RU2809243C2 - Antibodies b7-h4 and methods for their application - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: anti-B7-H4 antibody or an antigen-binding fragment and its use, a pharmaceutical composition that increases the immune response containing an anti-B7H4 antibody or an antigen-binding fragment, a nucleic acid encoding an antibody or an antigen-binding fragment. Also an expression vector containing a nucleic acid and a host cell for producing an antibody are disclosed.

EFFECT: increasing the immune response in a patient suffering from cancer.

11 cl

Description

CROSS REFERENCE TO SIMILAR APPLICATIONS

This application claims benefit from U.S. Provisional Patent Application No. 62/620,545, filed January 23, 2018, the entire contents of which are incorporated herein by reference.

LINK TO LIST OF SEQUENCES

The sequence listing, submitted on January 23, 2019, as a text file named "064467.005PCT_seqlist_ST25", created on January 23, 2019 and having a size of 357 MB, is incorporated herein by reference under Title 37 of the United States Code of Federal Regulations. (C.F.R.) § 1.52(e)(5).

FIELD OF THE INVENTION

The invention generally relates to the field of immunomodulation, and in particular to compositions and methods for modulating B7-H4 signal transduction to enhance or weaken the immune response.

BACKGROUND OF THE ART

The B7 family plays an important role in both the positive and negative regulation of immune responses, through the involvement of a number of different receptors on lymphocytes (Rahbar, R. et al ., Cancer Immunol. Res , 3(2):184-195(2015) ). The B7-H4 protein (also called VTCN1 or B7x or B7S1) is a member of the B7 family and inhibits T cell function. B7-H4 expression is also elevated in a number of different tumors and has been suggested to stimulate tumor growth. High expression of B7-H4 is found in many tumor tissues, providing a correlation of expression levels on tumor cells with adverse clinicopathological features, including tumor aggressiveness.

B7-H4 biological activity has been associated with attenuated CD4+ T cell inflammatory responses and with a correlation between tumor-associated macrophages expressing B7-H4 and FoxP3+ regulatory T cells (Tregs) in the tumor microenvironment. Because B7-H4 is expressed on the surface of tumor cells and tumor-associated macrophages in a variety of cancers, therapeutic blockade of B7-H4 may beneficially alter the tumor microenvironment allowing antigen-specific clearance of tumor cells (Podojil, JR. and Miller, SD, Immunol Rev, 276(1):40-51 (2017)).

Complementary DNA (cDNA) encoding the human B7-H4 protein was identified and cloned from placental cDNA (Sica, GL et al. (2003) "B7-H4, A Molecule Of The B7 Family, Negatively Regulates T Cell Immunity"(" B7-H4, a B7 family molecule negatively regulates T cell immunity"), Immunity 18:849-861; Zang, X. et al. (2003) "B7x: A Widely Expressed B7 Family Member That Inhibits T Cell Activation"(" B7x: a widely expressed member of the B7 family that inhibits T cell activation (Proc. Natl. Acad. Sci. (USA) 100:10388-10392). B7-H4 is discussed in US Patent Nos. 7,931,896; 7875702; 7847081; and 7622565.

Anti-B7-H4 antibodies are described in US Pat. Nos. 9,574,000; 7888477; 7737255; 7619068 and 6962980.

The human B7-H4 protein contains 282 amino acid residues, which have been classified as comprising an amino-terminal extracellular domain, a large hydrophobic transmembrane domain, and a very short intracellular domain (consisting of only 2 amino acid residues). Like other members of the B7 family, B7-H4 possesses a pair of Ig-like regions in the extracellular domain. The B7-H4 protein has the general structure of a type I transmembrane protein. This protein has minimal (about 25%) homology with other members of the B7 family (Zang, X. et al. (2003) "B7x: A Widely Expressed B7 Family Member That Inhibits T Cell Activation"("B7x: a widely expressed member of the B7 family that inhibits T cell activation"), Proc. Natl. Acad. Sci. (USA) 100:10388-10392).

B7-H4 is also expressed on tumor-associated macrophages (TAMs). MAOIs inhibit the antitumor immune response through the release of humoral mediators and also protect tumors from immune recognition by interfering with the cell-mediated immune response through cell surface expression of inhibitory molecules such as B7-H4. Tumor-associated macrophages are derived from resident macrophages or from monocytes mobilized by the tumor microenvironment and polarized at the tumor site. Tumor infiltration by tumor-associated macrophages has been associated with poor patient survival; and targeting tumor-associated macrophages represents a promising anti-cancer strategy. Several approaches have already been developed to solve this problem, including depletion using clodronate-containing liposomes; inhibition of tumor mobilization by targeting CFSR-1 and CCL2; and “retraining” through activation by anti-CD40 class M antibodies of either the plasma protein histidine-rich glycoprotein (HRG) or the mannose receptor (Dangai, D. et al., Cancer Res, 73(15): 4820- 4829 (2013)).

Since B7-H4 is expressed on tumor cells and tumor-associated macrophages in various types of cancer, targeting therapeutic interventions against B7-H4 may have enormous synergistic results in beneficially altering the tumor microenvironment and eliminating cancer cells. Thus, there is a need for new B7-H4 immunomodulators.

Therefore, one object of the present invention is to provide compositions that inhibit B7-H4-mediated inhibitory signal transduction, thereby enhancing or stimulating an immunological response by inhibiting B7-H4 inhibitory signal transduction. Such compositions are useful for the treatment of cancer and infectious diseases.

Another object of the present invention is to provide compositions that enhance B7-H4 mediated inhibitory signal transduction, thereby stimulating a weakened immunological response.

SUMMARY OF THE INVENTION

Compositions and methods of their use for modulating B7-H4 signal transduction are presented. Such compositions are useful for the treatment of inflammatory diseases and lesions of an inflammatory nature, as well as autoimmune diseases. In one embodiment of the present invention, immunomodulatory agents are provided that reduce B7-H4 expression, ligand binding, cross-linking, signal transduction, or a combination thereof. In one embodiment of the present invention, immunomodulatory agents specifically bind to B7-H4. Immunomodulatory agents may be antibodies or antigen-binding fragments thereof, fusion proteins, aptamers, or agents that specifically bind B7-H4 and modulate signal transduction through the B7-H4 signaling cascade. Modulation involves enhancing signal transduction through the B7-H4 signaling cascade or inhibiting signal transduction through the B7-H4 signaling cascade.

In one embodiment, the present invention provides a method of treating an infection in a patient by administering an agent that inhibits or blocks an attenuated B7-H4 immune response in an amount that effectively increases neutrophil production and/or enhances innate immunity. The described agents can be used to enhance the immune response in a patient by inhibiting B7-H4-mediated suppression of the immune response. The immune response may be, for example, a primary immune response to an antigen or an enhancement of effector cell function, such as an increase in antigen-specific T cell proliferation, an increase in T cell cytokine production, stimulation of differentiation, an increase in neutrophil numbers, or a combination of these immune mechanisms. Exemplary agents include (i) a soluble B7-H4 polypeptide or fusion protein; (ii) a function blocking anti-B7-H4 antibody; (iii) an antibody that can be used to deplete B7-H4 positive cells; and (iv) combinations of these agents. In some embodiments of the present invention, the immunomodulatory agent is a B7-H4 antagonist.

In specific embodiments of the present invention, the agent is a B7-H4 fusion protein, for example a fusion protein that includes the B7-H4 extracellular domain or a functional variant thereof linked to an immunoglobulin domain. An exemplary fusion protein includes the amino acid sequence of SEQ ID NO:1 or a fragment thereof.

In other embodiments of the present invention, the agent is a B7-H4 protein or a functional fragment or variant thereof. For example, the B7-H4 protein or a functional fragment or variant thereof may have a sequence that is at least 80%, 90%, 95% or 100% identical to the sequence of SEQ ID NO:l. In one embodiment of the present invention, B7-H4 proteins or polypeptides modulate signal transduction through the B7-H4 signaling cascade by specifically binding to a B7-H4 ligand.

In other embodiments of the present invention, the agent is a soluble B7-H4 protein or a functional fragment or variant thereof. For example, a soluble B7-H4 protein may consist of a B7-H4 extracellular domain or a functional fragment or variant thereof.

Methods of enhancing an immune response in a subject generally involve administering to a subject in need thereof an effective amount of an immunomodulatory agent that reduces B7-H4 expression, ligand binding, cross-linking, signal transduction, or a combination thereof. Such a subject may have, for example, cancer or an infectious disease.

In some embodiments of the present invention, such an entity, cancer or disease, is characterized by increased expression of B7-H4, increased expression of a B7-H4 ligand, or a combination thereof. In specific embodiments of the present invention, the cancer is ovarian cancer, breast cancer, lung cancer, thyroid cancer, gastrointestinal cancer, acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), endometrial cancer, brain cancer, head and neck cancer, pancreatic cancer and bladder cancer. The agent can be administered simultaneously or in combination with a vaccine or a component thereof.

Also provided are immunomodulatory agents that increase B7-H4 expression, ligand binding, cross-linking, immunosuppressive signal transduction, or a combination thereof. Such agents can be used to weaken the immune response in a patient by inducing or enhancing an immune response weakened by B7-H4. Exemplary immunomodulatory agents include an anti-B7-H4 antibody activating function or a soluble B7-H4 polypeptide.

