CN114075288A

CN114075288A - anti-PD-L2 antibody

Info

Publication number: CN114075288A
Application number: CN202110935982.3A
Authority: CN
Inventors: 桂秋; 朱虹; 王亮亮; 徐宏江; 杨玲
Original assignee: Chia Tai Tianqing Pharmaceutical Group Co Ltd
Current assignee: Chia Tai Tianqing Pharmaceutical Group Co Ltd
Priority date: 2020-08-18
Filing date: 2021-08-16
Publication date: 2022-02-22

Abstract

The present invention relates to anti-PD-L2 antibodies and antigen-binding fragments thereof, such as mouse, chimeric and humanized monoclonal antibodies and antigen-binding fragments thereof. The invention also provides nucleic acids encoding the above antibodies and antigen-binding fragments thereof, as well as expression vectors and cells for expressing the antibodies and antigen-binding fragments thereof. Methods of using such antibodies and antigen-binding fragments thereof are also provided, for example, methods of using chimeric and humanized anti-PD-L2 antibodies and antigen-binding fragments thereof are provided.

Description

anti-PD-L2 antibody

Technical Field

The present invention is in the field of biotechnology, specifically to antibodies and antigen-binding fragments thereof that specifically bind to PD-L2, and to methods of using such antibodies and antigen-binding fragments.

Background

The tumor immunotherapy is an important anti-tumor therapy means, and the key point of the method is to activate the anti-tumor reaction of T lymphocytes of tumor patients and improve the killing function of the T lymphocytes on tumor cells. Programmed death receptor 1(PD-1) is a protein receptor expressed on immune cells (including T cells and B cells) and has two ligands, namely programmed death-1 ligand 1(PD-L1, B7-H1 or CD274) and programmed death-1 ligand 2(PD-L2, B7-DC or CD273), and after being combined with the ligand, PD-1 can transmit an inhibitory signal to negatively regulate T cell immune response and inhibit TCR-mediated T cell activation, cytokine production and T cell proliferation. Therefore, the function of the PD-1/PD-L1 and PD-1/PD-L2 signal pathways in tumor immune escape and the application of the pathways in tumor immunotherapy become research hotspots.

Both PD-L2 and PD-L1 are type I transmembrane proteins composed of IgV-like domain, IgC-like domain, transmembrane region and tail cytoplasmic region, and the genes encoding human PD-L2 and PD-L1 are located on chromosome 9p24.2, but the sequences, tissue distribution, receptor-ligand binding specificity and affinity of PD-L2 and PD-L1 and the involved signaling pathways all show great differences. PD-L1 is widely distributed on leukocytes and non-hematopoietic cells in lymphoid and non-lymphoid tissues and on various cancer cells, and is upregulated during tumorigenesis by PD-L1; PD-L2 is typically expressed on antigen presenting cells, including dendritic cells, macrophages and bone marrow derived mast cells (Zhong et al (2007) Eur. J. Immunol 37: 2405-2410). PD-L2 can be induced to express in various immune cells and non-immune cells under the stimulation of specific microenvironment, such as squamous cell carcinoma of head and neck, breast cancer, gastric cancer and other tumor cells (Zou et al (2015) Nat Rev Immunol 8: 467-477). The result shows that the interaction of PD-1/PD-L2 is blocked, the tumor specific T cell immunity can be enhanced, and the immune system is helped to eliminate tumor cells. More importantly, high expression of PD-L2 was detected in a number of Human tumor samples that did not express or that expressed low in PD-L1 (Baptista et al (2016) Human pathology 47: 78-84; Yearley et al (2017) clean cancer research 23: 3158-. Furthermore, in 172 patients with recurrent or metastatic cervical squamous cell carcinoma who received Keytruda treatment, both PD-L1 and PD-L2 expressed 27.5% of the ORRs of positive patients, while the ORRs of PD-L1-positive PD-L2-negative patients were only 11.4% of the ORRs, the former being 2-fold higher (Yearley et al (2017) clinical research 23:3158-3167), suggesting that simultaneous inhibition of PD-L1 and PD-L2 may increase the clinical response rate, while PD-L2 may serve as a supplementary indicator for predicting the clinical efficacy of PD-1 inhibitors.

Since 2010, many antibodies against PD-1 or PD-L1 have been in clinical trials or approved on the market, and these antibodies show significant antitumor activity both in vitro and in vivo, such as Pembrolizumab, Atezolizumab, Durvalumab, but no antibody drug against PD-L2 has been in clinical studies to date. Therefore, it is of great interest to develop a therapeutic antibody that specifically binds to human PD-L2. In addition, PD-L2 plays an important role as a molecular marker in the targeted therapy and prognosis diagnosis of various tumors, so that a monoclonal antibody which is highly specifically combined with human PD-L2 needs to be prepared to guide the diagnosis and treatment of tumors.

Disclosure of Invention

In one aspect, the invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds programmed death-1 ligand 2 (PD-L2). In some embodiments, the isolated antibody or antigen-binding fragment thereof of the invention is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the isolated antibody or antigen binding fragment thereof of the invention is a monoclonal antibody, Fab fragment, F (ab')2 fragment, Fd fragment, Fv fragment, dAb, isolated CDR, single chain Fv molecule, and recombinant polypeptide, fusion protein, bispecific molecule, or a combination thereof. In some embodiments, an isolated antibody or antigen-binding fragment thereof of the invention binds to human PD-L2; in other embodiments, an isolated antibody or antigen-binding fragment thereof of the invention binds to human PD-L2 and binds to cynomolgus monkey (cynomolgous) PD-L2. In another embodiment, the isolated antibody or antigen-binding fragment thereof of the invention blocks the binding of PD-L2 to its receptor PD-1.

In one embodiment, the isolated antibody or antigen binding fragment thereof of the invention binds to PD-L2 and comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, wherein (1) heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 1, 2, 3 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 1, 2, 3, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (2) the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 7, 8 and 9 or an amino acid sequence corresponding to SEQ ID NOs: 7, 8, 9, an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (3) the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 13, 14, 15 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 13, 14, 15, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in seq id No. 13, 14, 15; or (4) heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 comprise SEQ ID NOs: 19, 20, 21 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 19, 20, 21, an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity;

wherein, SEQ ID NO: 2 is ridsxseth, X ═ D or E; SEQ ID NO: 8 is midpxsetr, X ═ D or E; SEQ ID NO: 14 is vinpvxvggis, X ═ N or Q; SEQ ID NO: 20 is LISPYXGVTT, X ═ N or Q.

In one embodiment, the isolated antibody or antigen binding fragment thereof of the invention binds PD-L2 and comprises light chain CDR1, light chain CDR2, and light chain CDR3, wherein (1) light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5,6 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 4, 5,6, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in seq id No. 4, 5, 6; (2) the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 10, 11, 12 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 10, 11, 12, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in seq id No. 10, 11, 12; (3) the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 16, 17, 18 or an amino acid sequence substantially identical to SEQ ID NOs: 16, 17, 18, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in seq id no; or (4) the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 22, 23, 24 or an amino acid sequence substantially identical to SEQ ID NOs: 22, 23, 24, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity;

wherein, SEQ ID NO: 22 is kssqslllxsgnqknylt, X ═ N or Q.

In one embodiment, the isolated antibody or antigen binding fragment thereof of the invention binds to PD-L2 and comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NOs: 1, 7, 13, or 19 or an amino acid sequence substantially identical to SEQ ID NOs: 1, 7, 13, or 19, an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; heavy chain CDR2 comprises SEQ ID NOs: 2, 8, 14, or 20 or an amino acid sequence substantially identical to SEQ ID NOs: 2, 8, 14, or 20, an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; heavy chain CDR3 comprises SEQ ID NOs: 3, 9, 15, or 21 or a sequence substantially identical to the amino acid sequence set forth in SEQ ID NOs: 3, 9, 15, or 21, an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; and light chain CDR1 comprises SEQ ID NOs: 4, 10, 16, or 22 or an amino acid sequence substantially identical to SEQ ID NOs: 4, 10, 16, or 22, an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; the light chain CDR2 comprises SEQ ID NOs: 5, 11, 17, or 23 or an amino acid sequence substantially identical to the amino acid sequence set forth in SEQ ID NOs: 5, 11, 17, or 23, an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; the light chain CDR3 comprises SEQ ID NOs: 6, 12, 18, or 24 or an amino acid sequence substantially identical to the amino acid sequence set forth in SEQ ID NOs: 6, 12, 18, or 24, an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical;

wherein, SEQ ID NO: 2 is ridsxseth, X ═ D or E; SEQ ID NO: 8 is midpxsetr, X ═ D or E; SEQ ID NO: 14 is vinpvxvggis, X ═ N or Q; SEQ ID NO: 20 is LISPYXGVTT, X ═ N or Q; SEQ ID NO: 22 is kssqslllxsgnqknylt, X ═ N or Q.

In one embodiment, the isolated antibody or antigen binding fragment thereof of the invention binds to PD-L2 and comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise SEQ ID NOs: 1, 2, 3 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 1, 2, 3, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; and, the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 4, 5,6 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 4, 5,6, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in seq id No. 4, 5, 6;

wherein, SEQ ID NO: 2 is ridsxseth, X ═ D or E.

In one embodiment, the isolated antibody or antigen binding fragment thereof of the invention binds to PD-L2 and comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise SEQ ID NOs: 7, 8 and 9 or an amino acid sequence corresponding to SEQ ID NOs: 7, 8, 9, an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; and, the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 10, 11, 12 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 10, 11, 12, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in seq id No. 10, 11, 12;

wherein, SEQ ID NO: 8 is midpxsetr, X ═ D or E.

In one embodiment, the isolated antibody or antigen binding fragment thereof of the invention binds to PD-L2 and comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise SEQ ID NOs: 13, 14, 15 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 13, 14, 15, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in seq id No. 13, 14, 15; and, the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 16, 17, 18 or an amino acid sequence substantially identical to SEQ ID NOs: 16, 17, 18, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in seq id no;

wherein, SEQ ID NO: 14 is vinpvxvggis, X ═ N or Q.

In one embodiment, the isolated antibody or antigen binding fragment thereof of the invention binds to PD-L2 and comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise SEQ ID NOs: 19, 20, 21 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 19, 20, 21, an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; and, the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 22, 23, 24 or an amino acid sequence substantially identical to SEQ ID NOs: 22, 23, 24, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity;

wherein, SEQ ID NO: 20 is LISPYXGVTT, X ═ N or Q; SEQ ID NO: 22 is kssqslllxsgnqknylt, X ═ N or Q.

In one embodiment, the isolated antibody or antigen binding fragment thereof of the invention binds to PD-L2 and comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NOs: 25, 27, 29, 31, 33, 35, 36, 38, 39, 41, 42, or 44, or an amino acid sequence substantially identical to SEQ ID NOs: 25, 27, 29, 31, 33, 35, 36, 38, 39, 41, 42, or 44 has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

In one embodiment, the isolated antibody or antigen binding fragment thereof of the invention binds to PD-L2 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NOs: 26, 28, 30, 32, 34, 37, 40, 43, or 45, or an amino acid sequence substantially identical to SEQ ID NOs: 26, 28, 30, 32, 34, 37, 40, 43, or 45, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

In one embodiment, the isolated antibody or antigen binding fragment thereof of the invention binds PD-L2 and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NOs: 25, 27, 29, 31, 33, 35, 36, 38, 39, 41, 42, or 44, or an amino acid sequence substantially identical to SEQ ID NOs: 25, 27, 29, 31, 33, 35, 36, 38, 39, 41, 42, or 44, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; and, the light chain variable region comprises SEQ ID NOs: 26, 28, 30, 32, 34, 37, 40, 43, or 45, or an amino acid sequence substantially identical to SEQ ID NOs: 26, 28, 30, 32, 34, 37, 40, 43, or 45, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

In one embodiment, the isolated antibody or antigen binding fragment thereof of the invention binds PD-L2 and comprises a heavy chain variable region and a light chain variable region, wherein (1) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 25 and 26 or an amino acid sequence substantially identical to SEQ ID NO: 25 and 26, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (2) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 27 and 28 or an amino acid sequence substantially identical to SEQ ID NO: 27 and 28, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (3) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 29 and 30 or an amino acid sequence substantially identical to SEQ ID NO: 29 and 30, an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (4) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 31 and 32 or an amino acid sequence substantially identical to SEQ ID NO: 31 and 32, an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; (5) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 33 and 34 or an amino acid sequence substantially identical to SEQ ID NO: 33 and 34, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (6) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 35 and 34 or an amino acid sequence substantially identical to SEQ ID NO: 35 and 34, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (7) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 36 and 37 or an amino acid sequence substantially identical to SEQ ID NO: 36 and 37, an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (8) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 38 and 37 or an amino acid sequence substantially identical to SEQ ID NO: 38 and 37, an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (9) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 39 and 40 or an amino acid sequence substantially identical to SEQ ID NO: 39 and 40, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (10) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 41 and 40 or an amino acid sequence substantially identical to SEQ ID NO: 41 and 40, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (11) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 42 and 43 or an amino acid sequence substantially identical to SEQ ID NO: 42 and 43, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (12) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 44 and 43 or an amino acid sequence substantially identical to SEQ ID NO: 44 and 43, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (13) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 42 and 45 or an amino acid sequence substantially identical to SEQ ID NO: 42 and 45, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; or (14) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 44 and 45 or an amino acid sequence substantially identical to SEQ ID NO: 44 and 45, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

In one embodiment, an isolated antibody or antigen-binding fragment thereof of the invention binds to PD-L2 and comprises a heavy chain comprising the amino acid sequence of SEQ ID NOs: 46, 50, 54, 58, 62, 64, 68, 70, 74, 76, 80, or 82, or an amino acid sequence substantially identical to SEQ ID NOs: 46, 50, 54, 58, 62, 64, 68, 70, 74, 76, 80, or 82, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

In one embodiment, an isolated antibody or antigen-binding fragment thereof of the invention binds to PD-L2 and comprises a light chain comprising the amino acid sequence of SEQ ID NOs: 47, 51, 55, 59, 63, 69, 75, 81, or 83, or an amino acid sequence substantially identical to SEQ ID NOs: 47, 51, 55, 59, 63, 69, 75, 81, or 83, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

In one embodiment, an isolated antibody or antigen-binding fragment thereof of the invention binds to PD-L2 and comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NOs: 46, 50, 54, 58, 62, 64, 68, 70, 74, 76, 80, or 82, or an amino acid sequence substantially identical to SEQ ID NOs: 46, 50, 54, 58, 62, 64, 68, 70, 74, 76, 80, or 82, an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; and, the light chain comprises SEQ ID NOs: 47, 51, 55, 59, 63, 69, 75, 81, or 83, or the amino acid sequence set forth in SEQ ID NOs: 47, 51, 55, 59, 63, 69, 75, 81, or 83, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

In one embodiment, an isolated antibody or antigen-binding fragment thereof of the invention binds to PD-L2, comprising a heavy chain and a light chain, wherein (1) the heavy chain and the light chain comprise SEQ ID NOs: 46 and 47 or an amino acid sequence substantially identical to SEQ ID NO: 46 and 47, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (2) the heavy and light chains comprise SEQ ID NOs: 50 and 51 or an amino acid sequence substantially identical to SEQ ID NO: 50 and 51, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (3) the heavy and light chains comprise SEQ ID NOs: 54 and 55 or an amino acid sequence substantially identical to SEQ ID NO: 54 and 55, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (4) the heavy and light chains comprise SEQ ID NOs: 58 and 59 or an amino acid sequence substantially identical to SEQ ID NO: 58 and 59, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (5) the heavy and light chains comprise SEQ ID NOs: 62 and 63 or an amino acid sequence substantially identical to SEQ ID NO: 62 and 63, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (6) the heavy and light chains comprise SEQ ID NOs: 64 and 63 or an amino acid sequence substantially identical to SEQ ID NO: 64 and 63, an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; (7) the heavy and light chains comprise SEQ ID NOs: 68 and 69 or an amino acid sequence substantially identical to SEQ ID NO: 68 and 69, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (8) the heavy and light chains comprise SEQ ID NOs: 70 and 69 or an amino acid sequence substantially identical to SEQ ID NO: 70 and 69, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; (9) the heavy and light chains comprise SEQ ID NOs: 74 and 75 or an amino acid sequence substantially identical to SEQ ID NO: 74 and 75, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; (10) the heavy and light chains comprise SEQ ID NOs: 76 and 75 or an amino acid sequence substantially identical to SEQ ID NO: 76 and 75, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (11) the heavy and light chains comprise SEQ ID NOs: 80 and 81 or an amino acid sequence corresponding to SEQ ID NO: 80 and 81, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (12) the heavy and light chains comprise SEQ ID NOs: 82 and 81 or an amino acid sequence substantially identical to SEQ ID NO: 82 and 81, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; (13) the heavy and light chains comprise SEQ ID NOs: 80 and 83 or an amino acid sequence substantially identical to SEQ ID NO: 80 and 83, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; or (14) the heavy and light chains comprise SEQ ID NOs: 82 and 83 or an amino acid sequence substantially identical to SEQ ID NO: 82 and 83, or a pharmaceutically acceptable salt thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

The antibodies of the invention further comprise a heavy chain comprising a heavy chain variable region and a heavy chain constant region, and a light chain comprising a light chain variable region and a light chain constant region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of the above heavy chain variable region and light chain variable region, respectively, and the heavy chain constant region can be a human IgG1, IgG2, or IgG4 constant region, preferably an IgG1 constant region; the light chain constant region can be a human kappa constant region or a human lambda constant region.

