq. ID 9, consisting of the amino acid His (H) or Gln (Q) at position 2, the amino acid Gln (Q) or Arg (R) at position 3, the amino acid Gly (G) or Asn (N) or Ser (S) at position 4, nothing or the amino acid Asn (N) at position 5, nothing or the amino acid Trp (W) at position 6, nothing or the amino acid Pro (P) at position 7, nothing or the amino acid Ile (I) or Leu (L) at position 8, and the amino acid Gln (Q) or Gly (G) at position 12. Additionally, LCDR3 is selected from the group consisting of Seq. ID 15 and 45.

[0083] In another embodiment, the invention provides the HCDR1 of Seq. ID 10. HCDR2 is Seq. ID 11, or is at least 81% or more identical to Seq. ID 11. HCDR2 comprises a sixteen amino acid sequence that is at least 81% identical to Seq. ID 11, Seq. ID 11 consisting of amino acid Gly (G) or Arg (R) at position 6, amino acid Phe (F) or Leu (L) at position 11, and amino acid Thr (T) or Ile (I) at position 13. Furthermore, HCDR2 is Seq. ID 17. HCDR3 is Seq. ID 12. Furthermore, HCDR3 is selected from the group consisting of Seq. ID 18 and 48.

[0084] In another embodiment, the human anti-PD-L2 antibody comprises a light chain consisting of the variable region LCDR1 of Seq. ID 7, the variable region LCDR2 of Seq. ID 8, and the variable region LCDR3 of Seq. ID 9, and a heavy chain consisting of the variable region HCDR1 of Seq. ID 10, the variable region HCDR2 of Seq. ID 11, and the variable region HCDR3 of Seq. ID 12.

[0085] In another embodiment, the human anti-PD-L2 antibody comprises a light chain consisting of variable region LCDR1 of Seq. ID 13 (which is 100% identical to Seq. ID 7), variable region LCDR2 of Seq. ID 14, and variable region LCDR3 of Seq. ID 15, and a heavy chain consisting of variable region HCDR1 of Seq. ID 16 (which is 100% identical to Seq. ID 10), HCDR2 of Seq. ID 17, and HCDR3 of Seq. ID 18.

[0086] In another embodiment, the human anti-PD-L2 antibody comprises a light chain consisting of variable region LCDR1 of Seq. ID 43, LCDR2 of Seq. ID 44 (which is 100% identical to Seq. ID 14), and LCDR3 of Seq. ID 45, and a heavy chain consisting of variable region HCDR1 of Seq. ID 46 (which is 100% identical to Seq. ID 10), HCDR2 of Seq. ID 17 (which is 100% identical to Seq. ID 17), and HCDR3 of Seq. ID 48.

[0087] In another embodiment, the human anti-PD-L2 antibody consists of a light chain and a heavy chain, wherein the light chain is Seq. ID 50 or has at least 92% or more identity with Seq. ID 50, and the heavy chain is Seq. ID 58 or has at least 97% or more identity with Seq. ID 58. The light chain is further selected from the group consisting of Seq. ID 51 and 56. The heavy chain is further selected from the group consisting of Seq. ID 59 and 64.

[0088] The present invention further provides a human anti-PD-L2 antibody consisting of the light chain of Seq. ID 51 and the heavy chain of Seq. ID 59.

[0089] The present invention also provides a human anti-PD-L2 antibody, which consists of the light chain of Seq. ID 56 and the heavy chain of Seq. ID 64.

[0090] The light chain of Seq. ID 50 consists of the variable region LCDR1 of Seq. ID 7, LCDR2 of Seq. ID 8, and LCDR3 of Seq. ID 9. In addition, the heavy chain of Seq. ID 58 consists of the variable region HCDR1 of Seq. ID 7, HCDR2 of Seq. ID 11, and HCDR3 of Seq. ID 12.

[0091] The light chain of Seq. ID 51 consists of the variable region LCDR1 of Seq. ID 7, the LCDR2 of Seq. ID 14, and the LCDR3 of Seq. ID 15. In addition, the heavy chain of Seq. ID 59 consists of the variable region HCDR1 of Seq. ID 10, the HCDR2 of Seq. ID 17, and the HCDR3 of Seq. ID 18.

[0092] The light chain of Seq. ID 56 consists of the variable region LCDR1 of Seq. ID 43, the LCDR2 of Seq. ID 14, and the LCDR3 of Seq. ID 45. In addition, the heavy chain of Seq. ID 64 consists of the variable region HCDR1 of Seq. ID 10, the HCDR2 of Seq. ID 17, and the HCDR3 of Seq. ID 48.

[0093] In yet another embodiment, the human anti-PD-L2 antibody comprises the light chain of Seq. ID 51 and the heavy chain of Seq. ID 59.

[0094] In yet another embodiment, the human anti-PD-L2 antibody comprises the light chain of Seq. ID 56 and the heavy chain of Seq. ID 64.