Methods of attenuating the immune response in a subject generally include administering to the subject in need thereof an effective amount of an immunomodulatory agent that increases B7-H4 expression, ligand binding, cross-linking, negative signaling, or a combination thereof. In some embodiments of the present invention, such a subject has inflammation or an autoimmune disease. In a specific embodiment of the present invention, such a subject has rheumatoid arthritis.

In some embodiments of the present invention, such a subject, or disease, or condition is characterized by reduced expression of B7-H4.

Any of the described methods may involve administering an immunomodulatory agent to a subject, either as monotherapy or in combination with one or more additional therapeutic agents.

In one embodiment, the present invention provides a method of treating leukemia in a patient by administering to the subject an effective amount of a pharmaceutical composition comprising a B7-H4 monoclonal antibody, a soluble B7-H4 polypeptide, a B7-H4 fusion protein, or a combination thereof, to inhibit or reduce B7-H4 signal transduction in leukemia cells and thereby inhibit the survival of leukemia cells or stimulate an antitumor immune response to leukemia cells. Said leukemia may be acute myeloid leukemia.

In one embodiment, the present invention provides a method for assessing or predicting the effectiveness of treatment with an anti-B7-H4 binding fragment molecule by analyzing cells of a subject in need of treatment to determine whether the cells express the B7-H4 protein, binding partners of the B7-H4 protein , or both. Exemplary cells that are analyzed include, but are not limited to, cancer cells obtained from a subject. Examples of cancer cells include, but are not limited to, acute myeloid leukemia (AML), endometrial cancer, brain cancer, head and neck cancer, and pancreatic cancer cells.

In one embodiment of the present invention, there is provided an anti-B7-H4 monoclonal antibody or an antigen-binding fragment thereof that has a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90% in sequence. 95%, 99% or 100% identical to the sequence of SEQ ID NO:3.

In one embodiment, the present invention provides a nucleic acid that encodes a light chain corresponding to the sequence of SEQ ID NO:3. In another embodiment, the present invention provides a nucleic acid that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identical to SEQ ID NO: 7.

In one embodiment of the present invention, there is provided an anti-B7-H4 monoclonal antibody or antigen binding fragment thereof that has a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90% in sequence. 95%, 99% or 100% identical to the sequence of SEQ ID NO:8.

In one embodiment, the present invention provides a nucleic acid encoding a heavy chain corresponding to the sequence of SEQ ID NO:8. In another embodiment, the present invention provides a nucleic acid that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identical to SEQ ID NO: 12.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof that has a light chain containing complementarity determining regions (CDRs) according to the sequences of SEQ ID NOs: 4, 5 and 6.

In another embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof having a heavy chain containing complementarity determining regions according to the sequences of SEQ ID NOs: 9, 10 and 11.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof having a light chain containing complementarity determining regions according to the sequences of SEQ ID NOs: 4, 5 and 6, and a heavy chain containing complementarity determining regions. complementarity, in accordance with the sequences of SEQ ID NO:9, 10 and 11.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% consistent , 99% or 100% identical to the sequence of SEQ ID NO:3, and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:8.

In another embodiment, the present invention provides an anti-B7-H4 monoclonal antibody or antigen binding fragment thereof comprising a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95 in sequence %, 99% or 100% identical to the sequence of SEQ ID NO:13.

In one embodiment of the present invention, the light chain of the antibody comprises complementarity determining regions with amino acid sequences corresponding to SEQ ID NOs: 14, 15 and 16.

In one embodiment, the present invention provides an antibody heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identical to SEQ ID NO:29.

In another embodiment, the present invention provides an antibody heavy chain comprising complementarity determining regions with amino acid sequences corresponding to SEQ ID NOs: 9, 11 and 30.

In one embodiment, the present invention provides an anti-B7-H4 monoclonal antibody or antigen binding fragment thereof comprising light chain complementarity determining regions of SEQ ID NOs: 14, 15 and 16, and heavy chain complementarity determining regions of according to the sequences SEQ ID NO:9, 11 and 30.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% consistent , 99% or 100% identical to the sequence of SEQ ID NO:13, and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:29.

In one embodiment, the present invention provides an anti-B7-H4 antibody light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% consistent in sequence. identical to the sequence of SEQ ID NO:46.

In another embodiment, the present invention provides an anti-B7-H4 antibody light chain comprising complementarity determining regions with amino acid sequences corresponding to SEQ ID NOs: 14, 16 and 47.

In one embodiment, the present invention provides an antibody heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identical to SEQ ID NO. :49.

In another embodiment, the present invention provides an antibody heavy chain comprising complementarity determining regions with amino acid sequences corresponding to SEQ ID NOs: 9, 11 and 47.

In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof comprising light chain complementarity determining regions according to SEQ ID NOs: 14, 16 and 47 and heavy chain complementarity determining regions according to SEQ ID NOs: :9, 11 and 50.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% consistent , 99% or 100% identical to the sequence of SEQ ID NO:46, and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:49.

In one embodiment, the present invention provides an antibody light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identical to SEQ ID NO. :52.

In another embodiment of the present invention, the light chain of an antibody comprises identity-determining regions with amino acid sequences corresponding to SEQ ID NOs: 53, 54 and 55.

In one embodiment, the present invention provides an antibody heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identical to SEQ ID NO. :65.

In another embodiment, the present invention provides an antibody heavy chain comprising identity determining regions with amino acid sequences corresponding to SEQ ID NOs: 66, 67 and 68.

In one embodiment, the invention provides an anti-B7-H4 antibody or an antigen-binding fragment thereof comprising light chain complementarity determining regions of SEQ ID NOs: 53, 54, and 55, and heavy chain complementarity determining regions of according to the sequences SEQ ID NO:66, 67 and 68.

In one embodiment, the present invention provides an antibody or antigen binding fragment thereof comprising a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100 in sequence % identical to the sequence of SEQ ID NO:52, and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identical to SEQ ID NO:65.

In one embodiment, the present invention provides an antibody light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identical to SEQ ID NO. :85.

In another embodiment, the present invention provides an antibody light chain comprising identity determining regions with amino acid sequences corresponding to SEQ ID NOs: 4, 86 and 87.

In one embodiment, the present invention provides an antibody heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identical to SEQ ID NO. :89.

In one embodiment, the present invention provides an antibody heavy chain comprising complementarity determining regions with amino acid sequences corresponding to SEQ ID NOs: 9, 90 and 91.

In another embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising light chain complementarity determining regions according to SEQ ID NOs: 4, 86 and 87 and heavy chain complementarity determining regions according to with the sequences SEQ ID NO:9, 90 and 91.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% consistent , 99% or 100% identical to the sequence of SEQ ID NO:85, and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO:89.

In one embodiment, the present invention provides an antibody light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identical to SEQ ID NO. :93.

In another embodiment, the present invention provides an antibody light chain comprising complementarity determining regions with amino acid sequences corresponding to SEQ ID NOs: 94, 95 and 96.

In one embodiment, the present invention provides an antibody heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identical to SEQ ID NO. :98.

In another embodiment, the present invention provides an antibody heavy chain comprising complementarity determining regions with amino acid sequences corresponding to SEQ ID NOs 9, 99 and 100.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising light chain complementarity determining regions of SEQ ID NOs: 94, 95, and 96 and heavy chain complementarity determining regions of according to SEQ ID NO:9, 99, and 100.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% consistent , 99% or 100% identical to the sequence of SEQ ID NO:93, and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:98.

In one embodiment, the present invention provides an antibody light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identical to SEQ ID NO. :102.

In another embodiment, the present invention provides an antibody light chain comprising complementarity determining regions with amino acid sequences corresponding to SEQ ID NOs: 103, 104 and 105.

In one embodiment, the present invention provides an antibody heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identical to SEQ ID NO. :107.

In another embodiment, the present invention provides an antibody heavy chain comprising complementarity determining regions with amino acid sequences corresponding to SEQ ID NOs: 9, 108 and 109.

In one embodiment of the present invention, the anti-B7-H4 antibody or antigen binding fragment thereof comprises light chain complementarity determining regions according to SEQ ID NOs: 103, 104, and 105 and heavy chain complementarity determining regions according to sequences SEQ ID NO:9, 108 and 109.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% consistent , 99% or 100% identical to the sequence of SEQ ID NO:102, and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO:107.

In one embodiment, the present invention provides an antibody light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identical to SEQ ID NO. :111.

In another embodiment, the present invention provides an antibody light chain comprising complementarity determining regions with amino acid sequences corresponding to SEQ ID NOs: 95, 112 and 113.

In one embodiment, the present invention provides an antibody heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identical to SEQ ID NO. :115.

In one embodiment, the present invention provides an antibody heavy chain comprising complementarity determining regions with amino acid sequences corresponding to SEQ ID NOs: 116, 117 and 118.

In one embodiment of the present invention, the anti-B7-H4 antibody or antigen binding fragment thereof comprises light chain complementarity determining regions according to the sequences of SEQ ID NOs: 95, 112 and 113, and heavy chain complementarity determining regions according to the sequences SEQ ID NO:116, 117 and 118.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% consistent , 99% or 100% identical to the sequence of SEQ ID NO:111, and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO:115.

In one embodiment, the present invention provides an antibody light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identical to SEQ ID NO. :120.

In another embodiment, the present invention provides an antibody light chain comprising complementarity determining regions with amino acid sequences corresponding to SEQ ID NOs: 54, 55 and 121.

In one embodiment, the present invention provides an antibody heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identical to SEQ ID NO. :123.