The antibodies of the invention in some embodiments comprise or consist of two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, wherein each heavy chain comprises the above-described heavy chain variable region (HV), and a heavy chain constant region, wherein the heavy chain variable region (HV) comprises a Framework Region (FR) and the above-described heavy chain Complementarity Determining Regions (CDRs), and each light chain comprises the above-described light chain variable region (LV), and a light chain constant region, wherein the light chain variable region (LV) comprises FRs and the above-described light chain CDRs, wherein the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region, and the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region. The antibody of the invention may be a full length antibody of, for example, the IgG1, IgG2 or IgG4 isotype. In another embodiment, an antibody of the invention can be a single chain antibody (scFv), or an antibody fragment, such as a Fab, F (ab')2 fragment, Fd fragment, Fv fragment, dAb, or isolated CDR.

The antibodies or antigen-binding fragments thereof of the invention specifically bind to PD-L2 (e.g., human PD-L2 and monkey PD-L2) and block the binding of PD-L2 to its receptor PD-1. In some embodiments, the anti-PD-L2 antibodies or antigen-binding fragments thereof provided herein, in combination with anti-PD-L1 antibodies, are capable of blocking PD-L2/PD-1 and PD-L1/PD-1 interactions, promoting T cell activation, proliferation, and/or cytokine production, including but not limited to: one or more of IL-2, IFN gamma, TNF-alpha, IL-1, IL-4, IL-5, IL-6, IL-12, IL-17 and GM-CSF. Thus, in another aspect, the invention provides methods for modulating an immune response comprising contacting T cells and antigen presenting cells with an anti-PD-L2 antibody or antigen binding fragment thereof and an anti-PD-L1 antibody provided herein. In one embodiment, modulation of an immune response by an anti-PD-L2 antibody or antigen-binding fragment thereof provided herein in combination with an anti-PD-L1 antibody can be measured in a Mixed Lymphocyte (MLR) reaction. In one embodiment, the anti-PD-L2 antibodies provided herein in combination with the anti-PD-L1 antibody increase the level of cytokine production by lymphocytes in the MLR. In one embodiment, an anti-PD-L2 antibody provided herein in combination with an anti-PD-L1 antibody increases the level of IL-2 and/or IFN- γ production in MLR.

In one aspect, the invention provides an isolated antibody or antigen-binding fragment thereof that binds PD-L2, said antibody or antigen-binding fragment thereof produced by a hybridoma selected from the group consisting of the hybridomas designated herein as 25G4, 14E7, 24A3, and 7a 7. Thus, the invention also includes antibodies produced by hybridomas 25G4, 14E7, 24A3, and 7a7, as well as any hybridomas that produce the antibodies disclosed herein, or antigen-binding fragments thereof.

In one aspect, the invention provides an antibody or antigen-binding fragment thereof that binds to the same epitope on PD-L2 as any of the exemplary PD-L2 antibodies or antigen-binding fragments thereof of the invention. In one embodiment, the invention provides an antibody or antigen-binding fragment thereof that competes for binding to PD-L2 with any of the exemplary PD-L2 antibodies or antigen-binding fragments thereof of the invention.

In another aspect, the invention also provides recombinant polypeptides and fusion proteins comprising one or more antibodies or antigen-binding fragments thereof of the invention and at least one other functional fragment including, but not limited to, another peptide, protein or cytokine. In another aspect, the invention also provides a bispecific molecule comprising one or more antibodies or antigen-binding fragments thereof of the invention and at least one other antibody or antigen-binding fragment thereof, said bispecific molecule being capable of binding to at least two different binding sites or targets. As used herein, "bispecific molecule" encompasses molecules having two (i.e., bispecific molecules), three (i.e., trispecific molecules), four (i.e., tetraspecific molecules), or more specificities.

In one aspect, the invention also provides a pharmaceutical composition comprising an anti-PD-L2 antibody or antigen-binding fragment thereof of the invention, and one or more pharmaceutically acceptable carriers. Optionally, the pharmaceutical composition may further comprise one or more other antibodies, such as PD-L1 antibody. In another embodiment, the invention also provides a pharmaceutical composition comprising a recombinant polypeptide, fusion protein or bispecific molecule of the invention, and a pharmaceutically acceptable carrier.

In one aspect, the invention also provides an isolated nucleic acid encoding the anti-PD-L2 antibody or antigen-binding fragment thereof of the invention. The invention also provides expression vectors comprising the nucleic acids, and host cells comprising the expression vectors.

In one aspect, the invention also provides a method of making an anti-PD-L2 antibody or antigen-binding fragment thereof, comprising the steps of: (i) expressing the antibody or antigen-binding fragment thereof in a host cell, and (ii) isolating the antibody or antigen-binding fragment thereof from the host cell or cell culture thereof.

In other aspects, the invention provides methods for modulating an immune response in a subject, comprising administering to the subject a therapeutically effective amount of an anti-PD-L2 antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition thereof. In another embodiment, the present invention provides a method for treating, ameliorating, or preventing cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L2 antibody or antigen-binding fragment thereof of the present invention or a pharmaceutical composition thereof.

In one embodiment, the invention provides a method for enhancing an anti-tumor immune response in a subject, comprising administering to the subject a therapeutically effective amount of an anti-PD-L2 antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition thereof. In one embodiment, the present invention provides a method for reducing tumor size or inhibiting tumor cell growth in a subject comprising administering to the subject a therapeutically effective amount of an anti-PD-L2 antibody or antigen-binding fragment thereof of the present invention or a pharmaceutical composition thereof. In one embodiment, the present invention provides a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of an anti-PD-L2 antibody or antigen-binding fragment thereof of the present invention or a pharmaceutical composition thereof. In other embodiments, the method comprises administering to the subject a therapeutically effective amount of a nucleic acid encoding an anti-PD-L2 antibody or antigen-binding fragment thereof of the invention. In other embodiments, the method comprises administering to the subject a therapeutically effective amount of a recombinant polypeptide, fusion protein, or bispecific molecule of the invention.

In some embodiments, at least one additional antibody may be administered with the anti-PD-L2 antibody or antigen-binding portion thereof of the invention, e.g., a PD-L1 antibody, a PD-1 antibody, and/or a CTLA-4 antibody, preferably a PD-L1 antibody. In some embodiments, at least one additional therapy may be administered with the anti-PD-L2 antibody or antigen-binding fragment thereof of the present invention, including radiation therapy, chemotherapy, biologics, chemicals, vaccines, and other tumor treatment modalities. In certain embodiments, the subject is a human.

Such tumors or cancers include, but are not limited to: colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, renal cancer, prostate cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine malignancies, head and neck cancer, salivary gland cancer, thymus cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protruberans, merkeloid, glioblastoma, glioma, sarcoma, mesothelioma and myelodysplastic syndrome, or a combination thereof; and optionally, the tumor is in situ, metastatic, recurrent, and/or refractory.

In another embodiment, the present invention provides a method for treating an infectious disease in a subject, comprising administering to the subject a therapeutically effective amount of an anti-PD-L2 antibody or antigen-binding fragment thereof of the present invention or a pharmaceutical composition thereof. In other embodiments, the method comprises administering to the subject a therapeutically effective amount of a nucleic acid encoding an anti-PD-L2 antibody or antigen-binding fragment thereof of the invention. Infectious diseases include, but are not limited to: candidiasis, candidemia, aspergillosis, streptococcal pneumonia, streptococcal skin and oropharyngeal diseases, gram-negative sepsis, tuberculosis, mononucleosis, influenza, respiratory diseases caused by respiratory syncytial virus, malaria, schistosomiasis and trypanosomiasis.

In another aspect, the invention also provides a method of detecting PD-L2 expression in a sample, comprising contacting the sample with an anti-PD-L2 antibody or antigen-binding fragment of the invention under conditions that allow the formation of a complex between the anti-PD-L2 antibody or antigen-binding fragment of the invention and PD-L2, and detecting the formation of the complex.

Other features and advantages of the present disclosure will be more apparent based on the following detailed description and examples, which should not be construed as limiting. The contents of all documents, Genbank records, patents and published patent applications cited in this application are expressly incorporated herein by reference.

Drawings

FIG. 1 shows flow cytometry analysis of binding of anti-PD-L2 humanized antibody (Hz25G4-1.1) to DC/T cells at various concentrations

FIG. 2a shows the production of IFN- γ (pg/ml) in response to different concentrations of antibody in MLR, and FIG. 2b shows the production of IL-2(pg/ml) in response to different concentrations of antibody in MLR. For fig. 2a and 2b, the antibodies tested are, from left to right: anti-PD-L2 antibody (Hz25G4-1.1) + anti-PD-L1 antibody, anti-PD-L1 antibody + anti-PD-L2 antibody (Hz25G4-1.1), anti-PD-L2 antibody (Hz25G4-1.1), anti-PD-L1 antibody and blank control (IgG1), wherein anti-PD-L1 antibody is described in CN107001463 for humanized 5G11-IgG1, amino acid sequences of heavy and light chains are as in SEQ ID NO: 88 and SEQ ID NO: 89 is shown.

Detailed Description

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "PD-L2" includes PD-L2 variants, homologs, orthologs, and paralogs. For example, in some embodiments, an antibody specific for human PD-L2 protein may cross-react in some cases with PD-L2 protein of another species (e.g., monkey). In other embodiments, an antibody specific to human PD-L2 protein may be completely specific for human PD-L2 protein without cross-reacting with other species or other types of proteins, or only cross-reacting with PD-L2 protein of some other species but not all other species.

The terms "human PD-L2", "hPD-L2", or "human PD-L2", and the like, are interchangeable herein and refer to a protein having the amino acid sequence of human PD-L2, e.g., the amino acid sequence of Genbank accession number AAI 13679. The terms "monkey PD-L2" or "cyno PD-L2" and the like are used interchangeably herein to refer to a protein having the amino acid sequence of monkey PD-L2, e.g., the amino acid sequence of Uniprot accession G7PSE 6.

The term "antibody" as used herein refers to a binding protein having at least one antigen binding domain. The antibody or antigen binding fragment thereof of the present invention may be a whole antibody or any fragment thereof, including monoclonal antibodies or fragments thereof and antibody variants or fragments thereof. Examples of antibody fragments include Fab fragments, F (ab')2 fragments, Fv fragments, isolated CDR regions, single chain Fv (scfv), and any antibody fragment known in the art. Antibodies or fragments thereof also include recombinant polypeptides, fusion proteins, and bispecific molecules. The anti-PD-L2 antibody or antigen-binding fragment thereof disclosed herein can be of the IgG1, IgG2, IgG3, or IgG4 isotype. The term "isotype" refers to the class of antibodies encoded by the heavy chain constant region gene. In some embodiments, an anti-PD-L2 antibody or antigen-binding fragment thereof disclosed herein is an IgG1 isotype. The anti-PD-L2 antibodies or antigen-binding fragments thereof disclosed herein may be derived from any species, including but not limited to mouse, rat, rabbit, primate, llama, and human. The anti-PD-L2 antibody or antigen-binding fragment thereof of the invention can be a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

An "antigen-binding fragment" or "antibody-binding portion" of an antibody refers to one or more fragments of an antibody that retain the function of specifically binding to an antigen (e.g., PD-L2 protein). It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples encompassed within the term "antigen-binding portion/fragment" of an antibody include: (i) fab fragment: from V_L、V_HA monovalent fragment consisting of the CL and CH1 domains; (ii) a F (ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (ii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragments consisting of VH domains (see Ward et al, Nature.341:544-546 (1989)); (vi) an isolated Complementarity Determining Region (CDR); and (vii) nanobodies, a heavy chain variable region comprising a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, recombinant methods can be used to join VH and VL via a linker into a single protein chain in which VL and VH pair to form a monovalent molecule known as single chain Fv (scFv); see Bird et al, science 242: 423-; huston et al, Proc.Natl.Acad.Sci.85:5879-5883(1988), these single chain antibodies are also encompassed by the term antigen binding portion/fragment. These antibody fragments may be generated by those skilled in the artConventional techniques known to the skilled artisan, and fragments can be functionally screened by the same methods as full-length antibodies.

"mouse antibody" or "murine antibody" refers to an antibody in which the Framework (FR) and CDR regions in the variable region are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region is also derived from a mouse germline immunoglobulin sequence. The mouse/murine antibodies of the invention may include amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random mutation, point mutation, or by somatic mutation in vitro), but do not include antibodies in which CDR sequences from other mammalian germline are inserted into the mouse framework sequences.

"chimeric antibody" means an antibody having at least a portion of a heavy chain variable region and at least a portion of a light chain variable region derived from one species; and at least a portion of a constant region derived from another species. For example, in some embodiments, a chimeric antibody can comprise a murine variable region and a human constant region.

By "humanized antibody" is meant an antibody that contains CDRs derived from a non-human antibody and framework and constant regions derived from a human antibody. For example, an anti-PD-L2 antibody provided herein can comprise CDRs derived from one or more murine antibodies as well as human framework and constant regions. Thus, in some embodiments, a humanized antibody provided herein binds to the same epitope on PD-L2 as a murine antibody from which the CDRs of the antibody are derived. Provided herein are exemplary humanized antibodies. The invention provides humanized antibodies, comprising the heavy chain CDR and light chain CDR and any other human framework region sequence of anti PD-L2 antibody or its variants. The humanized antibodies provided herein, anti-PD-L2 antibodies or variants thereof comprising heavy and light chain CDRs provided herein and a framework region that is structurally similar to the framework region sequences provided herein. Additional modifications may also be made in the framework regions to improve the properties of the antibodies provided by the invention, including chemical modifications, point mutations, or mutations back to residues in the original germline sequence. For example, in some embodiments, one or more amino acids in the human framework regions of the VH and/or VL of the humanized antibodies provided herein are back mutated to the corresponding amino acids in the parent murine antibody.

As used herein, "derived," when used with respect to a molecule or polypeptide of a reference antibody or other binding protein, means a molecule or polypeptide that is capable of specifically binding the same epitope as the reference antibody or other binding protein.

"isolated" refers to a compound of interest (e.g., an antibody, antigen-binding fragment, or nucleic acid) that has been isolated from its natural environment.

"antibody that specifically binds to an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen". An antibody that "specifically binds human PD-L2" refers to an antibody that binds to human PD-L2 protein (and possibly PD-L2 protein of other non-human species) but does not substantially bind to non-PD-L2 protein. Preferably, the antibody binds human PD-L2 with "high affinity", i.e., a KD of 5.0X 10^-8M or less, 1.0X 10^-8M or less, preferably 5.0X 10^-9M or less, more preferably 1.0X 10^-9M or less.

The term "substantially does not bind" to a protein or cell means not binding to the protein or cell, or not binding to it with high affinity, i.e., binding to the protein or cell has a KD of 1.0X 10^-6M or higher, preferably 1.0X 10^-5M or higher, 1.0X 10^-4M or higher, more preferably 1.0X 10^-3M or higher, 1.0X 10^-2M or higher.

The term "high affinity" for IgG means that the KD for the bound antigen is 1.0X 10^-6M or less, 1.0X 10^-7M or less, preferably 1.0X 10^-8M or less, 5.0X 10^-9M or less, more preferably 1.0X 10^-9M or less. However, "high affinity" binding may differ for other antibody isotypes. For example, "high affinity" binding of IgM isotype means that the KD of the bound antigen is 1.0X 10^-6M or less, preferably 1.0X 10^-7M or less, more preferably 1.0X 10^-8M or less.

"identity" refers to the similarity between two nucleic acid sequences or between two polypeptide sequences. The sequence identity of the present invention is at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. Sequence comparison and percent identity determination between two sequences can be performed by the default settings of the BLASTN/BLASTP algorithm on the National Center For Biotechnology Institute website.