[0095] In another embodiment, the present invention provides the nucleotide sequence of Seq. ID 66, which encodes the light chain of the human anti-PD-L2 antibody of Seq. ID 50.

[0096] In another embodiment, the present invention provides the nucleotide sequence of Seq. ID 74, which encodes the heavy chain of the human anti-PD-L2 antibody of Seq. ID 58.

[0097] In another embodiment, the present invention provides the nucleotide sequence of Seq. ID 67, which encodes the light chain of the human anti-PD-L2 antibody of Seq. ID 51.

[0098] In another embodiment, the present invention provides the nucleotide sequence of Seq. ID 75, which encodes the heavy chain of the human anti-PD-L2 antibody of Seq. ID 59.

[0099] In another embodiment, the present invention provides the nucleotide sequence of Seq. ID 72, which encodes the light chain of the human anti-PD-L2 antibody of Seq. ID 56.

[0100] In another embodiment, the present invention provides the nucleotide sequence of Seq. ID 80, which encodes the heavy chain of the human anti-PD-L2 antibody of Seq. ID 64.

[0101] In an alternative embodiment, the invention provides a human anti-PD-L2 antibody, wherein: the light chain consists of the LCDR1 of Seq. ID 31, the LCDR2 of Seq. ID 32, and the LCDR3 of Seq. ID 33; and the heavy chain consists of the HCDR1 of Seq. ID 34 (which is 100% identical to Seq. ID 10), the HCDR2 of Seq. ID 35, and the HCDR3 of Seq. ID 36.

[0102] In another embodiment, the human anti-PD-L2 antibody consists of the light chain of Seq. ID 54 and the heavy chain of Seq. ID 62.

[0103] The light chain of Seq. ID 54 consists of the variable region LCDR1 of Seq. ID 31, the LCDR2 of Seq. ID 32, and the LCDR3 of Seq. ID 33. In addition, the heavy chain of Seq. ID 62 consists of the variable region HCDR1 of Seq. ID 10, the HCDR2 of Seq. ID 35, and the HCDR3 of Seq. ID 36.

[0104] In another embodiment, the present invention provides the nucleotide sequence of Seq. ID 70, which encodes the light chain of the human anti-PD-L2 antibody of Seq. ID 54, and the nucleotide sequence of Seq. ID 78, which encodes the heavy chain of the human anti-PD-L2 antibody of Seq. ID 62.

[0105] In yet another alternative embodiment, the invention provides Seq. ID 25 or a LCDR1 that is at least 65% or more identical to Seq. ID 25. Additionally, LCDR1 is Seq. ID 37. LCDR2 is Seq. ID 26 or is at least 78% or more identical to Seq. ID 26. LCDR2 comprises a thirteen amino acid long sequence that is at least 85% or more identical to Seq. ID 26, consisting of the amino acid Lys (L) or Arg (R) at position 3, and the amino acid Ala (A) or Glu (G) at position 7. Additionally, LCDR2 is Seq. ID 38. LCDR3 is Seq. ID 27 or is at least 69% or more identical to Seq. ID 27. Additionally, LCDR3 is Seq. ID 39.

[0106] In yet another alternative embodiment, the invention provides LCDR1 of Seq. ID 28 and Seq. ID 40, which are 100% identical to Seq. ID 10. The invention also provides HCDR2 of Seq. ID 29 and Seq. ID 41, which are 100% identical to Seq. ID 17. The HCDR3 is selected from the group consisting of Seq. ID 30 and Seq. ID 42.

[0107] In another embodiment, the human anti-PD-L2 antibody comprises a light chain consisting of LCDR1 of Seq. ID 25, LCDR2 of Seq. ID 26, and LCDR3 of Seq. ID 27; and a heavy chain consisting of HCDR1 of Seq. ID 10, HCDR2 of Seq. ID 17, and HCDR3 of Seq. ID 30.

[0108] In another embodiment, the human anti-PD-L2 antibody comprises a light chain consisting of LCDR1 of Seq. ID 37, LCDR2 of Seq. ID 38, and LCDR3 of Seq. ID 39; and a heavy chain consisting of HCDR1 of Seq. ID 10, HCDR2 of Seq. ID 17, and HCDR3 of Seq. ID 42.

[0109] In another embodiment, the human anti-PD-L2 antibody consists of a light chain and a heavy chain, wherein the light chain is Seq. ID 53 or is at least 89% or more identical to Seq. ID 53; and the heavy chain is Seq. ID 61 or is at least 97% or more identical to Seq. ID 61. The light chain is Seq. ID 55. The heavy chain is Seq. ID 63.

[0110] The present invention also provides a human anti-PD-L2 antibody consisting of the light chain of Seq. ID 55 and the heavy chain of Seq. ID 63.

[0111] The light chain of Seq. ID 53 consists of the variable region LCDR1 of Seq. ID 25, LCDR2 of Seq. ID 26, and LCDR3 of Seq. ID 27. In addition, the heavy chain of Seq. ID 61 consists of the variable region HCDR1 of Seq. ID 10, HCDR2 of Seq. ID 17, and HCDR3 of Seq. ID 30.