In another embodiment, the present invention provides an antibody heavy chain comprising complementarity determining regions with amino acid sequences corresponding to SEQ ID NOs: 66, 124 and 125.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising light chain complementarity determining regions according to SEQ ID NOs: 54, 55 and 121 and heavy chain complementarity determining regions according to with the sequences SEQ ID NO:66, 124 and 125.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% consistent , 99% or 100% identical to the sequence of SEQ ID NO:120, and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO:123.

In one embodiment, the present invention provides an anti-B7-H4 antibody heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% complete in sequence. identical to the sequence of SEQ ID NO:128.

In another embodiment, the present invention provides an anti-B7-H4 antibody heavy chain comprising complementarity determining regions with amino acid sequences corresponding to SEQ ID NOs: 9, 129 and 130.

In one embodiment of the present invention, the anti-B7-H4 antibody or antigen binding fragment thereof comprises light chain complementarity determining regions according to the sequences SEQ ID NOs: 14, 15 and 16, and heavy chain complementarity determining regions according to the sequences SEQ ID NO:9, 129, 130.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% consistent , 99% or 100% identical to the sequence of SEQ ID NO:13, and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO:128.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising a light chain with an amino acid sequence corresponding to any of SEQ ID NO: 3, 13, 46, 52, 85, 93, 102, 111 or 120 .

In another embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising a heavy chain with an amino acid sequence corresponding to any of the sequences of SEQ ID NO: 8, 29, 49, 65, 89, 98, 107, 115, 123 or 128.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising a light chain with an amino acid sequence corresponding to any of SEQ ID NO: 3, 13, 46, 52, 85, 93, 102, 111 or 120 , and a heavy chain with an amino acid sequence corresponding to any of SEQ ID NO: 8, 29, 49, 65, 89, 98, 107, 115, 123 or 128.

Also provided is an anti-B7-H4 antibody or an antigen-binding fragment thereof containing three regions providing complementarity to a light chain with amino acid sequences selected from the group consisting of the sequences SEQ ID NO: 4, 5, 6, 14, 15, 16, 47, 53, 54, 55, 86, 87, 94, 95, 96, 103, 104, 105, 112, 113 or 121.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising three heavy chain complementarity regions with amino acid sequences selected from the group consisting of SEQ ID NO:9, 10, 11, 30 , 50, 66, 67, 68, 90, 91, 99, 100, 108, 109, 116, 117, 118, 124, 125, 129 or 130.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising three light chain complementarity regions with amino acid sequences selected from the group consisting of SEQ ID NOs: 4, 5, 6, 14 , 15, 16, 47, 53, 54, 55, 86, 87, 94, 95, 96, 103, 104, 105, 112, 113 and 121, and three heavy chain complementarity regions with amino acid sequences that are selected from the group consisting of the sequences SEQ ID NO: 9, 10, 11, 30, 50, 66, 67, 68, 90, 91, 99, 100, 108, 109, 116, 117, 118, 124, 125, 129 and 130.

In another embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising a light chain variable domain of at least 50%, 60%, 70%, 80%, 85%, 90% of the same sequence. 95%, 99%, or 100% identical to any of SEQ ID NO: 19, 20, 21, 22, or 23, and a heavy chain variable domain that is at least 50%, 60%, 70%, 80 identical in sequence %, 85%, 90%, 95%, 99% or 100% identical to any of SEQ ID NO:34, 35, 36 or 37.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen-binding fragment thereof, comprising a light chain comprising an amino acid sequence corresponding to any of SEQ ID NOs: 24, 25, 26, 27 or 28, and a heavy chain containing an amino acid sequence corresponding to any of SEQ ID NO:38, 39, 40 or 41.

In another embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising a light chain with an amino acid sequence corresponding to any of SEQ ID NO: 24, 25, 26, 27 or 28, and a heavy chain with an amino acid sequence corresponding to any of the sequences of SEQ ID NO:42, 43, 44 or 45.

Also provided is an anti-B7-H4 antibody or an antigen-binding fragment thereof containing a light chain variable domain, the sequence of which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to any of SEQ ID NO: 58, 59, 60 or 61, and a heavy chain variable domain of which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95 identical in sequence %, 99% or 100% identical to any of SEQ ID NO:70, 71, 72, 73 or 74.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising a light chain with an amino acid sequence corresponding to any of the sequences of SEQ ID NO: 62, 63 or 64, and a heavy chain with an amino acid sequence corresponding to any of the sequences SEQ ID NO:75, 76, 77, 78 or 79.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising a light chain with an amino acid sequence corresponding to any of the sequences of SEQ ID NO: 62, 63 or 64, and a heavy chain with an amino acid sequence corresponding to any of the sequences SEQ ID NOs: 80, 81, 82, 83 or 84.

In another embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising six complementarity determining regions (CDRs), where these CDRs include three light chain CDRs of a polypeptide selected from the group consisting of the sequences of SEQ ID NO:4, 5, 6, 14, 15, 16, 49, 55, 56, 57, 88, 89, 96, 97, 97, 105, 106, 107, 114, 115 or 123, or a variant thereof, the sequence of which is at least , 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more identical to the sequences of SEQ ID NO: 4, 5, 6, 14, 15, 16, 49, 55, 56, 57, 88, 89, 96, 97, 97, 105, 106, 107, 114, 115 or 123, and three heavy chain CDRs of a polypeptide selected from the group consisting of the sequences SEQ ID NO: 9, 10, 11, 31, 52, 68, 69, 70, 92, 93, 101, 102, 118, 119, 120, 126, 127, 131 or 132, or a variant thereof, the sequence of which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more identical to SEQ ID NO:9, 10, 11, 31, 52, 68, 69, 70, 92, 93, 101, 102, 118 , 119, 120, 126, 127, 131 or 132, and wherein said antibody or antigen binding fragment thereof binds B7-H4.

In another embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of the sequences SEQ ID NO:24, 25, 26 , 27, 28, 62, 63 or 64, or a variant thereof, the sequence of which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identical to SEQ ID NO:24, 25, 26, 27, 28, 62, 63 or 64, and the two heavy chains include a polypeptide selected from the group consisting of the sequences SEQ ID NO:38, 39, 40, 41, 75, 76, 77, 78 or 79, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO:38 , 39, 40, 41, 75, 76, 77, 78 or 79, and wherein said antibody or antigen binding fragment thereof binds to B7-H4.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of the sequences SEQ ID NO:24, 25, 26 , 27, 28, 62, 63 or 64, or a variant thereof, the sequence of which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identical to SEQ ID NO:24, 25, 26, 27, 28, 62, 63 or 64, and the two heavy chains include a polypeptide selected from the group consisting of the sequences SEQ ID NO:42, 43, 44, 45, 80, 81, 82, 83 or 84, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO:42 , 43, 44, 45, 80, 81, 82, 83 or 84, and wherein said antibody or antigen binding fragment thereof binds B7-H4.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of the sequences SEQ ID NO:24, 25, 26 , 27 or 28, or a variant thereof, the sequence of which is at least on. 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more identical to the sequences of SEQ ID NO:24, 25, 26, 27 or 28, and two heavy chains include a polypeptide selected from a group consisting of the sequences of SEQ ID NO:38, 39, 40 or 41, or a variant thereof, the sequence of which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more identical to the sequences of SEQ ID NO: 38, 39, 40 or 41, and wherein the antibody or antigen binding fragment thereof binds to B7-H4.

In another embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of the sequences SEQ ID NO:24, 25, 26 , 27 or 28, or a variant thereof, the sequence of which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identical to the sequences of SEQ ID NO:24, 25, 26, 27 or 28, and the two heavy chains comprise a polypeptide selected from the group consisting of the sequences of SEQ ID NO:42, 43, 44 or 45, or a variant thereof, which is at least 50% the same sequence as 60 %, 70%, 80%, 85%, 90%, 95%, 99% or more identical to the sequences of SEQ ID NO:42, 43, 44 or 45, and wherein the antibody or antigen binding fragment thereof binds to B7-H4.

In one embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of the sequences SEQ ID NO:62, 63 or 64 , or a variant thereof, the sequence of which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more identical to the sequences of SEQ ID NO:62, 63 or 64, and the two heavy chains comprise a polypeptide selected from the group consisting of the sequences of SEQ ID NO: 75, 76, 77, 78 or 79, or a variant thereof, the sequence of which is at least 50%, 60%, 70%, 80 %, 85%, 90%, 95%, 99% or more identical to the sequences of SEQ ID NO: 75, 76, 77, 78 or 79, and wherein the antibody or antigen binding fragment thereof binds to B7-H4.

In another embodiment, the present invention provides an anti-B7-H4 antibody or antigen binding fragment thereof comprising two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of the sequences SEQ ID NO:62, 63 or 64 , or a variant thereof, the sequence of which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more identical to the sequences of SEQ ID NO:62, 63 or 64, and the two heavy chains comprise a polypeptide selected from the group consisting of the sequences of SEQ ID NO:80, 81, 82, 83 or 84, or a variant thereof, the sequence of which is at least 50%, 60%, 70%, 80 %, 85%, 90%, 95%, 99% or more identical to the sequences of SEQ ID NO:80, 81, 82, 83 or 84, and wherein the antibody or antigen binding fragment thereof binds to B7-H4.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, a molecule is said to be capable of “immunospecifically binding” another molecule if such binding exhibits the specificity and affinity of the antibody for its structurally similar antigen. Antibodies are believed to be capable of immunospecifically binding to a target site or conformation ("epitope") of an antigen if such binding involves the antigen recognition site of an immunoglobulin molecule. An antibody that immunospecifically binds to a particular antigen may bind to other antigens with lower affinity if the other antigen has some sequence or conformational similarity that is recognized by the antigen recognition site, which is determined, for example, by an immunoassay, BIACORE® system assay, or other assays known in the art, but does not bind to a completely unrelated antigen. However, in some embodiments of the present invention, the antibodies (and antigen-binding fragments thereof) do not cross-react with other antigens. Antibodies can also bind to other molecules in a manner that is not immunospecific, such as FcR receptors, due to binding domains in other regions/domains of the molecule that do not include the antigen recognition site, such as the Fc region.