An antibody that "competes for binding" refers to an antibody that partially or completely blocks the binding of other antibodies to the target. Whether and to what extent two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody blocks the binding of the other antibody to the target, can be determined using competition assays known in the art, e.g., solid phase direct or indirect Radioimmunoassays (RIA), solid phase direct or indirect Enzyme Immunoassays (EIA), sandwich competition assays, and the like. In certain embodiments, an antibody competes with another antibody for binding to a target and blocks binding of the other antibody to the target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

Two or more antibodies "bind to the same epitope" means that the antibodies bind to the same segment of amino acid residues, as determined by a given method. Techniques for determining whether an antibody binds to the same epitope on "PD-L2" as an antibody described herein include, for example, epitope mapping methods, such as, antigen: x-ray analysis of crystals of antibody complexes and hydrogen/deuterium exchange mass spectrometry (HDX-MS).

The term "EC 50," also known as the half effective concentration, refers to the concentration of antibody that achieves 50% of the maximum effect after a particular exposure time. The term "IC 50," also known as the half maximal inhibitory concentration, refers to the concentration of an antibody that inhibits a specific biological or biochemical function by 50% relative to the absence of the antibody. Both EC50 and IC50 may be measured by ELISA or FACS analysis or any other method known in the art.

"KD" refers to the equilibrium dissociation constant, which is obtained from the ratio of KD to ka (i.e., KD/ka) and expressed as molar concentration (M). KD value of antibodyCan be determined using methods well established in the art. A preferred method for determining the KD of an antibody is by using surface plasmon resonance techniques, preferably using a biosensor system such as

Surface plasmon resonance system analysis.

"patient" or "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, preferably mammals, such as non-human primates, sheep, dogs, cats, cows and horses.

An "effective dose" or "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. A therapeutically "effective amount" or "effective dose" of a drug or therapeutic agent refers to an amount that, when used alone or in combination with another therapeutic agent, results in a reduction in the severity of disease symptoms, an increase in the frequency or duration of disease-free symptoms, or prevents or ameliorates damage due to disease or inhibits the development or recurrence of disease.

"cancer" refers to a collection of cells that proliferate in an abnormal manner, including solid and non-solid tumors.

Unless otherwise stated, "about" in the context of the present invention means within + -5%, preferably within + -2%, more preferably within + -1% of the specified numerical range given. For example, a pH of about 5.5 means a pH of 5.5. + -. 5%, preferably a pH of 5.5. + -. 2%, more preferably a pH of 5.5. + -. 1%. The use of the singular includes the plural unless specifically stated otherwise. Unless specifically stated otherwise, the words "a" or "an" mean "at least one" or "at least one", the meaning of the phrase "at least one" is equivalent to the meaning of "one or more", and/or the use of "means" and "or".

Aspects of the invention are described in more detail below.

anti-PD-L2 monoclonal antibody

Preferably, the antibodies of the invention are antibodies whose structural and chemical properties are as described below.

The anti-PD-L2 antibody or antigen-binding fragment thereof comprises heavy and light chain CDR regions, wherein exemplary heavy and light chain CDR sequences are provided in tables 1 and 2, respectively; the anti-PD-L2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, with exemplary heavy chain variable regions and light chain variable regions provided in table 3 below. Some antibodies have the same CDRs and some have the same VH or VL. The heavy chain constant region of the antibody can be a human IgG1 heavy chain constant region and the light chain constant region of the antibody can be a human kappa light chain constant region. These antibodies may also comprise a murine IgG1 heavy chain constant region, or a human or murine IgG2 or IgG4 heavy chain constant region, and/or a human or murine λ light chain constant region.

TABLE 1 amino acid sequence listing of heavy chain CDR regions

TABLE 2 amino acid sequence listing of light chain CDR regions

TABLE 3 amino acid sequence listing of heavy/light chain variable regions (CDR regions are underlined in order)

TABLE 4 amino acid sequence listing of heavy/light chains (variable regions underlined)

VH and/or VL sequences (or CDR sequences) of other anti-PD-L2 antibodies that bind to human PD-L2 can be "mixed and paired" with VH and/or VL sequences (or CDR sequences) of the antibodies of the invention. Preferably, the VH sequences in a particular VH/VL pair may be replaced by structurally similar VH sequences when the VH and VL chains (or CDRs thereof) are mixed and paired. Likewise, the VL sequences in a particular VH/VL pair are preferably replaced with structurally similar VL sequences.

Thus, in one embodiment, an antibody or antigen-binding fragment thereof of the invention comprises:

(a) a heavy chain variable region comprising an amino acid sequence listed in table 3; and

(b) a light chain variable region comprising the amino acid sequence set forth in table 3, or the VL of another PD-L2 antibody, wherein the antibody specifically binds human PD-L2.

In another embodiment, an antibody or antigen-binding fragment thereof of the invention comprises:

(a) the CDR1, CDR2 and CDR3 regions of the heavy chain variable region listed in Table 1; and

(b) the CDR1, CDR2 and CDR3 regions of the light chain variable region listed in table 2, or the light chain variable region CDR of another PD-L2 antibody, wherein the antibody specifically binds human PD-L2.

In another embodiment, the antibody or antigen binding fragment thereof of the invention comprises the heavy chain variable CDR2 region listed in table 1 and the CDRs of other PD-L2 antibodies, e.g., CDR1 and/or CDR3 of the heavy chain variable region of other PD-L2 antibodies, and/or CDR1, CDR2 and/or CDR3 of the light chain variable region of other PD-L2 antibodies.

Furthermore, it is well known in the art that the CDR3 domain is independent of the CDR1 and/or CDR2 domains, that the binding specificity of an antibody to the same antigen can be determined individually, and that multiple antibodies with the same binding specificity can be predicted to be produced based on this CDR3 sequence.

In another embodiment, the antibody or antigen-binding fragment thereof of the invention comprises CDR2 of the heavy chain variable region listed in table 1; and CDR3 of the heavy chain variable region listed in table 1 and/or CDR3 of the light chain variable region listed in table 2, or CDR3 of the heavy chain variable region and/or CDR3 of another PD-L2 antibody, wherein the antibody specifically binds human PD-L2. These antibodies preferably (a) compete for binding to PD-L2; (b) functional characteristics are reserved; (c) binds to the same epitope; and/or (d) has a similar binding affinity to the PD-L2 antibody of the invention. In another embodiment, the antibody of the invention further comprises CDR2 of the light chain variable region listed in table 2, or CDR2 of the light chain variable region of another PD-L2 antibody, wherein the antibody specifically binds human PD-L2. In another embodiment, the antibody of the invention further comprises CDR1 of the heavy chain variable region listed in table 1 and/or CDR1 of the light chain variable region listed in table 2, or CDR1 of the heavy chain variable region and/or CDR1 of the light chain variable region of another PD-L2 antibody, wherein the antibody specifically binds human PD-L2.

Conservative modifications

In another embodiment, the antibody of the invention comprises heavy and/or light chain variable region or CDR1, CDR2 and CDR3 sequences that are conservatively modified with one or more of the PD-L2 antibodies of the invention. It is understood in the art that some conservative sequence modifications do not abolish antigen binding.

Thus, in one embodiment, the antibody comprises a heavy chain variable region and/or a light chain variable region comprising CDR1, CDR2, and CDR3, respectively, wherein:

(a) the heavy chain variable region CDR1 sequence comprises the sequences listed in table 1, and/or conservative modifications thereof; and/or

(b) The heavy chain variable region CDR2 sequence comprises the sequences listed in table 1, and/or conservative modifications thereof; and/or

(c) The heavy chain variable region CDR3 sequence comprises the sequences listed in table 1, and/or conservative modifications thereof; and/or

(d) The light chain variable region CDR1 and/or CDR2 and/or CDR3 sequences comprise the sequences listed in table 2; and/or conservative modifications thereof; and is

(e) The antibody specifically binds to human PD-L2.

The antibodies of the invention have one or more of the following functional properties described above, e.g., high affinity for human PD-L2, and blocking the binding of PD-L2/PD-1.

The term "conservative sequence modification" as used herein refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having similar structural or chemical properties (e.g., a similar side chain). Families of amino acid residues with similar side chains are known in the art. These families of amino acid residues include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in a CDR region of an antibody of the invention can be substituted with other amino acid residues of the same side chain family, and the resulting antibody can be tested for retained function (i.e., the function described above) using the functional assays described herein.

Genetically engineered and modified antibodies

The antibody of the present invention may be prepared as a genetically engineered antibody using an antibody having one or more VH/VL sequences of the anti-PD-L2 antibody of the present invention as a starting material. One or more residues within one or both variable regions (i.e., VH and/or VL) (e.g., in one or more CDR regions and/or one or more framework regions) can be modified. Alternatively, residues in the constant region may be modified, for example to alter the effector function of the antibody.

In certain embodiments, CDR grafting can be used to genetically engineer the variable regions of antibodies. Antibodies interact with the target antigen primarily through amino acid residues located in the six heavy and light chain CDRs. Thus, the amino acid sequence within the CDRs of each antibody is more diverse than sequences outside the CDRs. Since the CDR sequences are responsible for the major antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular native antibody can be expressed by constructing expression vectors containing CDR sequences of the particular native antibody grafted onto framework sequences of a different antibody of different properties.

Accordingly, another embodiment of the invention relates to an isolated monoclonal antibody, or antigen binding fragment thereof, comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 having the sequences described above and/or a light chain variable region comprising CDR1, CDR2 and CDR3 having the sequences described above. Although these antibodies comprise the CDR sequences of the monoclonal antibodies of the invention, they may comprise different framework sequences. Such framework sequences can be obtained from public DNA databases or public references including germline antibody gene sequences. Such framework sequences are preferably those which are structurally similar to the framework sequences used for the antibodies of the invention. The CDR1, CDR2, and CDR3 sequences can be grafted into framework regions having the same sequence as the germline immunoglobulin gene for that framework sequence, or the CDR sequences can be grafted into framework regions having one or more mutations compared to the germline sequence. For example, in some cases, it may be beneficial to mutate residues in the framework regions that can maintain or enhance the antigen binding ability of the antibody (see, e.g., U.S. Pat. No. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

Another type of variable region modification is mutation of amino acid residues within the CDR1, CDR2, and/or CDR3 regions to improve one or more properties (e.g., affinity, physicochemical properties) of the antibody of interest. Mutations can be introduced by mutagenesis or PCR-mediated mutagenesis, and the effect of the mutation on antibody binding or other functional properties can be assessed by in vitro or in vivo assays known in the art. Amino acids may be substituted, added or deleted, and substitution is preferable. In addition, typically no more than one, two, three, four or five residues within each CDR region are altered.

In another embodiment, the present invention provides an isolated PD-L2 monoclonal antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, comprising: (a) a VH CDR1 region comprising a sequence of the invention, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; (b) a VH CDR2 region comprising a sequence of the invention, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; (c) a VH CDR3 region comprising a sequence of the invention, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; (d) a VL CDR1 region comprising a sequence of the invention, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; (e) a VL CDR2 region comprising a sequence of the invention, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; and (f) a VL CDR3 region comprising a sequence of the invention, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions.

Modified antibodies of the invention include those antibodies modified at the framework region residues of VH and/or VL to improve antibody properties. Typically, such framework region modifications may reduce the immunogenicity of the antibody. For example, one or more framework residues are "back mutated" to the corresponding germline sequence. These residues can be identified by comparing the antibody framework sequences to the germline sequences of the resulting antibody.

Another type of framework modification involves mutating one or more residues of the framework regions, or even one or more CDR regions, to remove T cell epitopes, thereby reducing the immunogenicity that an antibody may cause. This method, also known as "de-immunogenicity", is described in more detail in U.S. patent publication No. 20030153043.

In addition, modifications to the Fc region of the antibodies of the invention are also included, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. In addition, the antibodies of the invention may be chemically modified (e.g., to attach one or more chemical functional groups), or modified to alter glycosylation, to alter one or more functional properties of the antibody. In other embodiments, the Fc region is modified by pegylation (e.g., by reacting the antibody or fragment thereof with polyethylene glycol (PEG).

Pharmaceutical composition

In another aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the invention, formulated together with a pharmaceutically acceptable carrier. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active ingredients, such as another antibody or drug, e.g. another anti-PD-L2 antibody, an anti-PD-L1 antibody, an anti-PD-1 antibody, another anti-cancer drug, preferably comprising another anti-PD-L1 antibody.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial agents, isotonic agents, and combinations thereof that are physiologically compatible. The selection and use of a suitable "pharmaceutically acceptable carrier" is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003).

In some embodiments, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient may be packaged in a material to protect it from acids and other natural conditions that might inactivate it. "parenteral administration" means modes other than enteral and topical administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intramembranous, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, supradural and intrasternal injection and bolus injection. Alternatively, the antibody or antigen-binding fragment thereof of the invention may be administered by a parenteral route, e.g., topical, epidermal or mucosal administration, e.g., intranasal, oral, vaginal, rectal, sublingual or topical.

Various delivery systems are known and may be used to administer one or more antibodies or antigen-binding fragments thereof of the invention, or one or more antibodies or antigen-binding fragments thereof of the invention in combination with one or more other pharmaceutically active ingredients, such as another antibody or drug, e.g., another PD-L2 antibody, an anti-PD-L1 antibody, another anti-cancer drug.

Uses and methods of the invention

The antibodies or antigen-binding fragments thereof of the invention (nucleic acids encoding the antibodies or antigen-binding fragments thereof, pharmaceutical compositions, recombinant polypeptides, fusion proteins, or bispecific molecules) have a variety of in vitro and in vivo applications, e.g., for modulating an immune response in a subject, enhancing an anti-tumor immune response, reducing tumor size or inhibiting tumor cell growth, and/or treating (e.g., curing, inhibiting, ameliorating, delaying, or preventing recurrence of) a tumor or cancer. Accordingly, the present invention also provides a method of enhancing an anti-tumor immune response in a subject, comprising administering to the subject a therapeutically effective amount of an anti-PD-L2 antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition thereof. In one embodiment, the present invention also provides a method of reducing tumor size or inhibiting tumor cell growth in a subject comprising administering to the subject a therapeutically effective amount of an anti-PD-L2 antibody or antigen-binding fragment thereof of the present invention or a pharmaceutical composition thereof. In one embodiment, the invention also provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of an anti-PD-L2 antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition thereof. In other embodiments, the method comprises administering to the subject a therapeutically effective amount of a nucleic acid encoding an anti-PD-L2 antibody or antigen-binding fragment thereof of the invention.

In other embodiments, the method comprises administering to the subject a therapeutically effective amount of a recombinant polypeptide, fusion protein, or bispecific molecule of the invention. The anti-PD-L2 antibodies or antigen-binding fragments thereof described herein can be administered to a human subject, e.g., an anti-PD-L1 antibody, an anti-PD-1 antibody, and/or an anti-CTLA-4 antibody, preferably an anti-PD-L1 antibody, alone or in combination with one or more other antibodies. The anti-PD-L2 antibodies or antigen-binding fragments thereof described herein can be administered to a human subject alone or in combination with at least one other form of treatment, including radiation therapy, chemotherapy, biologics, chemicals, vaccines, and the like.

Tumors or cancers discussed herein include, but are not limited to: colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, renal cancer, prostate cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine malignancies, head and neck cancer, salivary gland cancer, thymus cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protruberans, merkeloid, glioblastoma, glioma, sarcoma, mesothelioma and myelodysplastic syndrome, or a combination thereof; and optionally, the tumor is in situ, metastatic, recurrent, and/or refractory.

In another embodiment, the invention also provides a method of treating an infectious disease in a subject, comprising administering to the subject a therapeutically effective amount of an anti-PD-L2 antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a nucleic acid encoding an antibody or antigen-binding fragment thereof of the invention. Infectious diseases include, but are not limited to: candidiasis, candidemia, aspergillosis, streptococcal pneumonia, streptococcal skin and oropharyngeal diseases, gram-negative sepsis, tuberculosis, mononucleosis, influenza, respiratory diseases caused by respiratory syncytial virus, malaria, schistosomiasis and trypanosomiasis.

The "combination" or "combination" of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or simultaneously as separate compositions, wherein each agent is in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents may be administered sequentially. Further, if multiple combination therapy administrations are performed and the agents are administered sequentially, the order of sequential administration at each time point may be reversed or remain the same, sequential administration may be combined with simultaneous administration or any combination thereof.

In another aspect, the present invention also provides a method of detecting PD-L2 expression in a sample, comprising contacting the sample with an anti-PD-L2 antibody or antigen-binding fragment thereof of the present invention under conditions that allow the formation of a complex between the anti-PD-L2 antibody or antigen-binding fragment thereof and PD-L2, and detecting the formation of the complex.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The invention is further illustrated by the following examples, which are not intended to be limiting. Those skilled in the art will readily identify a variety of noncritical parameters that may be changed or modified to produce substantially similar results.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology, within the skill of the art.