[0112] The light chain of Seq. ID 55 consists of the variable region LCDR1 of Seq. ID 37, the LCDR2 of Seq. ID 38, and the LCDR3 of Seq. ID 39. In addition, the heavy chain of Seq. ID 63 consists of the variable region HCDR1 of Seq. ID 10, the HCDR2 of Seq. ID 17, and the HCDR3 of Seq. ID 42.

[0113] In yet another embodiment, the human anti-PD-L2 antibody consists of the light chain of Seq. ID 55 and the heavy chain of Seq. ID 63.

[0114] In another embodiment, the present invention provides the nucleotide sequence of Seq. ID 69, which encodes the light chain of the human anti-PD-L2 antibody of Seq. ID 53.

[0115] In another embodiment, the present invention provides the nucleotide sequence of Seq. ID 77, which encodes the heavy chain of the human anti-PD-L2 antibody of Seq. ID 61.

[0116] In another embodiment, the present invention provides the nucleotide sequence of Seq. ID 71, which encodes the light chain of the human anti-PD-L2 antibody of Seq. ID 55.

[0117] In another embodiment, the present invention provides the nucleotide sequence of Seq. ID 79, which encodes the heavy chain of the human anti-PD-L2 antibody of Seq. ID 63.

[0118] In yet another alternative embodiment, the invention provides Seq. ID 1 or a LCDR1 that is at least 73% or more identical to Seq. ID 1. Additionally, LCDR1 is Seq. ID 19. LCDR2 is Seq. ID 2 or is at least 85% or more identical to Seq. ID 2. Additionally, LCDR2 is Seq. ID 20. LCDR3 is selected from the group consisting of Seq. ID 3 and Seq. ID 21.

[0119] In yet another alternative embodiment, the invention provides HCDR1 of Seq. ID 4 and Seq. ID 22, which are 100% identical to Seq. ID 10. HCDR2 is selected from the group consisting of Seq. ID 5 and Seq. ID 23, which are identical to Seq. ID 17. HCDR3 is selected from the group consisting of Seq. ID 6 and Seq. ID 24.

[0120] In another embodiment, the human anti-PD-L2 antibody comprises a light chain consisting of LCDR1 of Seq. ID 1, LCDR2 of Seq. ID 2, and LCDR3 of Seq. ID 3; and a heavy chain consisting of HCDR1 of Seq. ID 10, HCDR2 of Seq. ID 17, and HCDR3 of Seq. ID 6.

[0121] In another embodiment, the human anti-PD-L2 antibody comprises a light chain consisting of LCDR1 of Seq. ID 19, LCDR2 of Seq. ID 20, and LCDR3 of Seq. ID 21; and a heavy chain consisting of HCDR1 of Seq. ID 10, HCDR2 of Seq. ID 17, and HCDR3 of Seq. ID 24.

[0122] In yet another embodiment, the human anti-PD-L2 antibody consists of the light chain of Seq. ID 49 and the heavy chain of Seq. ID 57.

[0123] The present invention also provides a human anti-PD-L2 antibody consisting of the light chain of Seq. ID 52 and the heavy chain of Seq. ID 60.

[0124] The light chain of Seq. ID 49 consists of the variable region LCDR1 of Seq. ID 1, the LCDR2 of Seq. ID 2, and the LCDR3 of Seq. ID 3. In addition, the heavy chain of Seq. ID 57 consists of the variable region HCDR1 of Seq. ID 10, the HCDR2 of Seq. ID 17, and the HCDR3 of Seq. ID 6.

[0125] The light chain of Seq. ID 52 consists of the variable region LCDR1 of Seq. ID 19, the LCDR2 of Seq. ID 20, and the LCDR3 of Seq. ID 21. In addition, the heavy chain of Seq. ID 60 consists of the variable region HCDR1 of Seq. ID 10, the HCDR2 of Seq. ID 17, and the HCDR3 of Seq. ID 24.

[0126] In another embodiment, the present invention provides the nucleotide sequence of Seq. ID 65, which encodes the light chain of the human anti-PD-L2 antibody of Seq. ID 49.

[0127] In another embodiment, the present invention provides the nucleotide sequence of Seq. ID 73, which encodes the heavy chain of the human anti-PD-L2 antibody of Seq. ID 57.

[0128] In another embodiment, the present invention provides the nucleotide sequence of Seq. ID 68, which encodes the light chain of the human anti-PD-L2 antibody of Seq. ID 52.

[0129] In another embodiment, the invention provides the nucleotide sequence of Seq. ID 76, which encodes the heavy chain of the human anti-PD-L2 antibody of Seq. ID 60.

[0130] In another embodiment, the present invention provides a method for producing a recombinant monoclonal human anti-PD-L2 antibody. The antibody can be produced by techniques known in the art. The nucleotide sequences encoding the light and heavy chains of the antibody are cloned into a suitable vector for expression in a host cell. The method comprises the following steps: a. transforming a suitable host cell with an expression vector comprising a nucleotide sequence encoding the light and heavy chains of an antibody; b. Isolating / separating / collecting antibodies produced by host cells; and c. Antibody purification.