As used herein, a molecule is said to “bind in a physiospecific manner” to another molecule if such binding exhibits the specificity and affinity of the receptor for its structurally similar binding ligand. A molecule may be capable of binding to several other molecules in a physiospecific manner.

As used herein, the term “antibody” refers to an immunoglobulin molecule that has an antigen recognition site “variable region” and includes antigen-binding antibody fragments. The term "variable region" is intended to distinguish such an immunoglobulin domain from domains that are substantially common to the antibody (eg, the Fc domain of an antibody). The variable region includes a "hypervariable region", the residues of which are responsible for antigen binding. The hypervariable region includes amino acid residues from the “complementarity determining region” or “CDR” (i.e., typically approximately residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and at approximately residues 27-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain (Rabat et al., Sequences of Proteins of Immunological Interest ), 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)); and/or these residues from the “hypervariable loop” (i.e. residues 26-32 (LI), 50-52 ( L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain (Chothia and Lesk, 1987, J. Mol Biol. 196:901-917). "Framework region" or "FR" residues are variable domain residues other than hypervariable region residues as defined herein. The term "antibody" includes monoclonal antibodies, multi-specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (see, for example, Muyldermans el al., 2001, Trends Biochem. Sci. 26:230; Nuttall el al. , 2000, Cur. Pharm. Biotech. 1:253; Reichmann and Muyldermans, 1999, J. Immunol. Meth . 231: 25; International Publication No. WO 94/04678 and No. WO 94/25591; US Patent No. 6,005,079), single chain Fvs (scFv) (see, for example, Pluckthun in the Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994)) , single chain antibodies, disulfide-linked Fvs (sdFv), intracellular antibodies, diabodies, tribodies, tetrabodies, Bis-scFv, mini-antibodies, Fab2, Fab3 and anti-anti-idiotypic (anti-id) antibodies (including, for example, anti -id and anti-anti-Id antibodies to antibodies). In particular, such antibodies include immunoglobulin molecules of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgGi, IgG2, IgG3, IgG ₄ , IgAi and IgA ₂ ) or subclass.

As used herein, the term "antigen binding fragment" of an antibody refers to one or more portions of an antibody that contain antibody complementarity determining regions (CDRs) and additional framework residues that include the antigen recognition site of an antibody "variable region" and exhibit the ability to immunospecifically bind antigen . Such fragments include Fab', F(ab') ₂ , Fv, single chain (ScFv) and mutants thereof, natural variants and fusion proteins, including an antibody "variable region" antigen recognition site and a heterologous protein (eg, a toxin, an antigen recognition site for another antigen, enzyme, receptor or receptor ligand, etc.).

As used herein, the term “fragment” refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues of at least 20 consecutive amino acid residues of at least 25 consecutive amino acid residues of at least 40 consecutive amino acid residues of at least, of 50 consecutive amino acid residues, of at least 60 consecutive amino acid residues, of at least 70 consecutive amino acid residues, of at least 80 consecutive amino acid residues, of at least of at least 90 consecutive amino acid residues, of at least 100 consecutive amino acid residues, of at least 125 consecutive amino acid residues, of at least 150 consecutive amino acid residues, of at least 175 consecutive amino acid residues, of at least 200 consecutive amino acid residues, or of at least 250 consecutive amino acid residues.

As used herein, the term “modulate” refers to the ability to change an effect, outcome, or activity (eg, signal transduction). Such modulation can be agonistic or antagonistic. Antagonistic modulation may be partial (i.e., attenuating but not abolishing) or it may completely abolish such activity (i.e., neutralizing). Modulation may involve internalization of the receptor following antibody binding or reduction of receptor expression on the target cell. Agonistic modulation may enhance or otherwise increase or enhance activity (eg, signal transduction). In a further embodiment of the present invention, such modulation may change the nature of the interaction between the ligand and its structurally similar receptor so as to change the nature of the signal transduction caused. For example, these molecules may, by binding to a ligand or receptor, alter the ability of such molecules to bind to other ligands or receptors and thereby alter their overall activity. In some embodiments of the present invention, such modulation will provide a change in measured immune system activity of at least 10%, a change in such activity of at least 50%, or at least a 2-fold, 5-fold, 10-fold or at least 100-fold change in such activity.

The term “substantially” when used in relation to binding or exhibited effect is intended to indicate that the observed effect is physiologically or therapeutically relevant. Thus, for example, a molecule is capable of substantially blocking the activity of a ligand or receptor if the degree of blockade is physiologically or therapeutically significant (for example, if the degree of blockade is greater than 60% complete blockade, greater than 70% complete blockade, greater than 75% complete blockade, greater than 80% complete blockade , more than 85% complete block, more than 90% complete block, more than 95% complete block or more than 97% complete block). Similarly, a molecule is considered to have substantially the same immunospecificity and/or characteristics as another molecule if its immunospecificity and characteristics are more than 60% identical, more than 70% identical, more than 75% identical, more than 80% identical, more than 85% identical, more than 90% identical, more than 95% identical, or more than 97% identical.

As used herein, “co-stimulatory” signals include positive co-stimulatory signals (eg, signals that result in increased activity) and negative co-stimulatory signals (eg, signals that lead to inhibition of activity).

The term “derivative” refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to the same target of the parent or reference antibody, but which differs in amino acid sequence from the parent or reference antibody or antigen-binding fragment thereof by including one, two, three, four , five or more amino acid substitutions, additions, deletions or modifications relative to the parent or reference antibody or antigen binding fragment thereof. In some embodiments of the present invention, such derivatives will have substantially the same immunospecificity and/or characteristics, or the same immunospecificity and characteristics, as the parent or reference antibody, or antigen binding fragment thereof. Substitutions or additions of amino acids in such derivatives may include amino acid residues that are naturally occurring (ie, encoded by DNA) or not naturally occurring. The term "derivative" includes, for example, chimeric or humanized variants, as well as variants having altered CHI, hinge, CH2, CH3 or CH4 regions, to form, for example, antibodies, etc., having variant Fc regions, that exhibit increased or decreased effector or binding characteristics.

As used herein, a “chimeric antibody” is a molecule in which different parts of the antibody are derived from different immunoglobulin molecules, such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region.

As used herein, the term “humanized antibody” refers to an immunoglobulin comprising a human framework region and one or more complementarity determining regions (CDRs) from a non-human (usually murine or rat) immunoglobulin. The non-human immunoglobulin providing the CDR is called a “donor” and the human immunoglobulin providing the framework region is called an “acceptor”. Constant regions do not have to be present, but if present, they must be substantially identical to the human immunoglobulin constant regions, that is, at least about 85-99%, or about 95% or more identical. Therefore, all portions of the humanized immunoglobulin, with the possible exception of the CDRs, are substantially identical to the corresponding portions of the normal human immunoglobulin sequences. A humanized antibody is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody will not include a generic chimeric antibody because, for example, the entire variable region of the chimeric antibody is non-human.

The term "endogenous concentration" refers to the level at which a molecule is expressed naturally ( i.e., in the absence of expression vectors or recombinant promoters) by a cell (such a cell may be a normal cell, a cancer cell, or an infected cell).

As used herein, the terms “treat,” “therapeutic,” “treatment,” and “therapeutic use” refer to eliminating, reducing, or reducing the intensity of one or more symptoms of a disease or disorder. As used herein, the term “therapeutically effective amount” refers to that amount of a therapeutic agent that is sufficient to achieve clinically significant elimination, reduction, or amelioration of such symptoms. An effect is clinically significant if its magnitude is sufficient to affect the health or prognosis of the recipient subject. A therapeutically effective amount may refer to an amount of a therapeutic agent sufficient to delay or minimize the onset of a disease, such as delaying or minimizing the spread of cancer. A therapeutically effective amount may also refer to an amount of a therapeutic agent that provides a therapeutic effect in treating or managing a disease.

As used herein, the term “prophylactic agent” refers to an agent that can be used to prevent a disorder or disease before any symptoms of such disorder or disease are detected. A "prophylactically effective" amount is an amount of prophylactic agent sufficient to provide such protection. A prophylactically effective amount may also refer to an amount of a prophylactic agent that provides a prophylactic effect in preventing a disease.

As used herein, the term “cancer” refers to a neoplasm or tumor resulting from abnormal, uncontrolled cell growth. As used herein, cancer specifically includes leukemias and lymphomas. The term "cancer" refers to a disease involving cells that are capable of metastasizing to distant sites and exhibiting phenotypic features different from those of non-cancerous cells, for example, the formation of colonies in a three-dimensional substrate such as soft agar, or the formation of tubular networks or network-like matrices in three-dimensional basement membrane or extracellular matrix preparation. Non-cancerous cells do not form colonies in soft agar, but form distinct sphere-like structures in three-dimensional preparations of basement membrane or extracellular matrix.

As used herein, the term “immune cell” refers to any cell of hematopoietic origin, including (but not limited to) T cells, B cells, monocytes, dendritic cells and macrophages.