Examples

Example 1: production of anti-PD-L2 monoclonal antibody

To generate antibodies against human PD-L2, a first immunization of 500. mu.g protein/mouse was performed using an equal volume of recombinant hPD-L2-His protein (ACROBIOSystems, Cat.: PD2-H5220) emulsified in complete Freund's adjuvant. Using equal volumesRecombinant hPD-L2-mFc protein (ACROBIOSystems, Cat: PD2-H52A5) emulsified in incomplete Freund's adjuvant was boosted with 500. mu.g of protein/mouse, and Balb/c mice were immunized subcutaneously every 2 weeks for 6 weeks. A fourth immunization was performed by intramuscular injection of hPDL2-mFc protein (20. mu.g/mouse). 3 days prior to fusion, immunization was boosted by subcutaneous injection of antigen without adjuvant. Splenocytes (1X 10) from immunized mice were obtained using Polyethylene Glycol 1500(Roche, Cat. No.: 7181)⁸) And SP2/0 myeloma cells (1.5X 10)⁷) And (4) fusing. After fusion, cells were dispensed into 96-well plates at 0.1 ml/well and 5% CO at 37 ℃₂The incubator of (1) is used for incubation. The culture supernatants are usually checked for binding by ELISA between days 9 and 15 of feeding with hPD-L2-His (ACROBIOSystems, Cat: PD2-H5220), the procedure being described in example 4.

The selected mixed clones capable of binding hPD-L2 were subjected to limiting dilution, and one positive mixed clone was distributed to multiple 96-well plates and cultured in RPMI 1640(Hyclone, catalog No.: SH30809.01) containing 10% fetal bovine serum. After 10 days, the subclones to be subjected to limiting dilution were also subjected to ELISA test to examine the binding of the culture supernatant to hPD-L2-His (ACROBIOSystems, Cat.: PD2-H5220), and then a positive subclone capable of binding to hPD-L2 was selected.

Example 2: anti-PD-L2 antibody cDNA sequence cloning and humanization

Cloning of immunoglobulin cDNA

First strand cDNA was synthesized using a reverse transcription kit (Thermo Fisher, cat # K1652) using total RNA isolated from a hybridoma cell line producing human PD-L2 antibody using an RNA extraction kit (Takara, cat # 9767) as a template according to the manufacturer's instructions. The antibody-related sequences were then amplified in vitro by a PCR reaction using degenerate mouse IgG primers in a 50ml volume reaction. The PCR reaction was carried out in a T100TM thermal cycler (Bio-Rad, Cat. No.: 184-2000).

The PCR mixture was electrophoresed in a 1% agarose/Tris-acetate gel, and DNA fragments having a desired size were excised from the gel and purified. The purified PCR product was cloned into pMD-19T vector (Takara, Cat: 6013) and then transformed into DH5a competent E.coli cells (Takara, Cat: 9057). Clones were screened by colony PCR and selected for DNA sequencing. Obtaining the heavy chain variable region sequence and the light chain variable region sequence of the antibody: 25G4(SEQ ID NOS: 25-26), 14E7(SEQ ID NOS: 27-28), 24A3(SEQ ID NOS: 29-30), 7A7(SEQ ID NOS: 31-32) are shown in Table 3.

Construction and expression of chimeric 7A7, 14E7, 24A3 and 25G4 antibodies

Light chains of chimeric 25G4(SEQ ID NOS: 47, 49), 14E7(SEQ ID NOS: 51, 53), 24A3(SEQ ID NOS: 55, 57) and 7A7(SEQ ID NOS: 59, 61) were constructed by chemically synthesizing genes of mouse variable regions in which cDNAs of VL regions were ligated to human kappa light chain constant regions, respectively; the cDNAs of the VH regions were ligated to the constant regions of human IgG1, respectively, to construct the heavy chains of chimeric 25G4(SEQ ID NOS: 46, 48), 14E7(SEQ ID NOS: 50, 52), 24A3(SEQ ID NOS: 54, 56) and 7A7(SEQ ID NOS: 58, 60).

100 μ g of each chimeric heavy chain expression plasmid and chimeric light chain expression plasmid were transfected into Expi-CHO cells (200mL, density 10)⁶one/mL), cultured for 6 days. The chimeric antibody in the supernatant was then purified using a protein A column (Genscript, Cat: L00695-16). "Xi" in the present invention means a chimeric antibody, for example, Xi7A7 means a chimeric 7A7 antibody.

Antibody humanization design

Antibody humanization was performed using a CDR-grafting method (see, for example, U.S. Pat. No. 5225539), and VH and VL amino acid sequences of mouse 25G4, 14E7, 24A3 and 7A7 antibodies were imported to the International immune genetic information System-related tools Web site (http:// www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign. cgi), respectively, from which human germline antibody sequences having high homology with mouse 25G4, 14E7, 24A3 and 7A7 antibodies were selected as human antibody templates grafted with Complementary Determining Regions (CDRs) in VH and VL of mouse 25G4, 14E7, 24A3 and 7A7 antibodies. For 25G4, the selected human VH templates were a combination of IGHV1-46 x 01 and IGHJ 4x 01, and the selected human VL templates were a combination of IGKV1-39 x 01 and IGKJ1 x 01. For 14E7, the selected human VH templates were a combination of IGHV1-46 x 01 and IGHJ 4x 01, and the selected human VL templates were a combination of IGKV1-5 x 01 and IGKJ1 x 01. For 24a3, the selected human VH templates were a combination of IGHV1-46 x 01 and IGHJ 4x 01, and the selected human V templates L were a combination of IGKV4-1 x 01 and IGKJ1 x 01. For 7a7, the selected human VH templates were a combination of IGHV1-46 x 01 and IGHJ 4x 01, and the selected human VL templates were a combination of IGKV1-39 x 01 and IGKJ1 x 01.

The amino acid sequences of the CDRs of the above human antibody template were substituted with CDRs of mouse 25G4(SEQ ID NOS: 1-6), 14E7(SEQ ID NOS: 7-12), 24A3(SEQ ID NOS: 13-18) and 7A7(SEQ ID NOS: 19-24) antibodies, and the essential amino acid sequences of VH and VL of mouse 25G4, 14E7, 24A3 and 7A7 antibodies were grafted into the above human germline antibody template VH and VL to obtain a functional humanized antibody. For VH and VL of the 25G4, 14E7, 24A3 and 7a7 antibodies, several sites of framework amino acids of the above-described human antibody template were back-mutated to the corresponding amino acid sequences in the mouse 25G4, 14E7, 24A3 and 7a7 antibodies. Part of the CDRs are mutated on the basis of the above-mentioned mutation to improve the properties of the antibody, such as physicochemical properties or drug properties. Obtaining humanized antibodies Hz25G4-1.1, Hz25G4-1.2, Hz14E7-1.1, Hz14E7-1.2, Hz24A3-1.1, Hz24A3-1.2, Hz7A7-1.1, Hz7A7-1.2, Hz7A7-2.1 and Hz7A7-2.2, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the Hz25G4-1.1 are respectively SEQ ID NO: 33 and SEQ ID NO: 34; the amino acid sequences of the heavy chain variable region and the light chain variable region of Hz25G4-1.2 are SEQ ID NO: 35 and SEQ ID NO: 34; the amino acid sequences of the heavy chain variable region and the light chain variable region of Hz14E7-1.1 are respectively SEQ ID NO: 36 and SEQ ID NO: 37; the amino acid sequences of the heavy chain variable region and the light chain variable region of Hz14E7-1.2 are respectively SEQ ID NO: 38 and SEQ ID NO: 37; the amino acid sequences of the heavy chain variable region and the light chain variable region of Hz24A3-1.1 are respectively SEQ ID NO: 39 and SEQ ID NO: 40; the amino acid sequences of the heavy chain variable region and the light chain variable region of Hz24A3-1.2 are respectively SEQ ID NO: 41 and SEQ ID NO: 40; the amino acid sequences of the heavy chain variable region and the light chain variable region of Hz7A7-1.1 are respectively SEQ ID NO: 42 and SEQ ID NO: 43; the amino acid sequences of the heavy chain variable region and the light chain variable region of Hz7A7-1.2 are respectively SEQ ID NO: 44 and SEQ ID NO: 43; the amino acid sequences of the heavy chain variable region and the light chain variable region of Hz7A7-2.1 are respectively SEQ ID NO: 42 and SEQ ID NO: 45, a first step of; the amino acid sequences of the heavy chain variable region and the light chain variable region of Hz7A7-2.2 are respectively SEQ ID NO: 44 and SEQ ID NO: 45.

construction and expression of humanized 25G4, 14E7, 24A3 and 7A7 antibodies

Synthesizing DNA encoding VL and VH of humanized 25G4, 14E7, 24A3 and 7A7 antibodies, and ligating the humanized VL region gene synthesis fragments to a human kappa light chain constant region by a double enzyme digestion reaction to construct light chains of humanized 25G4-1.1 and 25G4-1.2(SEQ ID NOS: 63, 67), 14E7-1.1 and 14E7-1.2(SEQ ID NOS: 69, 73), 24A3-1.1 and 24A3-1.2(SEQ ID NOS: 75, 79), 7A7-1.1 and 7A7-1.2(SEQ ID NOS: 81, 86), 7A7-2.1 and 7A7-2.2(SEQ ID NOS: 83, 87); the humanized VH gene synthetic fragments were ligated to human IgG1 constant regions by a double restriction enzyme reaction to construct the heavy chains of humanized 25G4-1.1(SEQ ID NOS: 62, 65), 25G4-1.2(SEQ ID NOS: 64, 66), 14E7-1.1(SEQ ID NOS: 68, 71), 14E7-1.2(SEQ ID NOS: 70, 72), 24A3-1.1(SEQ ID NOS: 74, 77), 24A3-1.2(SEQ ID NOS: 76, 78), 7A7-1.1 and 7A7-2.1(SEQ ID NOS: 80, 84), 7A7-1.2 and 7A7-2.2(SEQ ID NOS: 82, 85).

100 μ g of each of the humanized heavy chain expression plasmid and the humanized light chain expression plasmid were transfected into Expi-CHO cells (200mL, density 10)⁶one/mL), cultured for 6 days. The humanized antibody in the supernatant was then purified using a protein A column (Genscript, Cat: L00695-16). "Hz" in the present invention means a humanized antibody, for example, Hz7A7-1.1 means a humanized 7A7-1.1 antibody.

Example 3: binding affinity analysis of anti-PD-L2 antibody to PD-L2

The binding kinetics of the anti-PD-L2 antibody to PD-L2 was examined by Biacore T200(GE) using the following experimental procedures: anti-human IgG antibodies were attached to the reference flow cell and the experimental flow cell of the CM5 chip by amino coupling. At the beginning of each cycle, anti-PD-L2 antibody was injected onto the experimental flow cell to be captured. The PD-L2 protein was then diluted with running buffer and PD-L2 protein was injected serially in the reference and experimental wells. Reaction signals were detected in real time using Biacore to obtain binding and dissociation curves. Finally, the data from the experiment were analyzed using the BiaEvaluation Software 2.0 Software in a 1:1(Langmuir) binding model to determine the association rate constant ka (kon) and dissociation rate constant KD (koff), which was calculated by KD ═ KD/ka. Affinity data for anti-PD-L2 antibodies to human PD-L2 antigen (hPD-L2-His: ACRObiosystems, Cat No: PD2-H5220), cynomolgus monkey PD-L2 antigen (Cyno PD-L2-His: Chinesium, Cat No: 90249-C08H) are shown in tables 5 and 6.

TABLE 5 binding affinity of anti-PD-L2 chimeric antibodies to PD-L2

TABLE 6 binding affinity of anti-PD-L2 humanized antibodies to PD-L2

NA: unreached detection line

Example 4: ELISA-based binding assays for anti-PD-L2 antibodies

The binding ability of anti-PD-L2 antibody to PD-L2 was analyzed using PD-L2-His as an antigen based on ELISA method, the experimental procedure was as follows: a96-well plate was coated with 2. mu.g/ml of hPD-L2-His (ACRObiosystems, Cat.: PD2-H5220) in PBS buffer at 100. mu.l/well and incubated overnight at 4 ℃. The liquid in the 96-well plate was removed, 100. mu.L of blocking buffer (PBS buffer containing 1% (w/v) bovine serum albumin) was added to each well, and incubated at 37 ℃ for 1 hour. After washing the 96-well plate 3 times with washing buffer (PBS buffer containing 0.01% (w/v) Tween 20), 100. mu.l/well of anti-PD-L2 antibody (initial concentration of 30. mu.g/mL, serial dilutions at 1: 4; i.e., concentration of about 30. mu.g/mL, about 7.5. mu.g/mL, about 1.875. mu.g/mL, about 0.469. mu.g/mL, about 0.117. mu.g/mL, about 0.029. mu.g/mL, etc.) was added and incubated at room temperature for 2 hours. After washing the 96-well plate 3 times with the washing buffer, 100. mu.l/well of blocking buffer containing biotin (Thermo, cat. No.: 21336) was added and labeled at room temperature for 1 hour. After washing the 96-well plate for 3 times by the washing buffer, the second antibody Avidin-HRP is added for incubation in the darkFor 20 minutes. After washing the 96-well plate 3 times, 100. mu.l/well of TMB (TIANGEN, Cat.: PA107-01) as a substrate solution was added, and the 96-well plate was incubated at room temperature for 10 minutes, followed by addition of 100. mu.l/well of a stop solution (1M H)₂SO₄) To stop the reaction. The resulting colorimetric signal was read at 450nm using a multifunctional microplate reader (PerkinElmer, EnVision), the experimental data was analyzed using GraphPad Prism5, and EC50 was calculated. The binding of anti-PD-L2 humanized antibody to PD-L2 in an ELISA assay, EC50, is shown in Table 7.

TABLE 7 binding of anti-PD-L2 humanized antibody to PD-L2 in an ELISA assay EC50

Antibodies	Hz7A7-1.1	Hz7A7-1.2	Hz7A7-2.1	Hz7A7-2.2	Hz14E7-1.1
						EC50(ng/mL)	308.2	679.6	1125	NA	115.4
Antibodies	Hz14E7-1.2	Hz24A3-1.1	Hz24A3-1.2	Hz25G4-1.1	Hz25G4-1.2
						EC50(ng/mL)	33.28	166.5	223	68.93	87.4

NA: unreached detection line

Example 5: flow-based cell binding assay for anti-PD-L2 antibodies

The binding ability of anti-PD-L2 antibodies to cells expressing PD-L2 was analyzed by binding experiments with a U2OS cell line (PD-L2-U2OS) stably expressing human PD-L2. Mix 8x10³Each of the U2OS-PD-L2 cells was added to each well of a 96-well U-plate (Corstar, Cat: 3799), and an anti-PD-L2 antibody (initial concentration of 50. mu.g/ml, serially diluted 1: 5) was added to the cell suspension and incubated at room temperature for 1 to 2 hours. After the cells were washed 3 times with PBS buffer, 1:200 diluted Alexa Fluor 488-labeled goat anti-human secondary antibody (Jackson ImmunoResearch Inc, Cat. No. 109-545-088) was added to each well at 100. mu.l and incubated at room temperature for 30 minutes. The fluorescence signal was detected by flow cytometry (BD, Accuri C6) analysis after 3 PBS washes. The binding ability of the anti-PD-L2 antibody to PD-L2 on PD-L2-U2OS cells was measured by Mean Fluorescence Intensity (MFI) of staining. Data were analyzed using GraphPad Prism5 and EC50 values were calculated. Flow cell-based binding of anti-PD-L2 chimeric antibody and anti-PD-L2 humanized antibody to PD-L2 EC50 is shown in tables 8 and 9, respectively.

TABLE 8 flow-based cellular binding of anti-PD-L2 chimeric antibodies to PD-L2 EC50

Antibodies	Xi7A7	Xi14E7	Xi24A3	Xi25G4
					EC50(ng/mL)	429.6	1125	967	540

TABLE 9 binding of anti-PD-L2 humanized antibodies to PD-L2 flow-based cell-bound EC50

Antibodies	Hz7A7-1.1	Hz7A7-1.2	Hz7A7-2.1	Hz7A7-2.2	Hz14E7-1.1
						EC50(ng/mL)	613.1	832.7	924.3	1735	442.8
Antibodies	Hz14E7-1.2	Hz24A3-1.1	Hz24A3-1.2	Hz25G4-1.1	Hz25G4-1.2
						EC50(ng/mL)	NA	1048	619.8	482.8	NA

NA: unreached detection line

Example 6: cell-based antibody blocking assay for anti-PD-L2 antibodies

PD-L2 is a ligand for PD-1, and an anti-PD-L2 antibody can block the binding of PD-L2 to its receptor PD-1. Taking receptor cells (PD-1-Jurkat suspension cells stably expressing human PD-1) in exponential growth phase, and adjusting cell density to 6.4x10⁵one/mL of the cells were inoculated at 12.5. mu.L/well into 384-well plates and incubated at 37 ℃ for 15 minutes in a 5% CO2 incubator. 2.5. mu.L/well of anti-PD-L2 antibody (initial concentration 50. mu.g/ml, serial dilution at 1: 5) was added, incubated at 37 ℃ for 60 minutes in a 5% CO2 incubator. Taking ligand cells (stably expressing U2OS cell line of human PD-L2, PD-L2-U2OS) in exponential growth phase, and adjusting cell density to 9.6x10⁵one/mL of the cells were inoculated into the 384-well plate at 12.5. mu.L/well, and the plate was incubated at room temperature for 2 hours on a shaker. Each time2.5. mu.L of Bioassay Reagent (DiscoverX, Cat.: 12604-. The chemiluminescent signal was read by a multifunctional microplate reader EnVision (PerkinElmer, MNR0643,), the data was analyzed using GraphPad Prism5, and IC50 values were calculated. IC50 of the anti-PD-L2 chimeric antibody blocking the binding of PD-L2 to PD-1 is shown in Table 10, and IC50 of the anti-PD-L2 humanized antibody blocking the binding of PD-L2 to PD-1 is shown in Table 11.