[0131] In another embodiment, the present invention provides a kit comprising a human anti-PD-L2 antibody or antibody fragment thereof for detecting the presence of PD-L2 in a biological sample. The antibody can be further conjugated to a labeling molecule. Furthermore, the antibody can be attached to a substrate or device for use in assays such as ELISA.

[0132] In another embodiment, the present invention provides pharmaceutical compositions comprising a human anti-PD-L2 antibody or antibody fragment thereof and one or more pharmaceutically acceptable carriers. The pharmaceutical compositions may be liquid solutions, suspensions, emulsions, capsules, tablets, sustained-release formulations, and powders. Furthermore, these pharmaceutical compositions may be administered parenterally or non-parenterally.

[0133] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials and methods described below fall within the scope of the present invention in whole or in part. These specific compositions, materials and methods are not intended to limit the invention, but are merely to illustrate specific embodiments that fall within the scope of the present invention. Those skilled in the art can, without paying creative work and without departing from the scope of the invention, develop equivalent compositions, materials and methods. It should be understood that, while still remaining within the boundaries of the present invention, many changes can be made in the process described herein. The inventor's intention is to include such changes within the scope of the invention.

[0134] Example Example 1 Panning and screening of recombinant humanized antibody libraries a. Kingfisher method

[0135] The stored natural library phage suspension was diluted and blocked with 2.5% BSA, incubated with Dynabeads, and the phage incubated after negative screening was collected for background subtraction. According to the Kingfisher magnetic bead screening system method, the Dynabeads were blocked and coated by binding and washing the magnetic beads and incubating the magnetic beads with 2.5% BSA. Positive screening was performed by incubating the phage suspension collected after negative screening with Dynabeads coated with biotin-labeled antigen and blocked, followed by binding and washing according to the Kingfisher magnetic bead screening system method. The phage was eluted with trypsin. After elution, the library plate was infected, and the phage solution was thoroughly mixed with logarithmic SS320 cells and then incubated at 37°C for 30 minutes. It was then spread on 2YT-Car+-Tet+ plates and incubated at 37°C overnight. The eluted phage solution was diluted with a 10-fold logarithmic gradient of SS320 cells, incubated at 37°C for 30 minutes, mixed, and 2 μL was dropped onto a plate and incubated at 37°C overnight. The phage input and output were calculated, and the input phage was prepared by scraping the phage from the plate. Four rounds of screening were performed. Each round of screening used three antigen concentration gradients to screen and obtain antibody clones with high affinity.

[0136] b. Immunotube method To subtract background, the stored naive library was blocked with 5% PBSM. Immunotubes were coated and blocked with diluted antigen overnight at 4°C, then washed according to the Immunotube Screening System protocol and blocked with 5% PBSM. Positive screening was performed by incubating the phage suspension with the protein-coated and blocked immunotubes, followed by binding and washing according to the Immunotube Screening System protocol. Elution, infection, and titration were performed as described in the Kingfisher method.

[0137] c. Use ELISA to detect the positive rate of the panning pool.

[0138] d. Primary screening of monoclonal antibodies by ELISA: Monoclonal antibodies collected from the second and third rounds of panning were screened by phage ELISA using the human antigen PD-L2-Q9BQ51-His, as well as by coating with human PD-L2-Q9BQ51-His. The negative control was 6G / 6H, and the corresponding output blocking solution was 6A / 6B. The positive control was 12G / 12H. Positive clones were identified based on a certain background value. This initial screening yielded 447 positive clones that bound to the human antigen PD-L2-Q9BQ51-His, of which 69 had unique sequences.

[0139] e. Affinity ranking by ELISA: 69 clones with unique sequences were serially diluted to prepare phage supernatants and subjected to affinity ELISA analysis using the corresponding human antigen PD-L2-Q9BQ51-His. The results showed that 54 clones with unique sequences had affinity for the human antigen PD-L2-Q9BQ51-His.

[0140] f. Final clone selection: Based on affinity, 36 clones were selected out of 54.

[0141] Example 2 IgG construction and production DNA of the 36 best cell-binding candidates was synthesized and inserted into pcDNA3.4 to prepare expression plasmids for full-length IgG, fully human IgG. The heavy and light chain expression plasmids were co-transfected into Expi293F cells. The recombinant IgG secreted into the culture medium was purified using protein A affinity chromatography.

[0142] Example 3 Human IgG analysis 1. Evaluation of Binding Activity of Selected Monoclonal Antibody Clones The binding activity of the selected 36 monoclonal antibody clones was evaluated by FACS using HuPD-L2-HEK293 cells, which are HEK293 cells that overexpress human PD-L2 (HuPD-L2). Binding activity was tested against the well-known and reference PD-L2 antibody - 24F.10C12.

[0143] Table 3: Sample layout of FACS binding of candidate antibodies on huPD L2 HEK293 cells.