As used herein, the term “inflammatory molecules” refers to molecules that lead to an inflammatory response, including (but not limited to) cytokines and metalloproteases such as (including but not limited to), IL-lp (interleukin-lp), TNF-alpha (tumor necrosis factor-alpha), TGF-beta (transforming growth factor-beta), IFN-gamma (interferon-gamma), IL-18 (interleukin-18), IL-17 (interleukin-17), IL -6 (interleukin-6), IL-23 (interleukin-23), IL-22 (interleukin-22), IL-21 (interleukin-21) and MMPs (matrix metalloproteinases).

As used herein, the term “valency” refers to the range of binding sites present on a molecule.

As used herein, the terms “immunological” or “immune” response represent the development of a beneficial humoral (antibody-mediated) and/or cellular (antigen-specific T-cell or secretion product) response directed against the peptide in the recipient patient. Such a response may be an active response induced by administration of an immunogen, or a passive response induced by administration of an antibody or T cells primed with antigen. The cellular immune response is elicited by the presentation of polypeptide epitopes in association with MHC class I or class II molecules to activate antigen-specific CD4+ T helper cells and/or cytotoxic CD8+ T cells. The response may also include activation of monocytes, macrophages, NK cells (natural killer cells), basophils, dendritic cells, astrocytes, microglia cells, eosinophils, activation or mobilization of neutrophils or other components of the innate immune system. The presence of a cell-mediated immunological response can be determined using a proliferation assay (CD4+ T cells) or a CTL (cytotoxic T lymphocyte) assay. The relative contribution of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be determined by separately isolating antibodies and T cells from an immunized syngeneic animal and measuring the protective or therapeutic effect in a second subject.

An "immunogenic agent" or "immunogen" is capable of inducing an immunological response against itself when administered to a mammal, optionally in combination with an adjuvant.

The terms "individual", "host", "subject" and "patient" are used interchangeably in this application and refer to mammals, including but not limited to humans, rodents such as mice and rats, and other laboratory animals.

As used herein, the term “polypeptide” refers to a chain of amino acids of any length, regardless of modification (eg, phosphorylation or glycosylation). The term polypeptide includes proteins and their fragments. Polypeptides may be "exogenous", meaning that they are "heterologous", i.e. foreign to the host cell being used, such as a human polypeptide produced by a bacterial cell. Polypeptides are described herein as sequences of amino acid residues. These sequences are written from left to right in the direction from the amino acid end to the carboxyl end. According to standard nomenclature, amino acid sequences of residues are designated by either a three-letter or one-letter code as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys , C), glutamine (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (lie, I), leucine (Leu, L), lysine (Lys , K), methionine (Met, M), phenylalanine (PHE, F), proline (Pro, P), serine (Ser, S), threonine (THR, T), tryptophan (TRP, W), tyrosine (Tyr, Y) and valine (Val, V).

As used herein, the term “variant” refers to a polypeptide or polynucleotide that differs from the reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. In general, the differences are limited in such a way that the sequences of the reference polypeptide and the variant are generally similar and identical in many areas. The amino acid sequence of the variant and the reference polypeptide may differ by one or more modifications (eg, substitutions, additions and/or deletions). The substituted or inserted amino acid residue may or may not be encoded by the genetic code. A variant of a polypeptide may occur naturally, such as an allelic variant; or it may be a variant that is not known to occur in nature.

Modifications and changes may be made to the structure of the polypeptides of the present invention, but the resulting molecule will have similar characteristics to the polypeptide (eg, conservative amino acid substitution). For example, some amino acids can be replaced by other amino acids in a specific sequence without noticeable loss of activity. Since it is the ability to interact and the nature of the polypeptide that determine the biological functional activity of this polypeptide, it is possible to make certain substitutions in the amino acid sequences of the polypeptide and, nevertheless, obtain a polypeptide with similar properties.

When making such changes, the hydropathic amino acid index can be taken into account. In general, the art understands the importance of the hydropathic amino acid index in imparting a biological interaction function to a polypeptide. It is known that some amino acids can be replaced by other amino acids having a similar hydropathic index or index, but this will result in a polypeptide with similar biological activity. Each amino acid was assigned a hydropathic index based on its hydrophobicity and charge characteristics. The following are the hydropathic amino acid indices: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (- 1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5), asparagine (-3.5), lysine (-3.9) and arginine (-4.5).

The relative hydropathic nature of an amino acid is thought to determine the secondary structure of the resulting polypeptide, which in turn determines the interaction of that polypeptide with other molecules such as enzymes, substrates, receptors, antibodies, antigens, and co-factors. It is known in the art that an amino acid can be replaced with another amino acid having a similar hydropathic index and still produce a functionally equivalent polypeptide. With such changes, it is preferable to replace amino acids whose hydropathic indices are within ± 2, amino acids with a hydropathic index within ± 1 are especially preferred, and the most preferred are amino acids whose hydropathic index is within ± 0.5.

Substitution of such amino acids can also be made on the basis of hydrophilicity, especially in cases where the biological functional equivalent of the polypeptide or peptide thus created is intended for use in immunological embodiments of the present invention. The amino acid residues were assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 ± 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be replaced by another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent and, in particular, an immunologically equivalent polypeptide. With such changes, it is preferable to replace amino acids whose hydrophilicity values are within ± 2, amino acids whose hydrophilicity values are within ± 1 are especially preferred, and the most preferred are amino acids whose hydropathic index is within ± 0.5.

As noted above, amino acid substitutions are generally based on the relative similarity of the side chain amino acid substituents, such as their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into account the various above characteristics are well known to those skilled in the art and include (parent residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp : Glu, Cys, Ser), (Gin : Asn), (Glu : Asp), (Gly : Ala), (His : Asn, Gin), (He : Leu, Val), (Leu : Ile, Val ), (Lys : Arg), (Met : Leu, Tyr), (Ser : Thr), (Thr : Ser), (Trp : Tyr), (Tyr : Trp, Phe) and (Val : Ile, Leu). Embodiments of the present invention thus provide functional or biological equivalents of the polypeptide as set forth above. In particular, embodiments of the polypeptides may include variants that are approximately 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identical to the sequence of the polypeptide, of interest.

The term "percentage (%) sequence identity" is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to nucleotides or amino acids in a reference nucleic acid sequence, after sequence alignment and the introduction of gaps, if necessary, to achieve the maximum percentage sequence identity. Alignment to determine percent sequence identity can be achieved in a variety of ways within the ordinary skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign software (DNASTAR). Appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared, can be determined by known methods.

In this context, the percentage sequence identity of a given nucleotide or amino acid sequence C with a given nucleic acid sequence D (which can alternatively be stated as a given sequence C that has or contains a certain percentage sequence identity with a given sequence D) is calculated as follows:

100 x W/Z,

where W is the number of nucleotides or amino acids counted by the sequence alignment program as identical matches in this program's C and D alignment;

Z is the total number of nucleotides or amino acids in D. It should be borne in mind that where the length of the sequence C is not equal to the length of the sequence D, the percent sequence identity of C to D will not be equal to the percent sequence identity of D to C.

As used herein, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier such as phosphate buffered saline, water, and emulsions such as oil-in-water emulsion or water-in-oil emulsion, as well as various types of wetting agents.

II. Compositions

High levels of B7-H4 expression found in numerous tumor tissues, such as human ovarian cancer, indicate a key role for B7-H4 in mediating immune suppression. In addition, tumor-associated macrophages (TAMs) expressing B7-H4 have been found to suppress tumor-associated antigen-specific T-cell immunity (Kryczek, I. et al. (2006) “B7-H4 Expression Identifies A Novel Suppressive Macrophage Population in Human Ovarian Carcinoma"("B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma") J. Exp. Med. 203(4):871-881). The intensity of B7-H4 expression in MAO significantly correlates with the number of regulatory T cells in the tumor. Moreover, B7-H4 expressed on MAO is associated with poor patient outcome (Kryczek, I. et al. (2006) “B7-H4 Expression Identifies a Novel Suppressive Macrophage Population in Human Ovarian Carcinoma.” identifies a novel suppressive macrophage population in human ovarian carcinoma,” J. Exp. Med. 203(4):871–881). In addition, B7-H4 can be expressed on myeloid-derived suppressor cells (MDSCs), where it may have an immunosuppressive effect during viral infection (Garg, A et al. (2017) "Human Immunodeficiency Virus Type-1 Myeloid Derived Suppressor Cells Inhibit Cytomegalovirus Inflammation through Interleukin-27 and B7-H4"("Human immunodeficiency virus type 1 myeloid-derived suppressor cells inhibit cytomegalovirus inflammation through interleukin-27 and B7-H4") Sci. Rep. Mar 24; 7: 44485). Additionally, in a study of uterine cancer, B7-H4 expression in the tumor microenvironment was associated with increased BMSC infiltration (Vanderstraeten, A. et al. (2014), “Mapping the immunosuppressive environment in uterine tumors: implications for immunotherapy.” environment in uterine tumors: implications for immunotherapy", Cancer Immunol. Immunother. Jun; 63(6):545-57). Thus, B7-H4 may be expressed on tumor cells, MAOs, and/or BMSCs, where it may induce immunosuppressive signaling in cancer.