TABLE 10 IC50 of anti-PD-L2 chimeric antibody blocking the binding of PD-L2 and PD-1

Antibodies	Xi7A7	Xi14E7	Xi24A3	Xi25G4
					IC50(ng/mL)	NA	342	220.8	679.8

TABLE 11 IC50 of anti-PD-L2 humanized antibody blocking the binding of PD-L2 and PD-1

Antibodies	Hz7A7-1.1	Hz7A7-1.2	Hz7A7-2.1	Hz7A7-2.2	Hz14E7-1.1
						IC50(ng/mL)	585.4	373.5	144.3	NA	111.7
Antibodies	Hz14E7-1.2	Hz24A3-1.1	Hz24A3-1.2	Hz25G4-1.1	Hz25G4-1.2
						IC50(ng/mL)	2663	1953	1423	1005	700.4

NA: unreached detection line

Example 7: binding of anti-PD-L2 humanized antibodies to DC/T cells

FACS analysis of binding of anti-PD-L2 antibodies to DC/T cells Using CD4⁺T cell isolation kit (Mitenyi Biotech, Cat: 130-094-131) for isolation of human CD4 from human PBMC⁺T cells. Immature Dendritic Cells (DCs) were derived from monocytes isolated from human PBMCs using a human monocyte isolation kit (Stemcell, Cat: 19319), and the cells were cultured in a dendritic cell-induced differentiation medium (Stemcell, Cat: 10985) for 5 days and then induced in a dendritic cell-induced maturation medium (Stemcell, Cat: 10985) for 2 days to obtain mature DCs. Contacting mature DC cells with CD4⁺T cell cells are mixed according to the proportion of 1:4 of cell number, then anti-PD-L2 antibodies with the concentrations of 4 mug/mL, 0.8 mug/mL, 0.16 mug/mL, 0.032 mug/mL and 0.0064 mug/mL are added, the temperature is kept at 37 ℃, after a 5% CO2 incubator is incubated for 96 hours, supernatant is collected, goat 488 anti-human labeled fluorescent secondary antibody diluted according to the proportion of 1:200 is added into the cells, and fluorescent signals are analyzed and detected through a flow cytometer (BD, Accuri C6) so as to detect the binding condition of the antibodies and immune check points on the cells. The results are shown in FIG. 1, that the anti-PD-L2 antibody Hz25G4-1.1 can bind to an immune checkpoint on cells.

Example 8: effect of anti-PD-L2 humanized antibodies on T cell activation in Mixed lymphocyte reaction

The Mixed Lymphocyte Reaction (MLR) was used to demonstrate the effect of anti-PD-L2 humanized antibody, or anti-PD-L2 humanized antibody in combination with PD-L1 antibody, in blocking the binding of PD-1 to its ligand in lymphocyte effector cells, and to determine the secretion of IFN-. gamma.and IL-2 by T cells. Using CD4⁺T cell isolation kit (Mitenyi Biotech, Cat. No.: 130-094-131) for purification of human CD4 from human PBMC⁺T cells. Immature Dendritic Cells (DCs) were derived from monocytes isolated from human PBMCs using a human monocyte isolation kit (Stemcell, Cat. No.: 19319). The cells were cultured in a dendritic cell-induced differentiation medium (Stemcell, Cat.: 10985) for 5 days, and then induced in a dendritic cell-induced maturation medium (Stemcell, Cat.: 10985) for 2 days to obtain mature DCs. To establish the MLR, 10 was added for each reaction⁵Purified CD4⁺T cells and 10⁴Individual allogeneic mature DC cells, in a total volume of 200. mu.L. The test samples were added separately and the cells were incubated for 96 hours at a constant temperature of 37 ℃ in an incubator with 5% CO 2. The experimental samples were divided into: (1) hz25G4-1.1+ anti-PD-L1 antibody (wherein, the concentration of Hz25G4-1.1 is 4 mug/mL, 0.8 mug/mL, 0.16 mug/mL, 0.032 mug/mL, 0.0064 mug/mL, 0.16 mug/mL of anti-PD-L1 antibody respectively); (2) anti-PD-L1 antibody + Hz25G4-1.1 (wherein, the concentration of the anti-PD-L1 antibody is 4 mug/mL, 0.8 mug/mL, 0.16 mug/mL, 0.032 mug/mL, 0.0064 mug/mL, 4 mug/mL for Hz25G4-1.1 respectively); (3) hz25G4-1.1 (concentrations 4. mu.g/mL, 0.8. mu.g/mL, 0.16. mu.g/mL, 0.032. mu.g/mL, 0.0064. mu.g/mL, respectively); (4) anti-PD-L1 antibody (concentration of 4. mu.g/mL, 0.8. mu.g/mL, 0.16. mu.g/mL, 0.032. mu.g/mL, 0.0064. mu.g/mL, respectively); (5) blank IgG1 (concentrations of 4. mu.g/mL, 0.8. mu.g/mL, 0.16. mu.g/mL, 0.032. mu.g/mL, 0.0064. mu.g/mL, respectively). Detection kit (R) Using IL-2ELISA&D, catalog number: DY202-05) and hIFN-gamma detection ELISA kit (R)&D, catalog number: DY285) measures IFN-. gamma.and IL-2 levels in the medium. The PD-L1 antibody in this example is described in CN107001463 as humanized 5G11-IgG1, and the amino acid sequences of the heavy and light chains are as set forth in SEQ ID NO: 88 and SEQ ID NO: 89 is shown. The results are shown in fig. 2a and 2b, and the anti-PD-L2 humanized antibody Hz25G4-1.1 combined with the PD-L1 antibody promotes the secretion of IFN-gamma and IL-2 by T cells, shows synergistic effect and is obvious in dose dependence.

Example 9: detection of thermostability of anti-PD-L2 antibody based on NanoDSF

High throughput protein stability analyzer promemeus nt.48(NanoTemper) is a device based on differential scanning fluorescence (nanoDSF) technology for detecting various types of data such as protein molecular structure stability, aggregation stability, colloidal dispersion stability, etc. The melting temperature (Tm) and the polymerization temperature (Tagg) of the anti-PD-L2 chimeric antibody and the anti-PD-L2 humanized antibody were measured using the apparatus, and the results are shown in Table 12 and Table 13, indicating that the anti-PD-L2 antibody has good thermal stability.

TABLE 12 thermostability of anti-PD-L2 chimeric antibodies

Antibodies	Xi7A7	Xi14E7	Xi24A3	Xi25G4
					Tm(℃)	65.6	65.61	60.71	65.85
Tagg(℃)	66.94	67.76	61.26	67.53

TABLE 13 thermostability of anti-PD-L2 humanized antibodies

Antibodies	Hz7A7-1.1	Hz7A7-1.2	Hz7A7-2.1	Hz7A7-2.2	Hz14E7-1.1
						Tm(℃)	69.22	68.45	68.59	68.48	65.89
Tagg(℃)	70.09	69.16	69.26	69.14	84.11
						Antibodies	Hz14E7-1.2	Hz24A3-1.1	Hz24A3-1.2	Hz25G4-1.1	Hz25G4-1.2
Tm(℃)	66.77	66.29	66.02	69.83	69.78
						Tagg(℃)	83.82	79.65	76.91	70.86	71.20

Although the invention has been described in detail above with reference to a general description and specific embodiments, it will be apparent to those skilled in the art that modifications or improvements may be made on the basis of the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Ningda Ningqing pharmaceutical industry group, Inc

<120> anti-PD-L2 antibody

<150> CN202010833373.2

<151> 2020-08-18

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Gln Gln Tyr Arg Pro Tyr Pro Phe Thr

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Ser Gly Gly Asn Tyr Gly Gly Asp Tyr

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Lys Ala Ser Gln Ser Val Ser Asn Glu Val Ala

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Gln Gln His Phe Ser Ser Pro Leu Thr

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Gly Tyr Ser Phe Ser Gly Tyr Thr Met Asn

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Lys Ser Ser Gln Ser Leu Leu Xaa Ser Gly Asn Gln Lys Asn Tyr Leu

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Trp Ala Ser Thr Arg Glu Ser

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Gln Asn Asp Asp Ser Tyr Pro Leu Thr

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Gln Val Gln Leu Gln Gln Pro Gly Val Glu Val Val Arg Pro Gly Ala

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Pro Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Asn

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Trp Met Asn Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile

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Gly Arg Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Gln Phe

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Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr

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Ile Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Val Arg Glu Gly Ile Tyr Gly Pro Trp Phe Ala Tyr Trp Gly Gln Gly

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Thr Leu Val Thr Val Ser Ala

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Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

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Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

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Gly Ile Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asp Leu Glu Ser Gly Ile Pro Ala

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Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

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Pro Val Glu Thr Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asp

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Lys Asp Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

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Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala

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Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

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Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile

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Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Ala Arg Trp Ala Leu Gln Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

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Leu Thr Val Ser Ser

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Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Thr Val Gly

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Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Ala Ala

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Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

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Tyr Ser Ala Ser Asn Arg Tyr Pro Gly Val Pro Asp Arg Phe Thr Gly

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Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met His Ser

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Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Arg Pro Tyr Pro Phe

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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

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Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Val Arg Ser Gly Thr

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr

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Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Val Ile Asn Pro Val Asn Gly Gly Ile Ser Tyr Asn Glu Lys Phe

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Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr

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Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys

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Ile Arg Ser Gly Gly Asn Tyr Gly Gly Asp Tyr Trp Gly Gln Gly Thr

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Asp Val Val Val Thr Gln Thr Pro Lys Phe Leu Pro Val Thr Ala Glu

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Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Glu

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Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

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Tyr Tyr Ala Ser His Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

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Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Val

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Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln His Phe Ser Ser Pro Leu

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Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

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Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

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Thr Met Asn Trp Val Lys Gln Ser Leu Gly Lys Asn Leu Glu Trp Ile

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Gly Leu Ile Ser Pro Tyr Asn Gly Val Thr Thr Tyr Asn Gln Lys Phe

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Lys Gly Lys Ala Ser Leu Thr Val Asp Thr Ser Ser Asn Thr Ala Tyr

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Met Glu Leu Leu Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys

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Thr Pro Val Thr Val Ser Ser

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Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Thr Gly

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Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Thr Gly Gln

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Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

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Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

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Ile Asn Asn Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

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Asp Asp Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Asn

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Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Thr Asn Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Val Arg Glu Gly Ile Tyr Gly Pro Trp Phe Ala Tyr Trp Gly Gln Gly

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Thr Leu Val Thr Val Ser Ser

115

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

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Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

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Gly Ile Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asp Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asp

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Lys Asp Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Asn

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Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ser Glu Ser Glu Thr His Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Thr Asn Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Val Arg Glu Gly Ile Tyr Gly Pro Trp Phe Ala Tyr Trp Gly Gln Gly

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Thr Leu Val Thr Val Ser Ser

115

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

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Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Trp Ala Leu Gln Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

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Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

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Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Ala Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

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Tyr Ser Ala Ser Asn Arg Tyr Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Arg Pro Tyr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

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Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gly Met Ile Asp Pro Ser Glu Ser Glu Thr Arg Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Trp Ala Leu Gln Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu

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Val Thr Val Ser Ser

115

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr

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Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Val Asn Gly Gly Ile Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Ser Gly Gly Asn Tyr Gly Gly Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

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<213> Artificial Sequence (Artificial Sequence)

<400> 40

Asp Val Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Asn Glu

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser His Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Phe Ser Ser Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 41

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 41

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Val Gln Gly Gly Ile Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Gly Asn Tyr Gly Gly Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 42

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 42

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ser Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Ser Pro Tyr Asn Gly Val Thr Thr Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Val Asp Thr Ser Thr Asn Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Thr Val Pro Leu Arg Phe Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 43

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 43

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn

85 90 95

Asp Asp Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 44

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 44

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ser Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Ser Pro Tyr Gln Gly Val Thr Thr Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Val Asp Thr Ser Thr Asn Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Thr Val Pro Leu Arg Phe Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 45

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 45

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Gln Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn

85 90 95

Asp Asp Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 46

<211> 449

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 46

Gln Val Gln Leu Gln Gln Pro Gly Val Glu Val Val Arg Pro Gly Ala

1 5 10 15

Pro Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Asn

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Gln Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Ile Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Val Arg Glu Gly Ile Tyr Gly Pro Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 47

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 47

Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Ile Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asp Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Thr Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asp

85 90 95

Lys Asp Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 48

<211> 1350

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

caggtgcagc tgcagcagcc tggagtggag gtggtgagac caggagcccc tgtgaagctg 60

tcttgcaagg cttccggcta caccttcacc acaaactgga tgaattgggt gaagcagagg 120

ccaggaaggg gactggagtg gatcggacgc atcgacccca gcgattctga gacacactac 180

aaccagcagt ttaaggacaa ggccaccctg acagtggata agtccagcaa taccgcttat 240

atccagctgt cttccctgac atccgaggac agcgccgtgt actattgcgt gagggagggc 300

atctacggcc catggttcgc ctattgggga cagggcaccc tggtgacagt gtccgctgcc 360

tctacaaagg gcccttccgt gtttccactg gctccctcca gcaagtctac ctccggagga 420

acagccgctc tgggatgtct ggtgaaggac tatttcccag agcccgtgac cgtgtcttgg 480

aactccggcg ccctgacctc tggagtgcac acatttcctg ctgtgctgca gtcttccggc 540

ctgtactccc tgagctctgt ggtgaccgtg ccatccagct ctctgggcac ccagacatat 600

atctgcaacg tgaatcacaa gccatctaat acaaaggtgg acaagaaggt ggagcccaag 660

tcctgtgata agacccatac atgcccccct tgtcctgctc cagagctgct gggaggacca 720

tccgtgttcc tgtttccacc caagcctaag gacaccctga tgatcagcag aaccccagag 780

gtgacatgcg tggtggtggc agtgtctcac gaggatcccg aggtgaagtt taactggtac 840

gtggatggcg tggaggtgca taatgctaag acaaagccaa gggaggagca gtacaacagc 900

acctatcggg tggtgtctgt gctgacagtg ctgcaccagg actggctgaa cggcaaggag 960

tataagtgca aggtgtccaa taaggccctg cccgctccta tcgagaagac catcagcaag 1020

gccaagggcc agcctagaga gccacaggtg tacacactgc ctccatcccg cgacgagctg 1080

accaagaacc aggtgagcct gacatgtctg gtgaagggct tctatcccag cgatatcgct 1140

gtggagtggg agtctaatgg ccagcctgag aacaattaca agaccacacc ccctgtgctg 1200

gacagcgatg gctctttctt tctgtattct aagctgaccg tggataagtc ccgctggcag 1260

cagggcaacg tgtttagctg ttctgtgatg catgaggctc tgcacaatca ttacacacag 1320

aagtccctga gcctgtctcc tggcaagtga 1350

<210> 49

<211> 657

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

gacatcgtga tgacccagtc tccagcttcc ctggccgtgt ccctgggaca gagggctaca 60

atcagctgca gggcttccga gagcgtggat aactacggca tctctctgat gcactggtat 120

cagcagaagc ctggccagcc ccctaagctg ctgatctaca gggccagcga cctggagtct 180

ggcatccctg ctagattctc tggatccgga agcaggaccg acttcaccct gacaatcaat 240

ccagtggaga ccgacgatgt ggccacatac tattgtcagc agtccgacaa ggatccaccc 300

accttcggcg ctggcacaaa gctggagctg aagcgtacgg tggccgctcc ttccgtgttc 360

atctttcccc cttccgatga gcagctgaag agcggcacag cttctgtggt gtgcctgctg 420

aacaacttct acccaaggga ggccaaggtg cagtggaagg tggacaacgc tctgcagagc 480

ggcaattctc aggagtccgt gaccgagcag gacagcaagg attctacata ttccctgtcc 540

agcaccctga cactgtccaa ggccgattac gagaagcaca aggtgtatgc ttgcgaggtg 600

acccatcagg gcctgtcttc ccccgtgaca aagagcttta atagaggcga gtgttga 657

<210> 50

<211> 447

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 50

Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile

35 40 45

Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Ala Leu Gln Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 51