[0144]

[0145] Table 4: FACS binding assay results relative to positive control Ab 24F.10C12 (P08047), IgG1 (negative control) and other negative controls.

[0146]

[0147] All 36 antibodies showed good binding to HuPD-L2 compared to the positive reference antibody 24F.10C12.

[0148] 2. Evaluation of the Blocking Activity of Selected Monoclonal Antibody Clones The blocking activity of the selected 36 monoclonal antibody clones was assessed by FACS against the well-known and reference PD-L2 antibody - 24F.10C12 using HuPD-L2-HEK293 cells and the ligand human PD-1-Q15116-Fc.

[0149] As shown in FIG1 , only one of the 36 antibody clones did not show any blocking activity, and the remaining 35 showed good blocking activity against the ligand.

[0150] 3. Selection of the final anti-PD-L2 antibody clone for further evaluation Based on binding and blocking activity efficiency, only 8 clones out of 35 were selected for further analysis, namely A57, A96, A115, A117, A125, A131, A133 and A154. The amino acid sequences of the light and heavy chains of the selected 8 candidates are provided in Table 1.

[0151] 4. PD-1-NF-AT-Luciferase-Jurkat Reporter Assay A PD-1-NF-AT-luciferase-Jurkat reporter assay was performed using CD3L PD L2 CHO cells with the positive control antibody 24F.10C12 (negative control) and candidate anti-PD-L2 antibodies - A57, A96, A115, A117, A125, A131, A133, and A154.

[0152] Figure 2 All candidate antibodies showed blocking activity in the PD-1 NF AT luciferase Jurkat reporter system. However, the results showed that the blocking effect of the candidate molecules in the PD-1 NF AT luciferase Jurkat reporter system was less than that of the positive antibody 24F.10C12.

[0153] 5. EC50 value of candidate antibodies The EC50 value is the antibody concentration that produces half-maximal binding, as determined by direct and saturable binding of the antibody to two target antigens.

[0154] Table 5 provides the EC-50 values ​​for the positive control antibody 24F.10C12 and the candidate anti-PD-L2 antibodies - A57, A96, A115, A117, A125, A131, A133 and A154.

[0155] Table 5: EC50 values ​​of candidate antibodies

[0156] The results showed that the candidate antibody exhibited a higher EC50 value than the positive control 24F.10C12, indicating that the candidate antibody required a higher concentration than the positive control 24F.10C12 to achieve half-maximal antigen binding.

[0157] Therefore, only three of the selected candidate antibodies were finally determined for in vivo studies in animal models to examine the effects of the antibodies on tumor resistance and tumor growth reduction. The selected antibodies were A96, A125, and A131.

[0158] 6. In vivo efficacy evaluation using animal mouse models Cell line-derived xenograft (CDX) models are established by subcutaneous, intravenous, or orthotopic inoculation of tumor cell lines into wild-type, immunodeficient, or humanized immune system mice. CDX models offer a short experimental cycle, high modeling success rate, and relatively low experimental costs. They are widely used as functional in vivo tumor models for drug target validation, pharmacodynamic evaluation, PK / PD studies, and evaluation of multidrug synergy.

[0159] The aim of this study was to validate the tumor inhibitory effects of the selected candidate antibodies - A96, A125 and A131 compared to the reference antibody 24F.10C12.

[0160] Briefly, tumor cell lines were appropriately expanded in vitro and then inoculated subcutaneously into the right back of HPD-1 mice to establish the tumor model. Tumors grew to 50–80 mm 3Afterwards, mice are randomized and administered with a reference antibody or candidate antibody according to the dosage regimen. Tumor size and signs are then measured 3-5 weeks after administration, and the corresponding therapeutic effects are evaluated.

[0161] The tumor cell line used was PD-L2-MC38 cells, which are murine colon adenocarcinoma cells that overexpress PD-L2.

[0162] The resuspended PD-L2-MC38 cells were injected subcutaneously into the right back of the mouse at a dose of 0.1 mL per mouse. As shown in Table 6.

[0163]

[0164]

[0165] Note: Tumor volume = 1 / 2 (long diameter × wide diameter × wide diameter); ip stands for intraperitoneal injection; BIW stands for twice a week.

[0166] After tumor inoculation, the effects of tumor growth and treatment on the animals' normal behavior were assessed, including activity, food and water consumption, weight gain / loss, eye and coat abnormalities, and other abnormalities. No abnormal behavior or death was observed in the animals during the experiment.

[0167] result: a) Body weight: The weight of mice was measured twice a week using an electronic balance.

[0168] b) Tumor Measurement: After experimental mice were grouped, tumor length and width diameter were measured twice weekly using a vernier caliper. When tumor volume reached 2000 mm3, tumor-bearing mice were euthanized with CO2, and the tumor masses were excised, weighed, and photographed.