Neutrophils are a major component of the host's innate defense against infection and also contribute to the development of autoimmune diseases and chronic inflammation. During infection, neutrophils quickly migrate to sites of inflammation, are activated and trigger a cascade of protective mechanisms, including phagocytosis, destruction and degeneration of microorganisms by antimicrobial and proteolytic proteins, as well as the formation of reactive oxygen species. Neutrophils are also involved in tissue destruction, remodeling, wound healing, and modulation of other inflammatory and adaptive immune components. Due to the short lifespan of neutrophils, their numbers must be constantly replenished during infection and inflammation by increasing their production from myeloid progenitor cells in the bone marrow. B7-H4 inhibits in vitro the growth of bone marrow-derived neutrophil progenitor cells, indicating an inhibitory function of B7-H4 in neutrophil formation (Zhu, G. et al ., Blood, 113: 1759-1767 (2009)). Thus, compositions are provided that modulate B7-H4 signal transduction.

Sequences B7-H4

In one embodiment of the present invention, B7-H4 polypeptides are provided. These polypeptides may include the full-length B7-H4 amino acid sequence or a fragment or variant thereof, or a fusion protein thereof.

In one embodiment of the present invention, human B7-H4 proteins or polypeptides thereof are provided. The sequences of the human B7-H4 protein are known in the art. For example, the consensus sequence of the B7-H4 protein is:

1 MASLGQILFW SIISIIIILA GAIALIIGFG ISGRHSITVT TVASAGNIGE DGILSCTFEP

61 DIKLSDIVIQ WLKEGVLGLV HEFKEGKDEL SEQDEMFRGR TAVFADQVIV GNASLRLKNV

121 QLTDAGTYKC YIITSKGKGN ANLEYKTGAF SMPEWVDYN ASSETLRCEA PRWFPQPTW

181 WASQVDQGAN FSEVSNTSFE LNSENVTMKV VSVLYNVTIN NTYSCMIEND IAKATGDIKV

241 TESEIKRRSH LQLLNSKASL CVSSFFAISW ALLPLSPYLM LK

(SEQ ID NO:l, Q7Z7D3 (VTCN1_HUMAN)), wherein amino acids 1-27 are the signal sequence (underlined in accordance with US Patent Publication No. 2016/0039905, which is incorporated by reference in its entirety). The work of Sica et al. it is stated that amino acids 1-20 constitute the signal sequence. The UniProtKB protein sequence database accession number Q7Z7D3 indicates that the extracellular domain includes amino acids 25-259.

In another embodiment of the present invention, murine B7-H4 proteins and polypeptides are provided. The sequences of the mouse B7-H4 protein are known in the art. For example, the consensus sequence of the mouse B7-H4 protein is:

MASLGQIIFWSIINIIIILAGAIALIIGFGISGKHFITVTTFTSAGNIGEDGTLSCTFEP DIKLNGIVIQWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVWGNASLRLKNV QLTDAGTYTCYIRTSKGKGNANLEYKTGAFSMPEINVDYNASSESLRCEAPRWFPQPTVA WASQVDQGANFSEVSNT SFELNSENVTMKWSVLYNVTINNTYSCMIENDIAKATGDIKV TDSEVKRRSQLQLLNSGPSPCVFSSAFVAGWALLSLSCCLMLR

(SEQ ID NO:2, Q7TSP5 (VTCN1_MOUSE), which is incorporated by reference in its entirety).

1. Sequences of anti-B7-H4 antibodies

a. B1A1 sequences

In one embodiment of the present invention, a murine monoclonal antibody is provided that is produced by hybridoma clone B1A1, contains two light chains and two heavy chains, and specifically binds to B7-H4.

Light chain

In one embodiment of the present invention, there is provided a murine monoclonal antibody or antigen binding fragment thereof that has a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99 in sequence % or 100% identical to the following amino acid sequence:

DIVMTOSHKFMSTSVGDRVSITC KASODVRTAVAW YOOKPGOSPKLLIY STSYRYT GV PDRFTGSGSGTEFTFTIS SVOAEDLAVYYC OOYYVT PLTF GAGTKLELK (SEQ ID NO:3), which specifically binds to B7-H4.

The complementarity determining regions (CDRs) from SEQ ID NO:3 are shown in bold and underlined:

CDR1 KASQDVRTAVA (SEQ ID NO:4);

CDR2 STSYRYT (SEQ ID NO:5); And

CDR3 QQYYVTPLT (SEQ ID NO:6).

In another embodiment, the present invention provides a nucleic acid that encodes a light chain (SEQ ID NO:3). An exemplary light chain encoding nucleic acid (SEQ ID NO:3) is:

GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTC AGTATCACCTGCAAGGCCAGTCAGGATGTGAGAACTGCTGTAGCCTGGTATCAACA GAAACCAGGACAATCTCCTAAACTACTGATTTACTCGACATCCTACCGGTACACTGG AGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGAATTCACTTTCACCATCAG CAGTGTGCAGG CTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATGTTACTCC GCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO:7).

Heavy chain

In one embodiment of the present invention, there is provided a murine monoclonal antibody or antigen binding fragment thereof that has a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99 in sequence % or 100% identical to the following amino acid sequence:

EVOLOOSGTVLARPGASVKMSCKASGYTFT SYWMH WIKORPGOGLEWI GAIYPGNSD TKYNOKFKDKAKLT AVTSASTAYMEL SSLTNEDSAVYYCT ST VRNVMD YW GOGTSV TVSS (SEQ ID NO:8), and specifically binds to B7-H4.

The complementarity determining regions (CDRs) from SEQ ID NO:8 are shown in bold and underlined:

CDR1 SYWMH (SEQ ID NO:9);

CDR2 AIYPGNSDTKYNQKFKDK (SEQ ID NO 10); And

CDR3 TVRNVMDY (SEQ ID NO:11).

In another embodiment of the present invention, a nucleic acid encoding a heavy chain is provided (SEQ ID NO:8). An exemplary heavy chain encoding nucleic acid (SEQ ID NO:8) is:

GAGGTTCAGCTCCAGCAGTCTGGGACTGT GCTGGCAAGGCCTGGGGCTTC AGTGAA GATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTACTGGATGCACTGGATAAA ACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAAATAGTG ATACTAAATACAACCAGAAGTTCAAGGACAAGGCCAAACTGACTGCAGTCACATCT GCCAGCACTGCCTA CATGGAGCTCAGCAGCCTGACAAATGAGGACTCTGCGGTCTA TTACTGTACATCTACGGTACGGAATGTTATGGACTACTGGGGTCAAGGAACCTCAGT CACCGTCTCCTCA (SEQ ID NO:12).

In one embodiment of the present invention, there is provided an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, which contains a light chain containing complementarity determining regions according to the sequences of SEQ ID NOs: 4, 5 and 6, and specifically binds to B7-H4.

In one embodiment of the present invention, there is provided an antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, which contains a heavy chain containing complementarity determining regions according to the sequences of SEQ ID NOs: 9, 10 and 11, and specifically binds to B7-H4.

In one embodiment of the present invention there is provided an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, which comprises a light chain containing complementarity determining regions according to the sequences of SEQ ID NOs: 4, 5 and 6, and a heavy chain, containing complementarity determining regions according to SEQ ID NOs:9, 10 and 11, and specifically binds to B7-H4.

In one embodiment of the present invention there is provided an antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, which contains a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90 in sequence %, 95%, 99%, or 100% identical to the sequence of SEQ ID NO:3, and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95 identical in sequence %, 99% or 100% identical to the sequence of SEQ ID NO:8, and specifically binds to B7-H4.

b. B1h1 sequences

In one embodiment of the present invention, the murine monoclonal antibody is produced by the B1H1 hybridoma clone and contains two light chains and two heavy chains.

Light chain

In one embodiment of the present invention, there is provided a murine monoclonal antibody or antigen binding fragment thereof that contains a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99 in sequence % or 100% identical to the following amino acid sequence:

EIOMTO SP SSMS ASLGDRITITCOATODIVKSLNW YOOKPGKPP SFLIYYTAOLAEGVP S RFSGSGSGSDYSLTI SNLESEDFA YY CL OFYEFPPTF GGGTKLEIK (SEQ ID NO:13).

The complementarity determining regions (CDRs) from SEQ ID NO:13 are shown in bold and underlined:

CDR1 QATQDIVKSLN (SEQ ID NO:14);

CDR2 YTAQLAE (SEQ ID NO:15); And

CDR3 LQFYEFPPT (SEQ ID NO:16).

In another embodiment, the present invention provides a nucleic acid that encodes a light chain (SEQ ID NO:13).

A typical nucleic acid that encodes a light chain (SEQ ID NO:13) is:

GAAATCCAGATGACCCAGTCTCCATCCTCTATGTCTGCATCTCTGGGAGACAGAATA ACCATCACTTGCCAGGCAACTCAAGACATTGTTAAGAGTTTAAACTGGTATCAACAA AAACCAGGGAAACCCCCTTCATTCCTGATCTATTATACAGCTCAACTGGCAGAAGGG GTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGTCAGACTATTCTCTGACAATCAGC AACCTGGAGTCTGAAGA TTTTGCAGACTATTACTGTCTACAGTTTTATGAGTTTCCTC CGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO:17).

In one embodiment of the present invention, there is provided a monoclonal antibody or an antigen binding fragment thereof that contains a light chain constant domain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% in sequence. 99% or 100% identical to the following amino acid sequence:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC (SEQ ID NO:18) and specifically binds to B7-H4.