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 51

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Thr Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Ala Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Pro Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met His Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Arg Pro Tyr Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 52

<211> 1344

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

caggtgcagc tgcagcagtc cggaccacag ctggtgagac ctggagcctc tgtgaagatc 60

tcctgcaagg cttccggcta cagcttcacc tcttattgga tgcactgggt gaagcagagg 120

ccaggacagg gactggactg gatcggcatg atcgacccca gcgattctga gacacggctg 180

aaccagaagt ttaaggacaa ggccaccctg acagtggata agtccagctc taccgcttac 240

atgcagctgt ccagccccac atccgaggac agcgccgtgt actattgtgc ccgctgggct 300

ctgcagtact tcgattattg gggccagggc accacactga ccgtgtcttc cgcctctaca 360

aagggccctt ccgtgtttcc actggctccc tccagcaagt ctacctccgg aggaacagcc 420

gctctgggat gtctggtgaa ggactatttc ccagagcccg tgaccgtgtc ttggaactcc 480

ggcgccctga cctctggagt gcacacattt cctgctgtgc tgcagtcttc cggcctgtac 540

tccctgagct ctgtggtgac cgtgccatcc agctctctgg gcacccagac atatatctgc 600

aacgtgaatc acaagccatc taatacaaag gtggacaaga aggtggagcc caagtcctgt 660

gataagaccc atacatgccc cccttgtcct gctccagagc tgctgggagg accatccgtg 720

ttcctgtttc cacccaagcc taaggacacc ctgatgatca gcagaacccc agaggtgaca 780

tgcgtggtgg tggcagtgtc tcacgaggat cccgaggtga agtttaactg gtacgtggat 840

ggcgtggagg tgcataatgc taagacaaag ccaagggagg agcagtacaa cagcacctat 900

cgggtggtgt ctgtgctgac agtgctgcac caggactggc tgaacggcaa ggagtataag 960

tgcaaggtgt ccaataaggc cctgcccgct cctatcgaga agaccatcag caaggccaag 1020

ggccagccta gagagccaca ggtgtacaca ctgcctccat cccgcgacga gctgaccaag 1080

aaccaggtga gcctgacatg tctggtgaag ggcttctatc ccagcgatat cgctgtggag 1140

tgggagtcta atggccagcc tgagaacaat tacaagacca caccccctgt gctggacagc 1200

gatggctctt tctttctgta ttctaagctg accgtggata agtcccgctg gcagcagggc 1260

aacgtgttta gctgttctgt gatgcatgag gctctgcaca atcattacac acagaagtcc 1320

ctgagcctgt ctcctggcaa gtga 1344

<210> 53

<211> 645

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

gacatcgtga tgacccagtc ccagaagttc atgagcacca cagtgggcga tagagtgtct 60

atcacatgca aggcttccca gaacgtggga gctgctgtgg cttggtacca gcagaagcct 120

ggccagtccc caaagctgct gatctactcc gccagcaaca ggtatccagg agtgccagac 180

cggttcaccg gatctggatc cggcacagac ttcaccctga caatcagcaa tatgcactct 240

gaggacctgg ctgattattt ttgtcagcag taccgcccct atcctttcac ctttggcggc 300

ggcacaaagc tggagatcaa gcgtacggtg gccgctcctt ccgtgttcat ctttccccct 360

tccgatgagc agctgaagag cggcacagct tctgtggtgt gcctgctgaa caacttctac 420

ccaagggagg ccaaggtgca gtggaaggtg gacaacgctc tgcagagcgg caattctcag 480

gagtccgtga ccgagcagga cagcaaggat tctacatatt ccctgtccag caccctgaca 540

ctgtccaagg ccgattacga gaagcacaag gtgtatgctt gcgaggtgac ccatcagggc 600

ctgtcttccc ccgtgacaaa gagctttaat agaggcgagt gttga 645

<210> 54

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 54

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Val Arg Ser Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr

20 25 30

Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Val Asn Gly Gly Ile Ser Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ile Arg Ser Gly Gly Asn Tyr Gly Gly Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 55

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 55

Asp Val Val Val Thr Gln Thr Pro Lys Phe Leu Pro Val Thr Ala Glu

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Glu

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser His Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Val

65 70 75 80

Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln His Phe Ser Ser Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 56

<211> 1347

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 56

caggtgcagc tgcagcagtc cggagctgag gtggtgagga gcggcacctc tgtgaaggtg 60

tcttgcaagg cctccggcta cgctttcaca aactatctga tcgagtgggt gaagcagagg 120

ccaggacagg gactggagtg gatcggcgtg atcaaccctg tgaatggcgg catcagctac 180

aatgagaagt tcaagggcaa ggccaccctg acagctgaca agtccagctc taccgtgtac 240

atgcagctgt ccagcctgac atccgacgat tccgccgtgt acttttgtat caggtctggc 300

ggcaactacg gcggcgatta ttggggccag ggcaccacac tgaccgtgtc ttccgcctct 360

acaaagggcc cttccgtgtt tccactggct ccctccagca agtctacctc cggaggaaca 420

gccgctctgg gatgtctggt gaaggactat ttcccagagc ccgtgaccgt gtcttggaac 480

tccggcgccc tgacctctgg agtgcacaca tttcctgctg tgctgcagtc ttccggcctg 540

tactccctga gctctgtggt gaccgtgcca tccagctctc tgggcaccca gacatatatc 600

tgcaacgtga atcacaagcc atctaataca aaggtggaca agaaggtgga gcccaagtcc 660

tgtgataaga cccatacatg ccccccttgt cctgctccag agctgctggg aggaccatcc 720

gtgttcctgt ttccacccaa gcctaaggac accctgatga tcagcagaac cccagaggtg 780

acatgcgtgg tggtggcagt gtctcacgag gatcccgagg tgaagtttaa ctggtacgtg 840

gatggcgtgg aggtgcataa tgctaagaca aagccaaggg aggagcagta caacagcacc 900

tatcgggtgg tgtctgtgct gacagtgctg caccaggact ggctgaacgg caaggagtat 960

aagtgcaagg tgtccaataa ggccctgccc gctcctatcg agaagaccat cagcaaggcc 1020

aagggccagc ctagagagcc acaggtgtac acactgcctc catcccgcga cgagctgacc 1080

aagaaccagg tgagcctgac atgtctggtg aagggcttct atcccagcga tatcgctgtg 1140

gagtgggagt ctaatggcca gcctgagaac aattacaaga ccacaccccc tgtgctggac 1200

agcgatggct ctttctttct gtattctaag ctgaccgtgg ataagtcccg ctggcagcag 1260

ggcaacgtgt ttagctgttc tgtgatgcat gaggctctgc acaatcatta cacacagaag 1320

tccctgagcc tgtctcctgg caagtga 1347

<210> 57

<211> 645

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

gacgtggtgg tgacccagac acccaagttc ctgcctgtga ccgccgagga tagggtgacc 60

atcacatgca aggccagcca gtccgtgtcc aacgaggtgg cttggtacca gcagaagcca 120

ggccagtccc ccaagctgct gatctactat gcttcccaca ggtatacagg agtgcctgac 180

cggttcaccg gaagcggatc tggcacagat ttcaccttta caatctccag cgtgcaggtg 240

gaggacctgg ccgtgtactt ctgtcagcag catttttctt ccccactgac ctttggcgct 300

ggcacaaagc tggagctgaa gcgtacggtg gccgctcctt ccgtgttcat ctttccccct 360

tccgatgagc agctgaagag cggcacagct tctgtggtgt gcctgctgaa caacttctac 420

ccaagggagg ccaaggtgca gtggaaggtg gacaacgctc tgcagagcgg caattctcag 480

gagtccgtga ccgagcagga cagcaaggat tctacatatt ccctgtccag caccctgaca 540

ctgtccaagg ccgattacga gaagcacaag gtgtatgctt gcgaggtgac ccatcagggc 600

ctgtcttccc ccgtgacaaa gagctttaat agaggcgagt gttga 645

<210> 58

<211> 449

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 58

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ser Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser Leu Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Ser Pro Tyr Asn Gly Val Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Ser Leu Thr Val Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Thr Val Pro Leu Arg Phe Asp Val Trp Gly Ala Gly

100 105 110

Thr Pro Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 59

<211> 220

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 59

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Thr Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Thr Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Asn Asn Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Asp Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 60

<211> 1350

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 60

gaggtgcagc tgcagcagtc tggaccagag ctggtgaagc ctggagcttc catgaagatc 60

agctgcaagg cttctggcta cagcttctct ggctatacca tgaactgggt gaagcagtcc 120

ctgggcaaga atctggagtg gatcggcctg atcagcccct acaacggcgt gaccacatat 180

aatcagaagt ttaagggcaa ggcctccctg accgtggaca catccagcaa caccgcttac 240

atggagctgc tgtccctgac aagcgacgat tctgccgtgt actattgtgc taggggcgtg 300

acagtgccac tgcggttcga cgtgtggggc gccggcaccc ccgtgacagt gtcttccgcc 360

tctacaaagg gcccttccgt gtttccactg gctccctcca gcaagtctac ctccggagga 420

acagccgctc tgggatgtct ggtgaaggac tatttcccag agcccgtgac cgtgtcttgg 480

aactccggcg ccctgacctc tggagtgcac acatttcctg ctgtgctgca gtcttccggc 540

ctgtactccc tgagctctgt ggtgaccgtg ccatccagct ctctgggcac ccagacatat 600

atctgcaacg tgaatcacaa gccatctaat acaaaggtgg acaagaaggt ggagcccaag 660

tcctgtgata agacccatac atgcccccct tgtcctgctc cagagctgct gggaggacca 720

tccgtgttcc tgtttccacc caagcctaag gacaccctga tgatcagcag aaccccagag 780

gtgacatgcg tggtggtggc agtgtctcac gaggatcccg aggtgaagtt taactggtac 840

gtggatggcg tggaggtgca taatgctaag acaaagccaa gggaggagca gtacaacagc 900

acctatcggg tggtgtctgt gctgacagtg ctgcaccagg actggctgaa cggcaaggag 960

tataagtgca aggtgtccaa taaggccctg cccgctccta tcgagaagac catcagcaag 1020

gccaagggcc agcctagaga gccacaggtg tacacactgc ctccatcccg cgacgagctg 1080

accaagaacc aggtgagcct gacatgtctg gtgaagggct tctatcccag cgatatcgct 1140

gtggagtggg agtctaatgg ccagcctgag aacaattaca agaccacacc ccctgtgctg 1200

gacagcgatg gctctttctt tctgtattct aagctgaccg tggataagtc ccgctggcag 1260

cagggcaacg tgtttagctg ttctgtgatg catgaggctc tgcacaatca ttacacacag 1320

aagtccctga gcctgtctcc tggcaagtga 1350

<210> 61

<211> 663

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 61

gacatcgtga tgacccagag cccctccagc ctgacagtga ccacaggcga gaaggtgacc 60

atgtcttgca agtcttccca gtccctgctg aacagcggca atcagaagaa ctacctgacc 120

tggtatcagc agaagacagg ccagccccct aagctgctga tctactgggc ctctacaagg 180

gagtccggag tgccagaccg gttcaccgga agcggatctg gcacagactt caccctgaca 240

atcaacaatg tgcaggccga ggatctggcc gtgtactatt gtcagaatga cgattcctat 300

cccctgacct tcggcgctgg cacaaagctg gagctgaagc gtacggtggc cgctccttcc 360

gtgttcatct ttcccccttc cgatgagcag ctgaagagcg gcacagcttc tgtggtgtgc 420

ctgctgaaca acttctaccc aagggaggcc aaggtgcagt ggaaggtgga caacgctctg 480

cagagcggca attctcagga gtccgtgacc gagcaggaca gcaaggattc tacatattcc 540

ctgtccagca ccctgacact gtccaaggcc gattacgaga agcacaaggt gtatgcttgc 600

gaggtgaccc atcagggcct gtcttccccc gtgacaaaga gctttaatag aggcgagtgt 660

tga 663

<210> 62

<211> 449

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 62

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Asn

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Thr Asn Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Arg Glu Gly Ile Tyr Gly Pro Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 63

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 63

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Ile Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asp Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asp

85 90 95

Lys Asp Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 64

<211> 449

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 64

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Asn

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ser Glu Ser Glu Thr His Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Thr Asn Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Arg Glu Gly Ile Tyr Gly Pro Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 65

<211> 1350

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 65

caggtgcagc tggtgcagtc tggcgccgag gtgaagaagc caggcgcttc cgtgaaggtg 60

agctgcaagg cctctggcta caccttcacc acaaactgga tgaattgggt gagacaggct 120

ccaggacagg gactggagtg gatgggaagg atcgacccta gcgattctga gacacactac 180

gcccagaagt ttcagggcag ggtgaccatg acagtggaca agtccaccaa cacagtgtat 240

atggagctgt ccagcctgag gagcgaggat accgccgtgt actattgcgt gagggagggc 300

atctacggcc cttggttcgc ttattggggc cagggcaccc tggtgacagt gtcttccgcc 360

tctacaaagg gcccttccgt gtttccactg gctccctcca gcaagtctac ctccggagga 420

acagccgctc tgggatgtct ggtgaaggac tatttcccag agcccgtgac cgtgtcttgg 480

aactccggcg ccctgacctc tggagtgcac acatttcctg ctgtgctgca gtcttccggc 540

ctgtactccc tgagctctgt ggtgaccgtg ccatccagct ctctgggcac ccagacatat 600

atctgcaacg tgaatcacaa gccatctaat acaaaggtgg acaagaaggt ggagcccaag 660

tcctgtgata agacccatac atgcccccct tgtcctgctc cagagctgct gggaggacca 720

tccgtgttcc tgtttccacc caagcctaag gacaccctga tgatcagcag aaccccagag 780

gtgacatgcg tggtggtggc agtgtctcac gaggatcccg aggtgaagtt taactggtac 840

gtggatggcg tggaggtgca taatgctaag acaaagccaa gggaggagca gtacaacagc 900

acctatcggg tggtgtctgt gctgacagtg ctgcaccagg actggctgaa cggcaaggag 960

tataagtgca aggtgtccaa taaggccctg cccgctccta tcgagaagac catcagcaag 1020

gccaagggcc agcctagaga gccacaggtg tacacactgc ctccatcccg cgacgagctg 1080

accaagaacc aggtgagcct gacatgtctg gtgaagggct tctatcccag cgatatcgct 1140

gtggagtggg agtctaatgg ccagcctgag aacaattaca agaccacacc ccctgtgctg 1200

gacagcgatg gctctttctt tctgtattct aagctgaccg tggataagtc ccgctggcag 1260

cagggcaacg tgtttagctg ttctgtgatg catgaggctc tgcacaatca ttacacacag 1320

aagtccctga gcctgtctcc tggcaagtga 1350

<210> 66

<211> 1350

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 66

caggtgcagc tggtgcagtc tggcgccgag gtgaagaagc caggcgcttc cgtgaaggtg 60

agctgcaagg cctctggcta caccttcacc acaaactgga tgaattgggt gagacaggct 120

ccaggacagg gactggagtg gatgggaagg atcgacccta gcgagtctga gacacactac 180

gcccagaagt ttcagggcag ggtgaccatg acagtggaca agtccaccaa cacagtgtat 240

atggagctgt ccagcctgag gagcgaggat accgccgtgt actattgcgt gagggagggc 300

atctacggcc cttggttcgc ttattggggc cagggcaccc tggtgacagt gtcttccgcc 360

tctacaaagg gcccttccgt gtttccactg gctccctcca gcaagtctac ctccggagga 420

acagccgctc tgggatgtct ggtgaaggac tatttcccag agcccgtgac cgtgtcttgg 480

aactccggcg ccctgacctc tggagtgcac acatttcctg ctgtgctgca gtcttccggc 540

ctgtactccc tgagctctgt ggtgaccgtg ccatccagct ctctgggcac ccagacatat 600

atctgcaacg tgaatcacaa gccatctaat acaaaggtgg acaagaaggt ggagcccaag 660

tcctgtgata agacccatac atgcccccct tgtcctgctc cagagctgct gggaggacca 720

tccgtgttcc tgtttccacc caagcctaag gacaccctga tgatcagcag aaccccagag 780

gtgacatgcg tggtggtggc agtgtctcac gaggatcccg aggtgaagtt taactggtac 840

gtggatggcg tggaggtgca taatgctaag acaaagccaa gggaggagca gtacaacagc 900

acctatcggg tggtgtctgt gctgacagtg ctgcaccagg actggctgaa cggcaaggag 960

tataagtgca aggtgtccaa taaggccctg cccgctccta tcgagaagac catcagcaag 1020

gccaagggcc agcctagaga gccacaggtg tacacactgc ctccatcccg cgacgagctg 1080

accaagaacc aggtgagcct gacatgtctg gtgaagggct tctatcccag cgatatcgct 1140

gtggagtggg agtctaatgg ccagcctgag aacaattaca agaccacacc ccctgtgctg 1200

gacagcgatg gctctttctt tctgtattct aagctgaccg tggataagtc ccgctggcag 1260

cagggcaacg tgtttagctg ttctgtgatg catgaggctc tgcacaatca ttacacacag 1320

aagtccctga gcctgtctcc tggcaagtga 1350

<210> 67

<211> 657

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 67

gacatccaga tgacccagtc tccatccagc ctgagcgcct ctgtgggcga cagggtgacc 60

atcacatgcc gggcttccga gagcgtggat aactacggca tctccctgat gcactggtat 120

cagcagaagc caggcaaggc ccccaagctg ctgatctaca gggcttctga tctggagtcc 180

ggcgtgccta gcagattctc tggctccggc agccgcaccg actttaccct gacaatctct 240

tccctgcagc cagaggattt cgccacatac tattgtcagc agtccgacaa ggatccccct 300

acctttggcc agggcacaaa ggtggagatc aagcgtacgg tggccgctcc ttccgtgttc 360

atctttcccc cttccgatga gcagctgaag agcggcacag cttctgtggt gtgcctgctg 420

aacaacttct acccaaggga ggccaaggtg cagtggaagg tggacaacgc tctgcagagc 480

ggcaattctc aggagtccgt gaccgagcag gacagcaagg attctacata ttccctgtcc 540

agcaccctga cactgtccaa ggccgattac gagaagcaca aggtgtatgc ttgcgaggtg 600

acccatcagg gcctgtcttc ccccgtgaca aagagcttta atagaggcga gtgttga 657

<210> 68

<211> 447

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 68

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Ala Leu Gln Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 69