[0169] Figure 3A graphic representation of tumor growth 25 days after tumor cell inoculation is provided, and the reference antibody or candidate antibody or IgG negative control was administered at two doses of 2 mpk or 5 mpk as listed in Table 6. Tumor growth was measured by measuring length and width using a vernier caliper. As shown, the IgG negative control had no effect on tumor growth, and tumors continued to grow until days 16-17, and then began to slow down to reduce other physiological effects. Reference antibodies A96, A125, and A131 showed a reduction in tumor growth on days 10 and 25, with tumors equivalent to those of the negative control. This clearly shows that although the reference antibodies were initially effective, they later began to show reduced efficiency in tumor growth. At the 2 mpk dose, and more specifically at the 5 mpk dose, all candidate antibodies - A96, A125, and A131 - showed a significant reduction in tumor growth compared to the reference antibody. On day 25, the A96 antibody almost completely reduced the tumor.

[0170] This established that the candidate antibodies A96, A125 and A131 showed more efficacy compared to the reference antibodies A96, A125 and A131.

[0171] Furthermore, to show that the effect on tumor weight reduction was independent of overall mouse weight loss, the weight of all mice was monitored throughout the study. Figure 4 Shown are the weights of mice 25 days after tumor cell inoculation.

[0172] Furthermore, the tumor inhibition efficacy of the antibodies was calculated using the method provided below.

[0173] Calculation of relative tumor inhibition rate: Methods: TGI = 1 - T / C (%). T / C% refers to the relative tumor inhibition rate, which is the ratio of the relative tumor volume or tumor weight of the treatment group to the control group at a specific time point. T and C refer to the relative tumor volume (RTV) or tumor weight (TW) of the treatment and control groups, respectively, at a specific time point.

[0174] For this purpose, animal studies in mice were performed using R15-F7 (IgG1) (negative control), reference antibody or positive control (24F.10C12), A96, A125 and A131 at a dose of 5 mpk.

[0175] Table 7 provides the tumor inhibition rates at different time points for reference antibody 24F.10C12, candidate antibodies A96, A125, and A131 administered at a 5 mpk dose compared to an IgG1 negative control antibody.

[0176] Table 7: Tumor Inhibition Percentage / Ratio

[0177] These results show that the negative control antibody had no effect on tumor growth, resulting in an inhibition rate of 0. The reference antibody, 24F.10C12, exhibited tumor inhibition until day 20, but efficacy declined sharply thereafter, resulting in greater tumor regrowth than the negative control antibody. The maximum effect of 24F.10C12 on tumor weight was only approximately 31% on day 20.

[0178] The A96 candidate antibody showed significant tumor inhibition, reaching 80% inhibition by day 17, and tumors were reduced to 96% of their original weight by day 30.

[0179] The A131 and A125 candidate antibodies also reduced tumor weight. Compared to the reference antibody, A125 also showed 96% inhibition by day 30, and A131 also showed a significant tumor inhibition rate of approximately 76% by day 30.

[0180] Figure 5 A graphic representation of tumor weights at day 30 using a 5 mpk dose of A96, A125, A131, 24F.10C12, and R15-F7 (IgG1) is provided. The results clearly show that tumors regrew when treated with the reference antibody 24F.10C12. However, A96 and A125 almost completely reduced tumors, and A131 significantly reduced tumor growth compared to 24F.10C12.

[0181] These results clearly show that candidate antibodies A96, A125 and A131 are more potent than the reference antibody - 24F.10C12.

[0182] Table 8 summarizes the light and heavy chain sequence IDs of the candidate antibodies.

[0183] Table 8 The amino acid sequence numbers of each antibody and the complementarity determining regions (CDRs) of the light and heavy chains

[0184] Although specific exemplary embodiments have been described and shown in the accompanying drawings, it should be understood that such embodiments are merely illustrative of the invention and not limiting thereof, and that the invention is not limited to the specific constructions and arrangements shown and described; various other changes, combinations, omissions, modifications, and substitutions are possible in addition to those already enumerated in the preceding paragraphs. Those skilled in the art will appreciate that various modifications and adjustments of the embodiments just described may be configured without departing from the scope and spirit of the invention. Therefore, it should be understood that within the scope of the appended claims, the invention may be practiced in a manner other than as specifically described herein.

Claims

1. Human anti-PD-L2 antibody, wherein: The light chain consists of the variable regions LCDR1 of Seq. ID 7, LCDR2 of Seq. ID 8, and LCDR3 of Seq. ID 9. and The heavy chain consists of the variable region HCDR1 of Seq. ID 10, HCDR2 of Seq. ID 11, and HCDR3 of Seq. ID 12.

2. The human anti-PD-L2 antibody according to claim 1, wherein The LCDR1 consists of Seq. ID 7 or a nine amino acid long sequence with at least 89% identity to Seq. ID 7, wherein Seq. ID 7 consists of amino acid Asn (N) or Tyr (Y) at position 6.