Humanized light chain

In one embodiment of the present invention, there is provided a monoclonal anti-B7-H4 antibody or an antigen binding fragment thereof that contains a humanized light chain variable domain that is at least 50%, 60%, 70%, 80%, 85% of the same sequence. 90%, 95%, 99% or 100% identical to one of the following amino acid sequences:

Humanized B1H1 VL1:

DIQMTQSPSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFLIYYTAQLAEGVPS RFSGSGSGTDYTLTIS SLQSEDFATYYCLQFYEFPPTFGGGTKVEIK (SEQ ID NO:19)

Humanized B1H1 VL2:

DIQMTQSPSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFLIYYTAQLAEGVPS RFSGSGSGTDYTLTIS SLQPEDFATYYCLQFYEFPPTFGGGTKVEIK (SEQ ID NO:20)

Humanized B1H1 VL3:

DIQMTQSPSSLSASVGDRVTITCQATQDIVKSLNWYQQKPGKAPKFLIYYTAQLAEGVPS RFSGSGSGTDYTLTIS SLQPEDFATYYCLQFYEFPPTFGGGTKVEIK (SEQ ID NO:21)

Humanized B1H1 VL4:

DIQMTQSPSSLSSASVGDRVTITCQATQDIVKSLNWYQQKPGKPPKFLIYYTAQLAEGVPS

RF SSGSGSGTDFTLTISSLQPEDFATYYCLQFYEFPPTFGGGTKVEIK (SEQ ID NO:22)

Humanized B1H1 VL5:

DIQMTQ SPS SL SASVGDRVTITCQ ATQDIVKSLNW YQQKPGKPPKF AIYYTAQLAEGVPS RFSGSGSGTDYTLTIS SLQPEDFATYYCLQFYEFPPTFGGGTKVEIK (SEQ ID NO:23)

In one embodiment of the present invention, there is provided a monoclonal anti-B7-H4 antibody or antigen binding fragment thereof that contains a humanized light chain that is at least 50%, 60%, 70%, 80%, 85%, 90% consistent , 95%, 99% or 100% identical to one of the following amino acid sequences:

full length light chain B1H1 option 1:

SKAD YEKHKVYACEVTHQGL SSPVTK SFNRGEC (SEQ ID NO:24)

full length light chain B1H1 option 2:

SKAD YEKHKVYACEVTHQGL SSPVTK SFNRGEC (SEQ ID NO:25)

full length light chain B1H1 option 3:

SKAD YEKHKVYACEVTHQGL SSPVTK SFNRGEC (SEQ ID NO:26)

full length light chain B1H1 option 4:

SKADY EKHKVYACEVTHQGL SSPVTK SFNRGEC (SEQ ID NO:27)

full length light chain B1H1 option 5:

SKADY EKHKVYACEVTHQGL SSPVTK SFNRGEC (SEQ ID NO:28)

Heavy chain

In one embodiment of the present invention, there is provided a murine monoclonal antibody or antigen binding fragment thereof that contains a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99 in sequence % or 100% identical to the following amino acid sequence:

EVOLOOSGTVLARPGASVKMSCKVSGYPFT SYWMHW VKORPGOGLEWI GAIYPGKS DTEYNPNFKGK AKLTAVTSATTAYMELSSLTNEDSAVYYCTS TWTHYFDY WGOGTTL TVSS (SEQ ID NO:29), and specifically binds to B7-H4.

The complementarity determining regions (CDRs) from SEQ ID NO:29 are shown in bold and underlined:

CDR1 SYWMH (SEQ ID NO:9);

CDR2 AIYPGKSDTEYNPNFKG (SEQ ID NO:30); And

CDR3 TVRNVMDY (SEQ ID NO:11).

In another embodiment of the present invention, a nucleic acid encoding a heavy chain is provided (SEQ ID NO:29). An exemplary heavy chain encoding nucleic acid (SEQ ID NO:29) is:

GAGGTTCAGCTCCAGCAGTCTGGGACTGTTCTGGCAAGGCCTGGGGCTTCAGTGAAG ATGTCCTGCAAGGTTTCTGGCTACCCCTTTACCAGCTACTGGATGCACTGGGTAAAA CAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAAAAAGTGA CACTGAATACAACCCGAACTTCAAGGGCAAGGCCAAACTGACTGCAGTCACATCTG CCACCACTGCCTACAT GGAGCTCAGCAGCCTGACAAATGAGGACTCTGCGGTCTATT ACTGTACAAGTACCTGGACCCACTACTTTGACTACTGGGGCCAAGGCACCACTCTCA CAGTCTCCTCA (SEQ ID NO:31).

In one embodiment of the present invention, there is provided a murine monoclonal antibody or antigen binding fragment thereof that contains a heavy chain constant domain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% consistent in sequence. , 99% or 100% identical to the following amino acid sequence:

ASTKGPSVFPLA SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSL SSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHT CPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVV SVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQ VYTLPPSR DELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:32),

and specifically binds to B7-H4.

The underlined and bolded amino acids represent amino acids that differ from the mutant sequence.

Another embodiment of the present invention provides a mutant heavy chain constant domain that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the following amino acid sequences:

ASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSL SSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHT CPPCPAPELLGG PDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVV SVLTVLHQDWLNGKEYKCKV SNKALPLPEEKTISKAKGQPREPQ VYTLPP SRDELTKNQVSLTCLVKGFYP SDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:33)

The underlined and bolded amino acids represent amino acids that differ from the wild type sequence.

Humanized heavy chain

In one embodiment of the present invention, there is provided a monoclonal anti-B7-H4 antibody or antigen binding fragment thereof that contains a humanized heavy chain variable domain that is at least 50%, 60%, 70%, 80%, 85% the same in sequence. 90%, 95%, 99% or 100% identical to one of the following amino acid sequences:

Humanized B1H1 VH1:

EVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWIGAIYPGKSD TEYAPKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCTSTWTHYFDYWGQGTTVTV SS (SEQ ID NO:34)

Humanized B1H1 VH2:

VSS (SEQ ID NO:35)

Humanized B1H1 VH3:

VSS (SEQ ID NO:36)

Humanized B1H1 VH4:

VSS (SEQ ID NO:37)

In one embodiment of the present invention, there is provided a monoclonal anti-B7-H4 antibody or antigen binding fragment thereof that contains a humanized heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90% identical in sequence. , 95%, 99% or 100% identical to one of the following amino acid sequences:

Heavy chain B1H1 option 1:

EVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWIGAIYPGKSD TEYAPKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCTSTWTHYFDYWGQGTTVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPS SSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQ VYTLPP SRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:38)

Heavy chain B1H1 option 2:

QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWMGAIYPGKS DTEYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCTSTWTHYFDYWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSL SSWT VPSSSL GTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQ VYTLP PSRDELTKNQVSLTCLV KGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:39)

Heavy chain B1H1 option 3:

QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWMGAIYPGKS DTEYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTATYYCTSTWTHYFDYWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSL SSWT VPSSSLG TQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQ VYTLP PSRDELTKNQVSLTCLVK GFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:40)

Heavy chain B1H1 option 4:

QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYYMHWVRQAPGQGLEWMGAIYPGKS DTEYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTATYYCTSTWTHYFDYWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSL SSWT VPSSSLG TQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQ VYTLP PSRDELTKNQVSLTCLVK GFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:41)

In another embodiment of the present invention, the monoclonal antibody or antigen-binding fragment thereof comprises a humanized heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100 in sequence % identical to one of the following amino acid sequences:

B1H1 Heavy Chain Mutant 1:

EVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMHWVRQAPGQGLEWIGAIYPGKSD TEYAPKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCTSTWTHYFDYWGQGTTVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPS SSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPDVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYN STYRVVSVLTVLHQDWLNGKEYKCKV SNKALPLPEEKTISKAKGQPREPQ VYTL PPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:42);

B1H1 heavy chain mutant variant 2:

QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMFIWVRQAPGQGLEWMGAIYPGKS DTEYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTAVYYCTSTWmYFDYWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSL SSWT VPSS SLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPEL LGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVV SVLTVLHQDWLNGKEYKCKV SNKALPLPEEKTISKAKGQPREPQ VYT LPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:43);

B1H1 Heavy Chain Mutant 3:

QVQLVQSGAEVKKPGASVKVSCKVSGYPFTSYWMFIWVRQAPGQGLEWMGAIYPGKS DTEYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTATYYCTSTWTHYFDYWGQGTTYT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSL SSWT VPSS SLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPEL LGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVV SVLTVLHQDWLNGKEYKCKV SNKALPLPEEKTISKAKGQPREPQ VYT LPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:44); And

B1H1 heavy chain mutant variant 4:

QVQLVQSGAEVKKPGASVKVSCKVSGVPFTSYYMHWVRQAPGQGLEWMGAIYPGKS DTEYAQKFQGRVTLTADTSTSTAYMELSSLRSEDTATYYCTSTWTHYFDYWGQGTTVT

SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVL QSSGLYSL SSWT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPEL LGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYR VV SVLTVLHQDWLNGKEYKCKV SNKALPLPEEKTISKAKGQPREPQ VYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:45).

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, which comprises a light chain having complementarity determining regions (CDRs) according to the sequences of SEQ ID NOs: 14, 15 and 16.

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, which comprises a heavy chain containing complementarity determining regions (CDRs) according to the sequences of SEQ ID NOs:9, 11 and thirty.

In one embodiment, the present invention provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, which comprises a heavy chain having complementarity determining regions (CDRs) according to the sequences of SEQ ID NOs: 9, 11 and thirty.