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 69

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Ala Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Arg Pro Tyr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 70

<211> 447

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 70

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile Asp Pro Ser Glu Ser Glu Thr Arg Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Ala Leu Gln Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 71

<211> 1344

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 71

caggtgcagc tggtgcagag cggcgctgag gtgaagaagc caggagcttc tgtgaaggtg 60

tcctgcaagg cctctggcta ctccttcacc agctattgga tgcactgggt gaggcaggct 120

ccaggacagg gactggagtg gatgggaatg atcgacccta gcgattctga gacaagatac 180

gcccagaagt ttcagggccg ggtgaccatg acagtggaca agtccaccag cacagtgtat 240

atggagctgt ccagcctgag atctgaggac accgccgtgt actattgtgc ccgctgggct 300

ctgcagtact tcgattattg gggccagggc accctggtga cagtgtcttc cgcctctaca 360

aagggccctt ccgtgtttcc actggctccc tccagcaagt ctacctccgg aggaacagcc 420

gctctgggat gtctggtgaa ggactatttc ccagagcccg tgaccgtgtc ttggaactcc 480

ggcgccctga cctctggagt gcacacattt cctgctgtgc tgcagtcttc cggcctgtac 540

tccctgagct ctgtggtgac cgtgccatcc agctctctgg gcacccagac atatatctgc 600

aacgtgaatc acaagccatc taatacaaag gtggacaaga aggtggagcc caagtcctgt 660

gataagaccc atacatgccc cccttgtcct gctccagagc tgctgggagg accatccgtg 720

ttcctgtttc cacccaagcc taaggacacc ctgatgatca gcagaacccc agaggtgaca 780

tgcgtggtgg tggcagtgtc tcacgaggat cccgaggtga agtttaactg gtacgtggat 840

ggcgtggagg tgcataatgc taagacaaag ccaagggagg agcagtacaa cagcacctat 900

cgggtggtgt ctgtgctgac agtgctgcac caggactggc tgaacggcaa ggagtataag 960

tgcaaggtgt ccaataaggc cctgcccgct cctatcgaga agaccatcag caaggccaag 1020

ggccagccta gagagccaca ggtgtacaca ctgcctccat cccgcgacga gctgaccaag 1080

aaccaggtga gcctgacatg tctggtgaag ggcttctatc ccagcgatat cgctgtggag 1140

tgggagtcta atggccagcc tgagaacaat tacaagacca caccccctgt gctggacagc 1200

gatggctctt tctttctgta ttctaagctg accgtggata agtcccgctg gcagcagggc 1260

aacgtgttta gctgttctgt gatgcatgag gctctgcaca atcattacac acagaagtcc 1320

ctgagcctgt ctcctggcaa gtga 1344

<210> 72

<211> 1344

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 72

caggtgcagc tggtgcagag cggcgctgag gtgaagaagc caggagcttc tgtgaaggtg 60

tcctgcaagg cctctggcta ctccttcacc agctattgga tgcactgggt gaggcaggct 120

ccaggacagg gactggagtg gatgggaatg atcgacccta gcgagtctga gacaagatac 180

gcccagaagt ttcagggccg ggtgaccatg acagtggata agtccaccag cacagtgtat 240

atggagctgt ccagcctgag atctgaggac accgccgtgt actattgtgc ccgctgggct 300

ctgcagtact tcgattattg gggccagggc accctggtga cagtgtcttc cgcctctaca 360

aagggccctt ccgtgtttcc actggctccc tccagcaagt ctacctccgg aggaacagcc 420

gctctgggat gtctggtgaa ggactatttc ccagagcccg tgaccgtgtc ttggaactcc 480

ggcgccctga cctctggagt gcacacattt cctgctgtgc tgcagtcttc cggcctgtac 540

tccctgagct ctgtggtgac cgtgccatcc agctctctgg gcacccagac atatatctgc 600

aacgtgaatc acaagccatc taatacaaag gtggacaaga aggtggagcc caagtcctgt 660

gataagaccc atacatgccc cccttgtcct gctccagagc tgctgggagg accatccgtg 720

ttcctgtttc cacccaagcc taaggacacc ctgatgatca gcagaacccc agaggtgaca 780

tgcgtggtgg tggcagtgtc tcacgaggat cccgaggtga agtttaactg gtacgtggat 840

ggcgtggagg tgcataatgc taagacaaag ccaagggagg agcagtacaa cagcacctat 900

cgggtggtgt ctgtgctgac agtgctgcac caggactggc tgaacggcaa ggagtataag 960

tgcaaggtgt ccaataaggc cctgcccgct cctatcgaga agaccatcag caaggccaag 1020

ggccagccta gagagccaca ggtgtacaca ctgcctccat cccgcgacga gctgaccaag 1080

aaccaggtga gcctgacatg tctggtgaag ggcttctatc ccagcgatat cgctgtggag 1140

tgggagtcta atggccagcc tgagaacaat tacaagacca caccccctgt gctggacagc 1200

gatggctctt tctttctgta ttctaagctg accgtggata agtcccgctg gcagcagggc 1260

aacgtgttta gctgttctgt gatgcatgag gctctgcaca atcattacac acagaagtcc 1320

ctgagcctgt ctcctggcaa gtga 1344

<210> 73

<211> 645

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 73

gacatccaga tgacccagtc tccctccaca ctgagcgcct ctgtgggcga tagagtgacc 60

atcacatgca aggcctctca gaacgtggga gctgctgtgg cttggtacca gcagaagcct 120

ggcaaggccc caaagctgct gatctactcc gctagcaata ggtatccagg agtgccatcc 180

cggttctctg gctccggcag cggcaccgag tttaccctga caatctccag cctgcagcct 240

gacgatttcg ctacatacta ttgtcagcag taccgcccct atcctttcac ctttggccag 300

ggcacaaagg tggagatcaa gcgtacggtg gccgctcctt ccgtgttcat ctttccccct 360

tccgatgagc agctgaagag cggcacagct tctgtggtgt gcctgctgaa caacttctac 420

ccaagggagg ccaaggtgca gtggaaggtg gacaacgctc tgcagagcgg caattctcag 480

gagtccgtga ccgagcagga cagcaaggat tctacatatt ccctgtccag caccctgaca 540

ctgtccaagg ccgattacga gaagcacaag gtgtatgctt gcgaggtgac ccatcagggc 600

ctgtcttccc ccgtgacaaa gagctttaat agaggcgagt gttga 645

<210> 74

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 74

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Val Asn Gly Gly Ile Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Gly Asn Tyr Gly Gly Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 75

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 75

Asp Val Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Asn Glu

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser His Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Phe Ser Ser Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 76

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 76

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Val Gln Gly Gly Ile Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Gly Asn Tyr Gly Gly Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 77

<211> 1347

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 77

caggtgcagc tggtgcagtc tggcgccgag gtgaagaagc caggcgcttc cgtgaaggtg 60

agctgcaagg cctctggcta cgctttcacc aactatctga tcgagtgggt gagacaggct 120

ccaggacagg gactggagtg gatgggcgtg atcaaccctg tgaatggcgg catctcctac 180

gcccagaagt ttcagggccg cgtgaccatg acagctgaca agagcacctc tacagtgtat 240

atggagctgt ccagcctgag gtccgaggac acagccgtgt actattgtgc tcggagcggc 300

ggaaattacg gaggcgatta ttggggccag ggcaccctgg tgacagtgtc ttccgcctct 360

acaaagggcc cttccgtgtt tccactggct ccctccagca agtctacctc cggaggaaca 420

gccgctctgg gatgtctggt gaaggactat ttcccagagc ccgtgaccgt gtcttggaac 480

tccggcgccc tgacctctgg agtgcacaca tttcctgctg tgctgcagtc ttccggcctg 540

tactccctga gctctgtggt gaccgtgcca tccagctctc tgggcaccca gacatatatc 600

tgcaacgtga atcacaagcc atctaataca aaggtggaca agaaggtgga gcccaagtcc 660

tgtgataaga cccatacatg ccccccttgt cctgctccag agctgctggg aggaccatcc 720

gtgttcctgt ttccacccaa gcctaaggac accctgatga tcagcagaac cccagaggtg 780

acatgcgtgg tggtggcagt gtctcacgag gatcccgagg tgaagtttaa ctggtacgtg 840

gatggcgtgg aggtgcataa tgctaagaca aagccaaggg aggagcagta caacagcacc 900

tatcgggtgg tgtctgtgct gacagtgctg caccaggact ggctgaacgg caaggagtat 960

aagtgcaagg tgtccaataa ggccctgccc gctcctatcg agaagaccat cagcaaggcc 1020

aagggccagc ctagagagcc acaggtgtac acactgcctc catcccgcga cgagctgacc 1080

aagaaccagg tgagcctgac atgtctggtg aagggcttct atcccagcga tatcgctgtg 1140

gagtgggagt ctaatggcca gcctgagaac aattacaaga ccacaccccc tgtgctggac 1200

agcgatggct ctttctttct gtattctaag ctgaccgtgg ataagtcccg ctggcagcag 1260

ggcaacgtgt ttagctgttc tgtgatgcat gaggctctgc acaatcatta cacacagaag 1320

tccctgagcc tgtctcctgg caagtga 1347

<210> 78

<211> 1347

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 78

caggtgcagc tggtgcagtc tggcgccgag gtgaagaagc caggcgcttc cgtgaaggtg 60

agctgcaagg cctctggcta cgctttcacc aactatctga tcgagtgggt gagacaggct 120

ccaggacagg gactggagtg gatgggcgtg atcaatcctg tgcagggcgg catctcctac 180

gcccagaagt ttcagggccg cgtgaccatg acagctgaca agagcacctc tacagtgtat 240

atggagctgt ccagcctgag gtccgaggac acagccgtgt actattgtgc tcggagcggc 300

ggaaactacg gaggcgatta ttggggccag ggcaccctgg tgacagtgtc ttccgcctct 360

acaaagggcc cttccgtgtt tccactggct ccctccagca agtctacctc cggaggaaca 420

gccgctctgg gatgtctggt gaaggactat ttcccagagc ccgtgaccgt gtcttggaac 480

tccggcgccc tgacctctgg agtgcacaca tttcctgctg tgctgcagtc ttccggcctg 540

tactccctga gctctgtggt gaccgtgcca tccagctctc tgggcaccca gacatatatc 600

tgcaacgtga atcacaagcc atctaataca aaggtggaca agaaggtgga gcccaagtcc 660

tgtgataaga cccatacatg ccccccttgt cctgctccag agctgctggg aggaccatcc 720

gtgttcctgt ttccacccaa gcctaaggac accctgatga tcagcagaac cccagaggtg 780

acatgcgtgg tggtggcagt gtctcacgag gatcccgagg tgaagtttaa ctggtacgtg 840

gatggcgtgg aggtgcataa tgctaagaca aagccaaggg aggagcagta caacagcacc 900

tatcgggtgg tgtctgtgct gacagtgctg caccaggact ggctgaacgg caaggagtat 960

aagtgcaagg tgtccaataa ggccctgccc gctcctatcg agaagaccat cagcaaggcc 1020

aagggccagc ctagagagcc acaggtgtac acactgcctc catcccgcga cgagctgacc 1080

aagaaccagg tgagcctgac atgtctggtg aagggcttct atcccagcga tatcgctgtg 1140

gagtgggagt ctaatggcca gcctgagaac aattacaaga ccacaccccc tgtgctggac 1200

agcgatggct ctttctttct gtattctaag ctgaccgtgg ataagtcccg ctggcagcag 1260

ggcaacgtgt ttagctgttc tgtgatgcat gaggctctgc acaatcatta cacacagaag 1320

tccctgagcc tgtctcctgg caagtga 1347

<210> 79

<211> 645

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 79

gacgtggtca tgacccagag ccctgattct ctggccgtgt ccctgggaga gagggctaca 60

atcagctgca aggctagcca gtccgtgtcc aacgaggtgg cctggtacca gcagaagcca 120

ggccagcccc ctaagctgct gatctactat gcttctcaca ggtataccgg cgtgcccgac 180

aggttctccg gctctggctc cggcacagac ttcaccctga caatctccag cctgcaggct 240

gaggacgtgg ccgtgtacta ttgtcagcag catttctctt ccccactgac ctttggccag 300

ggcacaaagg tggagatcaa gcgtacggtg gccgctcctt ccgtgttcat ctttccccct 360

tccgatgagc agctgaagag cggcacagct tctgtggtgt gcctgctgaa caacttctac 420

ccaagggagg ccaaggtgca gtggaaggtg gacaacgctc tgcagagcgg caattctcag 480

gagtccgtga ccgagcagga cagcaaggat tctacatatt ccctgtccag caccctgaca 540

ctgtccaagg ccgattacga gaagcacaag gtgtatgctt gcgaggtgac ccatcagggc 600

ctgtcttccc ccgtgacaaa gagctttaat agaggcgagt gttga 645

<210> 80

<211> 449

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 80

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ser Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Ser Pro Tyr Asn Gly Val Thr Thr Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Val Asp Thr Ser Thr Asn Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Thr Val Pro Leu Arg Phe Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 81

<211> 220

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 81

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn

85 90 95

Asp Asp Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 82

<211> 449

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 82

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ser Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Ser Pro Tyr Gln Gly Val Thr Thr Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Val Asp Thr Ser Thr Asn Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Thr Val Pro Leu Arg Phe Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 83

<211> 220

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 83

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Gln Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn

85 90 95

Asp Asp Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 84

<211> 1350

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 84

caggtgcagc tggtgcagtc tggcgccgag gtgaagaagc caggcgcttc cgtgaaggtg 60

agctgcaagg cctctggcta cagcttctct ggctatacaa tgaactgggt gaggcaggct 120

ccaggacagg gactggagtg gatgggcctg atctcccctt acaatggcgt gaccacatat 180

gcccagaagt ttcagggcag agtgaccatg acagtggaca cctccacaaa caccgtgtac 240

atggagctgt ccagcctgcg cagcgaggat acagccgtgt actattgtgc taggggagtg 300

accgtgccac tgcggttcga cgtgtgggga cagggcacac tggtgaccgt gtcttccgcc 360

tctacaaagg gcccttccgt gtttccactg gctccctcca gcaagtctac ctccggagga 420

acagccgctc tgggatgtct ggtgaaggac tatttcccag agcccgtgac cgtgtcttgg 480

aactccggcg ccctgacctc tggagtgcac acatttcctg ctgtgctgca gtcttccggc 540

ctgtactccc tgagctctgt ggtgaccgtg ccatccagct ctctgggcac ccagacatat 600

atctgcaacg tgaatcacaa gccatctaat acaaaggtgg acaagaaggt ggagcccaag 660

tcctgtgata agacccatac atgcccccct tgtcctgctc cagagctgct gggaggacca 720

tccgtgttcc tgtttccacc caagcctaag gacaccctga tgatcagcag aaccccagag 780

gtgacatgcg tggtggtggc agtgtctcac gaggatcccg aggtgaagtt taactggtac 840

gtggatggcg tggaggtgca taatgctaag acaaagccaa gggaggagca gtacaacagc 900

acctatcggg tggtgtctgt gctgacagtg ctgcaccagg actggctgaa cggcaaggag 960

tataagtgca aggtgtccaa taaggccctg cccgctccta tcgagaagac catcagcaag 1020

gccaagggcc agcctagaga gccacaggtg tacacactgc ctccatcccg cgacgagctg 1080

accaagaacc aggtgagcct gacatgtctg gtgaagggct tctatcccag cgatatcgct 1140

gtggagtggg agtctaatgg ccagcctgag aacaattaca agaccacacc ccctgtgctg 1200

gacagcgatg gctctttctt tctgtattct aagctgaccg tggataagtc ccgctggcag 1260

cagggcaacg tgtttagctg ttctgtgatg catgaggctc tgcacaatca ttacacacag 1320

aagtccctga gcctgtctcc tggcaagtga 1350

<210> 85

<211> 1350

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 85

caggtgcagc tggtgcagtc tggcgccgag gtgaagaagc caggcgcttc cgtgaaggtg 60

agctgcaagg cctctggcta cagcttctct ggctatacaa tgaactgggt gaggcaggct 120

ccaggacagg gactggagtg gatgggcctg atctcccctt accagggcgt gaccacatat 180

gcccagaagt ttcagggcag agtgaccatg acagtggaca cctccacaaa taccgtgtac 240

atggagctgt ccagcctgcg cagcgaggat acagccgtgt actattgtgc taggggagtg 300

accgtgccac tgcggttcga cgtgtgggga cagggcacac tggtgaccgt gtcttccgcc 360

tctacaaagg gcccttccgt gtttccactg gctccctcca gcaagtctac ctccggagga 420

acagccgctc tgggatgtct ggtgaaggac tatttcccag agcccgtgac cgtgtcttgg 480

aactccggcg ccctgacctc tggagtgcac acatttcctg ctgtgctgca gtcttccggc 540

ctgtactccc tgagctctgt ggtgaccgtg ccatccagct ctctgggcac ccagacatat 600

atctgcaacg tgaatcacaa gccatctaat acaaaggtgg acaagaaggt ggagcccaag 660

tcctgtgata agacccatac atgcccccct tgtcctgctc cagagctgct gggaggacca 720

tccgtgttcc tgtttccacc caagcctaag gacaccctga tgatcagcag aaccccagag 780

gtgacatgcg tggtggtggc agtgtctcac gaggatcccg aggtgaagtt taactggtac 840

gtggatggcg tggaggtgca taatgctaag acaaagccaa gggaggagca gtacaacagc 900

acctatcggg tggtgtctgt gctgacagtg ctgcaccagg actggctgaa cggcaaggag 960

tataagtgca aggtgtccaa taaggccctg cccgctccta tcgagaagac catcagcaag 1020

gccaagggcc agcctagaga gccacaggtg tacacactgc ctccatcccg cgacgagctg 1080

accaagaacc aggtgagcct gacatgtctg gtgaagggct tctatcccag cgatatcgct 1140

gtggagtggg agtctaatgg ccagcctgag aacaattaca agaccacacc ccctgtgctg 1200

gacagcgatg gctctttctt tctgtattct aagctgaccg tggataagtc ccgctggcag 1260

cagggcaacg tgtttagctg ttctgtgatg catgaggctc tgcacaatca ttacacacag 1320

aagtccctga gcctgtctcc tggcaagtga 1350

<210> 86

<211> 663

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 86

gacatccaga tgacacagag cccttccagc ctgtccgcca gcgtgggcga tagggtgacc 60

atcacatgca agtcttccca gtctctgctg aactccggca atcagaagaa ctacctgacc 120

tggtatcagc agaagcccgg caaggcccct aagctgctga tctactgggc tagcaccagg 180

gagtctggcg tgccatcccg gttctctggc tccggcagcg gcacagactt taccctgaca 240

atcagctctc tgcagccaga ggatttcgct acctactatt gtcagaatga cgattcttat 300

cccctgacct ttggccaggg cacaaaggtg gagatcaagc gtacggtggc cgctccttcc 360

gtgttcatct ttcccccttc cgatgagcag ctgaagagcg gcacagcttc tgtggtgtgc 420

ctgctgaaca acttctaccc aagggaggcc aaggtgcagt ggaaggtgga caacgctctg 480

cagagcggca attctcagga gtccgtgacc gagcaggaca gcaaggattc tacatattcc 540

ctgtccagca ccctgacact gtccaaggcc gattacgaga agcacaaggt gtatgcttgc 600

gaggtgaccc atcagggcct gtcttccccc gtgacaaaga gctttaatag aggcgagtgt 660

tga 663

<210> 87

<211> 663

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 87

gacatccaga tgacacagag cccttccagc ctgtccgcca gcgtgggcga tagggtgacc 60

atcacatgca agtcttccca gtctctgctg cagtccggca accagaagaa ttacctgacc 120

tggtatcagc agaagcccgg caaggcccct aagctgctga tctactgggc tagcaccagg 180

gagtctggcg tgccatcccg gttctctggc tccggcagcg gcacagactt taccctgaca 240

atcagctctc tgcagccaga ggatttcgct acctactatt gtcagaacga cgattcttat 300

cccctgacct ttggccaggg cacaaaggtg gagatcaagc gtacggtggc cgctccttcc 360

gtgttcatct ttcccccttc cgatgagcag ctgaagagcg gcacagcttc tgtggtgtgc 420

ctgctgaaca acttctaccc aagggaggcc aaggtgcagt ggaaggtgga caacgctctg 480

cagagcggca attctcagga gtccgtgacc gagcaggaca gcaaggattc tacatattcc 540

ctgtccagca ccctgacact gtccaaggcc gattacgaga agcacaaggt gtatgcttgc 600

gaggtgaccc atcagggcct gtcttccccc gtgacaaaga gctttaatag aggcgagtgt 660

tga 663

<210> 88

<211> 446

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 88

Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Arg Gly Val Thr Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Thr Ile Thr Lys Asp Asn Ser Lys Asn Gln Val Val Leu

65 70 75 80

Thr Met Asn Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Leu Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 89

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 89

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Ala Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Asp Tyr Thr Ser Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

Claims

1. An isolated anti-PD-L2 antibody or antigen-binding fragment thereof, comprising:

(i) a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, wherein:

(1) the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 1, 2, 3 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 1, 2, 3, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in seq id no;

(2) the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 7, 8 and 9 or an amino acid sequence corresponding to SEQ ID NOs: 7, 8, 9, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in seq id no;

(3) the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 13, 14, 15 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 13, 14, 15, or a variant thereof, wherein the amino acid sequences are at least 80% identical; or

(4) The heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 19, 20, 21 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 19, 20, 21, having an amino acid sequence of at least 80% identity; and/or

(ii) A light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein:

(1) the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 4, 5,6 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 4, 5,6, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in seq id no;

(2) the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 10, 11, 12 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 10, 11, 12, or an amino acid sequence having at least 80% identity to the amino acid sequence depicted in seq id no;

(3) the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 16, 17, 18 or an amino acid sequence substantially identical to SEQ ID NOs: 16, 17, 18, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in seq id no; or

(4) The light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 22, 23, 24 or an amino acid sequence substantially identical to SEQ ID NOs: 22, 23, 24, or an amino acid sequence having at least 80% identity thereto;

2. The anti-PD-L2 antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein:

(1) the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 1, 2, 3 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 1, 2, 3, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in seq id no; and, the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 4, 5,6 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 4, 5,6, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in seq id no; wherein, SEQ ID NO: 2 is ridsxseth, X ═ D or E;

(2) the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 7, 8 and 9 or an amino acid sequence corresponding to SEQ ID NOs: 7, 8, 9, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in seq id no; and, the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 10, 11, 12 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 10, 11, 12, or an amino acid sequence having at least 80% identity to the amino acid sequence depicted in seq id no; wherein, SEQ ID NO: 8 is midpxsetr, X ═ D or E;

(3) the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 13, 14, 15 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 13, 14, 15, or a variant thereof, wherein the amino acid sequences are at least 80% identical; and, the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 16, 17, 18 or an amino acid sequence substantially identical to SEQ ID NOs: 16, 17, 18, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in seq id no; wherein, SEQ ID NO: 14 is vinpvxvggis, X ═ N or Q; or

(4) The heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 19, 20, 21 or a sequence substantially identical to the amino acid sequence shown in SEQ ID NOs: 19, 20, 21, having an amino acid sequence of at least 80% identity; and, the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 22, 23, 24 or an amino acid sequence substantially identical to SEQ ID NOs: 22, 23, 24, or an amino acid sequence having at least 80% identity thereto; wherein, SEQ ID NO: 20 is LISPYXGVTT, X ═ N or Q; SEQ ID NO: 22 is kssqslllxsgnqknylt, X ═ N or Q.

3. The anti-PD-L2 antibody or antigen-binding fragment thereof of any one of claims 1-2, which is murine, chimeric, or humanized.

4. The anti-PD-L2 antibody or antigen-binding fragment thereof of any one of claims 1-3, which comprises a heavy chain variable region and a light chain variable region, wherein:

(1) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 25 and 26 or an amino acid sequence substantially identical to SEQ ID NOs: 25 and 26, (2) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 27 and 28 or an amino acid sequence substantially identical to SEQ ID NOs: 27 and 28, (3) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 29 and 30 or a sequence substantially identical to the amino acid sequences shown in SEQ ID NOs: 29 and 30, (4) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 31 and 32 or an amino acid sequence substantially identical to SEQ ID NOs: 31 and 32, (5) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 33 and 34 or an amino acid sequence substantially identical to SEQ ID NOs: 33 and 34, (6) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 35 and 34 or an amino acid sequence substantially identical to SEQ ID NOs: 35 and 34, (7) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 36 and 37 or an amino acid sequence substantially identical to SEQ ID NOs: 36 and 37, (8) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 38 and 37 or an amino acid sequence substantially identical to SEQ ID NOs: 38 and 37, (9) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 39 and 40 or an amino acid sequence substantially identical to SEQ ID NOs: 39 and 40, (10) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 41 and 40 or an amino acid sequence substantially identical to SEQ ID NOs: 41 and 40, (11) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 42 and 43 or to SEQ ID NOs: 42 and 43, and (12) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 44 and 43 or an amino acid sequence substantially identical to SEQ ID NOs: 44 and 43, and (13) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 42 and 45 or amino acid sequences substantially identical to those shown in SEQ ID NOs: 42 and 45, or (14) the heavy chain variable region and the light chain variable region comprise SEQ ID NOs: 44 and 45 or amino acid sequences substantially identical to those shown in SEQ ID NOs: 44 and 45, or an amino acid sequence having at least 80% identity thereto.

5. The anti-PD-L2 antibody or antigen-binding fragment thereof of any one of claims 1-4, which comprises a heavy chain and a light chain, wherein:

(1) the heavy and light chains comprise SEQ ID NOs: 46 and 47 or a sequence identical to the amino acid sequences shown in SEQ ID NOs: 46 and 47, (2) the heavy and light chains comprise, respectively, the amino acid sequences set forth in SEQ ID NOs: 50 and 51 or amino acid sequences substantially identical to those shown in SEQ ID NOs: 50 and 51, (3) the heavy and light chains comprise, respectively, the amino acid sequences set forth in SEQ ID NOs: 54 and 55 or amino acid sequences substantially identical to those shown in SEQ ID NOs: 54 and 55, (4) the heavy and light chains comprise, respectively, the amino acid sequences set forth in SEQ ID NOs: 58 and 59 or an amino acid sequence substantially identical to SEQ ID NOs: 58 and 59, and (5) the heavy and light chains comprise the amino acid sequences of SEQ ID NOs: 62 and 63 or an amino acid sequence substantially identical to SEQ ID NOs: 62 and 63, and (6) the heavy and light chains comprise the amino acid sequences set forth in SEQ ID NOs: 64 and 63 or amino acid sequences substantially identical to those shown in SEQ ID NOs: 64 and 63, and (7) the heavy and light chains comprise the amino acid sequences set forth in SEQ ID NOs: 68 and 69 or an amino acid sequence substantially identical to SEQ ID NOs: 68 and 69, (8) the heavy and light chains comprise the amino acid sequences of SEQ ID NOs: 70 and 69 or an amino acid sequence substantially identical to SEQ ID NOs: 70 and 69, (9) the heavy and light chains comprise the amino acid sequences of SEQ ID NOs: 74 and 75 or an amino acid sequence substantially identical to SEQ ID NOs: 74 and 75, and (10) heavy and light chains comprising, respectively, SEQ ID NOs: 76 and 75 or an amino acid sequence substantially identical to SEQ ID NOs: 76 and 75, and (11) the heavy and light chains comprise the amino acid sequences set forth in SEQ ID NOs: 80 and 81 or a sequence substantially identical to the amino acid sequences shown in SEQ ID NOs: 80 and 81, (12) the heavy and light chains comprise, respectively, the amino acid sequences set forth in SEQ ID NOs: 82 and 81 or a sequence identical to the amino acid sequences shown in SEQ ID NOs: 82 and 81, and (13) the heavy and light chains comprise the amino acid sequences of SEQ ID NOs: 80 and 83 or an amino acid sequence substantially identical to SEQ ID NOs: 80 and 83, or (14) the heavy and light chains comprise, respectively, the amino acid sequences set forth in SEQ ID NOs: 82 and 83 or an amino acid sequence substantially identical to SEQ ID NOs: 82 and 83, which have at least 80% identity.

6. The anti-PD-L2 antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof is selected from a monoclonal antibody, a Fab fragment, a F (ab')2 fragment, a Fd fragment, an Fv fragment, a dAb, an isolated CDR, a single chain Fv molecule, or a combination thereof.

7. An isolated nucleic acid encoding the anti-PD-L2 antibody or antigen-binding fragment thereof of any one of claims 1-6.

8. An expression vector comprising the isolated nucleic acid of claim 7.

9. A host cell comprising the expression vector of claim 8.

10. An isolated anti-PD-L2 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to the same epitope as the anti-PD-L2 antibody or antigen-binding fragment thereof of any one of claims 1-6, or

The antibody or antigen-binding fragment thereof competes for binding to PD-L2 with the PD-L2 antibody or antigen-binding fragment thereof of any one of claims 1-6.

11. A recombinant polypeptide, fusion protein, or bispecific molecule comprising the anti-PD-L2 antibody or antigen-binding fragment thereof of any one of claims 1-6.

12. A pharmaceutical composition comprising the anti-PD-L2 antibody or antigen-binding fragment thereof of any one of claims 1-6, and one or more pharmaceutically acceptable carriers, optionally further comprising an anti-PD-L1 antibody.

13. A method for enhancing an anti-tumor immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of the anti-PD-L2 antibody or antigen-binding fragment thereof of any one of claims 1-6, the anti-PD-L2 antibody or antigen-binding fragment thereof of claim 10, or the pharmaceutical composition of claim 12.

14. A method for reducing tumor size, inhibiting tumor cell growth, or treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of the anti-PD-L2 antibody or antigen-binding fragment thereof of any one of claims 1-6, the anti-PD-L2 antibody or antigen-binding fragment thereof of claim 10, or the pharmaceutical composition of claim 12.

15. The method of claim 13 or 14, wherein the tumor or cancer includes but is not limited to: colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, renal cancer, prostate cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine malignancies, head and neck cancer, salivary gland cancer, thymus cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protruberans, merkeloid, glioblastoma, glioma, sarcoma, mesothelioma and myelodysplastic syndrome, or a combination thereof; and optionally, the tumor is in situ, metastatic, recurrent, and/or refractory.

16. A method for treating an infectious disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the anti-PD-L2 antibody or antigen-binding fragment thereof of any one of claims 1-6, the anti-PD-L2 antibody or antigen-binding fragment thereof of claim 10, or the pharmaceutical composition of claim 12, wherein the infectious disease includes, but is not limited to: candidiasis, candidemia, aspergillosis, streptococcal pneumonia, streptococcal skin and oropharyngeal diseases, gram-negative sepsis, tuberculosis, mononucleosis, influenza, respiratory diseases caused by respiratory syncytial virus, malaria, schistosomiasis and trypanosomiasis, or combinations thereof.

17. A method of detecting PD-L2 expression in a sample from a subject, the method comprising contacting the sample with the anti-PD-L2 antibody, or antigen-binding fragment thereof, of any one of claims 1-6 under conditions that allow the formation of a complex between the anti-PD-L2 antibody, or antigen-binding fragment thereof, of any one of claims 1-6 and PD-L2, and detecting the formation of the complex.

18. A method of making the anti-PD-L2 antibody or antigen-binding fragment thereof of any one of claims 1-6, the method comprising: (i) expressing the anti-PD-L2 antibody or antigen-binding fragment thereof of any one of claims 1-6 in a host cell, and (ii) isolating the anti-PD-L2 antibody or antigen-binding fragment thereof of any one of claims 1-6 from the host cell or cell culture thereof.