3. The human anti-PD-L2 antibody according to claim 1, wherein The LCDR1 is Seq. ID 43.

4. The human anti-PD-L2 antibody according to claim 1, wherein The LCDR2 consists of Seq. ID 8 or a thirteen amino acid long sequence having at least 85% identity with Seq. ID 8, wherein Seq. ID 8 consists of amino acid Gly (G) or Asp (D) at position 7, and amino acid Thr (T) or Asn (N) at position 10.

5. The human anti-PD-L2 antibody according to claim 1, wherein The LCDR2 is Seq. ID 14.

6. The human anti-PD-L2 antibody according to claim 1, wherein The LCDR3 consists of Seq. ID 9 or a fourteen amino acid long sequence with at least 50% identity to Seq. ID 9, wherein Seq. ID 9 consists of the amino acid His (H) or Gln (Q) at position 2, the amino acid Gln (Q) or Arg (R) at position 3, the amino acid Gly (G) or Asn (N) or Ser (S) at position 4, no amino acid or the amino acid Asn (N) at position 5, no amino acid or the amino acid Trp (W) at position 6, no amino acid or the amino acid Pro (P) at position 7, no amino acid or the amino acid Ile (I) or Leu (L) at position 8, and the amino acid Gln (Q) or Gly (G) at position 12.

7. The human anti-PD-L2 antibody according to claim 1, wherein The LCDR3 is selected from the group consisting of Seq. ID 15 and 45.

8. The human anti-PD-L2 antibody according to claim 1, wherein The HCDR1 is Seq. ID 10.

9. The human anti-PD-L2 antibody according to claim 1, wherein The HCDR2 consists of Seq. ID 11 or a sixteen amino acid long sequence with at least 81% identity to Seq. ID 11, wherein Seq. ID 11 consists of amino acid Gly (G) or Arg (R) at position 6, amino acid Phe (F) or Leu (L) at position 11, and amino acid Thr (T) or Ile (I) at position 13.

10. The human anti-PD-L2 antibody according to claim 1, wherein The HCDR2 is Seq. ID 17.

11. The human anti-PD-L2 antibody according to claim 1, wherein The HCDR3 is selected from the group consisting of Seq. ID 18 and 48.

12. The human anti-PD-L2 antibody according to claim 1, wherein The light chain consists of the variable regions LCDR1 of Seq. ID 7, LCDR2 of Seq. ID 14, and LCDR3 of Seq. ID 15; and The heavy chain consists of the variable region HCDR1 of Seq. ID 10, HCDR2 of Seq. ID 17, and HCDR3 of Seq. ID 18.

13. The human anti-PD-L2 antibody according to claim 1, wherein The light chain consists of the variable regions LCDR1 of Seq. ID 43, LCDR2 of Seq. ID 14, and LCDR3 of Seq. ID 45; and The heavy chain consists of the variable region HCDR1 of Seq. ID 10, HCDR2 of Seq. ID 17, and HCDR3 of Seq. ID 48.

14. The human anti-PD-L2 antibody according to claim 1, wherein The light chain is Seq. ID 50, and the heavy chain is Seq. ID 58.

15. The human anti-PD-L2 antibody according to claim 1, wherein The light chain is at least 92% identical to Seq. ID 50.

16. The human anti-PD-L2 antibody according to claim 1, wherein The light chain is selected from the group consisting of Seq. ID 51 and 56.

17. The human anti-PD-L2 antibody according to claim 1, wherein The heavy chain is at least 97% identical to Seq. ID 58.

18. The human anti-PD-L2 antibody according to claim 1, wherein The heavy chain was selected from the group consisting of Seq. ID 59 and 64.

19. The human anti-PD-L2 antibody according to claim 1, wherein The heavy chain is Seq. ID 51, and the heavy chain is Seq. ID 59.

20. The human anti-PD-L2 antibody of claim 1, wherein The light chain is Seq. ID 56, and the heavy chain is Seq. ID 64.

21. The human anti-PD-L2 antibody according to claim 1, wherein The nucleotide sequence encoding the light chain of the human anti-PD-L2 antibody is Seq. ID 66, and the nucleotide sequence encoding the heavy chain is Seq. ID 74.

22. The human anti-PD-L2 antibody of claim 1, wherein The nucleotide sequence encoding the light chain of the human anti-PD-L2 antibody is Seq. ID 67, and the nucleotide sequence encoding the heavy chain is Seq. ID 59.

23. The human anti-PD-L2 antibody according to claim 1, wherein The nucleotide sequence encoding the light chain of the human anti-PD-L2 antibody is Seq. ID 72, and the nucleotide sequence encoding the heavy chain is Seq. ID 80.

24. Human anti-PD-L2 antibody, wherein The light chain consists of variable regions LCDR1 of Seq. ID 31, LCDR2 of Seq. ID 32, and LCDR3 of Seq. ID 33; and The heavy chain consists of the variable region HCDR1 of Seq. ID 10, HCDR2 of Seq. ID 35, and HCDR3 of Seq. ID 36.

25. The human anti-PD-L2 antibody according to claim 24, wherein The light chain is Seq. ID 54, and the heavy chain consists of Seq. ID 62.

26. The human anti-PD-L2 antibody according to claim 24, wherein The nucleotide sequence encoding the light chain of the human anti-PD-L2 antibody is Seq. ID 70, and the nucleotide sequence encoding the heavy chain is Seq. ID 78.

27. Human anti-PD-L2 antibody, wherein The light chain consists of the variable regions LCDR1 of Seq. ID 25, LCDR2 of Seq. ID 26, and LCDR3 of Seq. ID 27; and The heavy chain consists of the variable region HCDR1 of Seq. ID 10, HCDR2 of Seq. ID 17, and HCDR3 of Seq. ID 30.

28. The human anti-PD-L2 antibody according to claim 27, wherein The LCDR1 is Seq. ID 25 or Seq. ID 37.

29. The human anti-PD-L2 antibody according to claim 27, wherein The LCDR2 comprises a thirteen amino acid long sequence that is at least 78% identical to Seq. ID 26, consisting of the amino acid Lys (L) or Arg (R) at position 3, and the amino acid Ala (A) or Glu (G) at position 7.

30. The human anti-PD-L2 antibody of claim 27, wherein The LCDR2 is Seq. ID 38.

31. The human anti-PD-L2 antibody of claim 27, wherein The LCDR3 is Seq. ID 27 or Seq. ID 39.

32. The human anti-PD-L2 antibody of claim 27, wherein The HCDR1 is Seq. ID 10.

33. The human anti-PD-L2 antibody of claim 27, wherein The HCDR2 is Seq. ID 17.

34. The human anti-PD-L2 antibody of claim 27, wherein The HCDR3 is Seq. ID 30 or Seq. ID 42.

35. The human anti-PD-L2 antibody of claim 27, wherein The light chain consists of the variable regions LCDR1 of Seq. ID 37, LCDR2 of Seq. ID 38, and LCDR3 of Seq. ID 39; and The heavy chain consists of the variable region HCDR1 of Seq. ID 10, HCDR2 of Seq. ID 17, and HCDR3 of Seq. ID 42.

36. The human anti-PD-L2 antibody of claim 27, wherein The light chain is Seq. ID 53, and the heavy chain consists of Seq. ID 61.

37. The human anti-PD-L2 antibody of claim 27, wherein The light chain is Seq. ID 55, and the heavy chain consists of Seq. ID 63.

38. The human anti-PD-L2 antibody of claim 27, wherein The nucleotide sequence encoding the light chain of the human anti-PD-L2 antibody is Seq. ID 69, and the nucleotide sequence encoding the heavy chain is Seq. ID 77.

39. The human anti-PD-L2 antibody of claim 27, wherein The nucleotide sequence encoding the light chain of the human anti-PD-L2 antibody is Seq. ID 71, and the nucleotide sequence encoding the heavy chain is Seq. ID 79.

40. Human anti-PD-L2 antibody, wherein The light chain consists of the variable region LCDR1 of Seq. ID 1 or Seq. ID 19, LCDR2 of Seq. ID 2 or Seq. ID 20, and LCDR3 of Seq. ID 3 or Seq. ID 21; and The heavy chain consists of the variable region HCDR1 of Seq. ID 10, HCDR2 of Seq. ID 17, and HCDR3 of Seq. ID 6 or Seq. ID 24.

41. The human anti-PD-L2 antibody of claim 40, wherein The light chain consists of the variable region LCDR1 of Seq. ID 1, LCDR2 of Seq. ID 2, and LCDR3 of Seq. ID 3; and The heavy chain consists of the variable region HCDR1 of Seq. ID 10, HCDR2 of Seq. ID 17, and HCDR3 of Seq. ID 6.

42. The human anti-PD-L2 antibody of claim 40, wherein The light chain consists of the variable regions LCDR1 of Seq. ID 19, LCDR2 of Seq. ID 20, and LCDR3 of Seq. ID 21; and The heavy chain consists of the variable region HCDR1 of Seq. ID 10, HCDR2 of Seq. ID 17, and HCDR3 of Seq. ID 24.

43. The human anti-PD-L2 antibody of claim 40, wherein The light chain is Seq. ID 49 or Seq. ID 52, and the heavy chain is Seq. ID 57 or Seq. ID 60.

44. The human anti-PD-L2 antibody of claim 40, wherein The light chain is Seq. ID 49, and the heavy chain is Seq. ID 57.

45. The human anti-PD-L2 antibody of claim 40, wherein The light chain is Seq. ID 52, and the heavy chain is Seq. ID 60.

46. ​​The human anti-PD-L2 antibody of claim 40, wherein The nucleotide sequence encoding the light chain of the human anti-PD-L2 antibody is Seq. ID 65, and the nucleotide sequence encoding the heavy chain is Seq. ID 73.

47. The human anti-PD-L2 antibody of claim 40, wherein The nucleotide sequence encoding the heavy chain of the human anti-PD-L2 antibody is Seq. ID 68, and the nucleotide sequence encoding the heavy chain is Seq. ID 76.

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