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, comprising a light chain of at least 50%, 60%, 70%, 80%, 85% sequence , 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO:13, and a heavy chain which is at least 50%, 60%, 70%, 80%, 85%, 90% identical in sequence, 95%, 99% or 100% identical to the sequence of SEQ ID NO:29.

In another embodiment, the present invention provides an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, comprising a light chain variable domain whose sequence is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to any of the sequences of SEQ ID NO: 19, 20, 21, 22 or 23, as well as a heavy chain variable domain that is at least 50% identical in sequence 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to any of SEQ ID NO:34, 35, 36 or 37.

In one embodiment, the present invention provides an anti-B7H4 antibody or antigen binding fragment thereof comprising a light chain having an amino acid sequence corresponding to any of SEQ ID NO: 24, 25, 26, 27 or 28, and a heavy chain having an amino acid sequence corresponding to any from SEQ ID NO:38, 39, 40 or 41.

In another embodiment, the present invention provides an anti-B7H4 antibody or antigen binding fragment thereof comprising a light chain whose amino acid sequence corresponds to any of SEQ ID NOs: 24, 25, 26, 27 or 28, and a heavy chain whose amino acid sequence corresponds to any from SEQ ID NO:42, 43, 44 or 45.

In one embodiment, the present invention provides an anti-B7H4 antibody or antigen binding fragment thereof comprising two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of the sequences SEQ ID NO:24, 25, 26, 27 or 28, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO:24, 25, 26, 27 or 28, and the two heavy chains include a polypeptide selected from the group consisting of the sequences of SEQ ID NO: 38, 39, 40 or 41, or a variant thereof, the sequence of which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more identical to the sequences of SEQ ID NO: 38, 39, 40 or 41, and wherein the antibody or antigen binding fragment thereof binds to B7-H4.

In one embodiment, the present invention provides an antibody or antigen binding fragment thereof that contains two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of the sequences SEQ ID NO:24, 25, 26, 27 or 28 , or a variant thereof, the sequence of which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more identical to the sequences of SEQ ID NO:24, 25, 26, 27 or 28, and the two heavy chains include a polypeptide selected from the group consisting of the sequences of SEQ ID NO: 42, 43, 44 or 45, or a variant thereof, the sequence of which is at least 50%, 60%, 70% , 80%, 85%, 90%, 95%, 99% or more identical to the sequences of SEQ ID NO:42, 43, 44 or 45, and wherein the antibody or antigen binding fragment thereof binds to B7-H4.

B1H3 sequences

In one embodiment of the present invention, the mouse monoclonal antibody is produced by the hybridoma clone B1H3 and contains two light chains and two heavy chains, and specifically binds to B7-H4.

Light chain

In one embodiment, the present invention provides a murine monoclonal anti-B7-H4 antibody or antigen binding fragment thereof that contains a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90% consistent , 95%, 99% or 100% identical to the following amino acid sequence:

EIOMTOSPSSMSASLGDTITITC OATODIVKSLN WYOOKPGKPPSFLI YYTTOLAE GVPS R SSGSGSGSDYSLTISNLDSEDF A YYCLOFYEFPPTFGGGTKLEIK (SEQ ID NO:46)

The complementarity determining regions (CDRs) from SEQ ID NO:46 are shown in bold and underlined:

CDR1 QATQDIVKSLN (SEQ ID NO:14);

CDR2 YYTTQLAE (SEQ ID NO:47); And

CDR3 LQFYEFPPT (SEQ ID NO:16).

In another embodiment, the present invention provides a nucleic acid that encodes a light chain (SEQ ID NO:46).

An exemplary light chain encoding nucleic acid (SEQ ID NO:46) is:

GAAATCCAGATGACCCAGTCTCCATCCTCTATGTCTGCATCTCTGGGAGACACAATA ACCATCACTTGCCAGGCAACTCAAGACATTGTTAAGAGTTTAAACTGGTATCAACAA AAACCAGGGAAACCCCCTTCATTCCTGATCTATTATACAACTCAACTGGCAGAAGGG GTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGTCAGACTATTCTCTGACAATCAGC AACCTGGACTCTGAAGATTT TGCAGACTATTACTGTCTACAGTTTTATGAGTTTCCTC CGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO:48).

Heavy chain

In one embodiment of the present invention, there is provided a murine monoclonal anti-B7-H4 antibody or antigen binding fragment thereof that contains a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90% identical in sequence. , 95%, 99% or 100% identical to the following amino acid sequence:

EVOLOOSGTVLARPGASVKMSCKASGYTFS SYWMH WVKORPGOGLEWI GAIYPGKS DTSYNOKFOG KAKLTAVTSASTAEMELTSLTNEDSAVYYCTS TWTHYFDY WGOGTTL TVSS (SEQ ID NO:49).

The complementarity determining regions (CDRs) from SEQ ID NO:49 are shown in bold and underlined:

CDR1 SYWMH (SEQ ID NO:9);

CDR2 AIYPGKSDTEYNPNFKG (SEQ ID NO:50); And

CDR3 TVRNVMDY (SEQ ID NO:11).

In another embodiment of the present invention, a nucleic acid encoding a heavy chain is provided (SEQ ID NO:49).

An exemplary heavy chain encoding nucleic acid (SEQ ID NO:49) is:

GAGGTTCAGCTCCAGCAGTCTGGGACTGTTCTGGCAAGGCCTGGGGCTTCAGTGAAG ATGTCCTGCAAGGCTTCTGGCTACACCTTTTCCAGCTACTGGATGCACTGGGTAAAA CAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAAAAAGTGA TACTAGCTACAACCAGAAGTTCCAGGGCAAGGCCAAACTGACTGCAGTCACATCTG CCAGCACTGCC TTCATGGAGCTCACCAGCCTGACAAATGAGGACTCTGCGGTCTATT ACTGTACAAGTACCTGGACCCACTACTTTGACTACTGGGGCCAAGGCACCACTCTCA CAGTCTCCTCA (SEQ ID NO:51).

In one embodiment, the present invention provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, which comprises a light chain having complementarity determining regions according to the sequences of SEQ ID NOs: 14, 16 and 47.

In one embodiment, the present invention provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, comprising a heavy chain having complementarity determining regions according to the sequences of SEQ ID NOs: 9, 11 and 50.

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, which contains a light chain having complementarity determining regions (CDRs) according to the sequences SEQ ID NO: 14, 16, and 47, and a heavy chain containing CDRs according to the sequences of SEQ ID NOs: 9, 11, and 50.

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, comprising a light chain of at least 50%, 60%, 70%, 80%, 85% sequence , 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO:46, and a heavy chain which is at least 50%, 60%, 70%, 80%, 85%, 90% identical in sequence, 95%, 99% or 100% identical to the sequence of SEQ ID NO:49.

B1H10 sequences

In one embodiment of the present invention, the mouse monoclonal anti-b7h4 antibody is produced by hybridoma clone B1H10 and contains two light chains and two heavy chains.

Light chain

In one embodiment of the present invention, there is provided a murine monoclonal anti-B7H4 antibody or antigen binding fragment thereof that contains a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95 in sequence %, 99% or 100% identical to the following amino acid sequence:

DIOMTOTT SSL S ASLGDRVTISCRASODISNYLNW YOOKPDGTIKLLIY YTSRLHS GVPS RFSGSGSGSDYSLTISNLEOEDIATYEC OOGNTLPWT FGGGTKLEFK (SEQ ID NO:52).

The complementarity determining regions (CDRs) from SEQ ID NO:52 are shown in bold and underlined:

CDR1 RASQDISNYLN (SEQ ID NO:53);

CDR2 YTSRLHS (SEQ ID NO:54); And

CDR3 QQGNTLPWT (SEQ ID NO 55).

In another embodiment, the present invention provides a nucleic acid that encodes a light chain (SEQ ID NO:52).

An exemplary light chain encoding nucleic acid (SEQ ID NO:52) is:

GATATCCAGATGACACAAACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTC ACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAG AAACCAGATGGAACTATTAAACTCCTGATCTATTACACATCAAGATTACATTCAGGA GTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGATCAGATTATTCTCTCACCATTAGC AACCTGGAGCAAGAAG ATATTGCCACTTACTTTTTGCCAACAGGGTAATACGCTTCCG TGGACGTTCGGTGGAGGCACCAAGCTGGAATTCAAA (SEQ IDNO: 56).

In one embodiment of the present invention, there is provided an anti-B7H4 monoclonal antibody or antigen binding fragment thereof that contains a light chain constant domain that is at least 50%, 60%, 70%, 80%, 85%, 90% the same in sequence. 95%, 99% or 100% identical to the following amino acid sequence:

SKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:57)

Humanized light chain B1H10

In another embodiment of the present invention, the anti-B7H4 monoclonal antibody or antigen binding fragment thereof comprises a humanized light chain variable domain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% consistent. , 99% or 100% identical to one of the following amino acid sequences:

Humanized B1H10 VL1:

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKTIKLLIYYTSRLHSGVPSR FSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKLEIK (SEQ ID NO:58)

Humanized B1H10 VL2:

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPS RFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKLEIK (SEQ ID NO:59)

Humanized B1H10 VL3:

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPS RFSGSGSGTDYTLTISSLQPEDFATYFCQQGQTLPWTFGQGTKLEIK (SEQ ID NO:60)

Humanized B1H10 VL4:

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPS RFSGSGSGTDYTLTISSLQPEDFATYFCQQGSTLPWTFGQGTKLEIK (SEQ ID NO:61)

In one embodiment of the present invention, there is provided a monoclonal anti-B7H4 antibody or antigen binding fragment thereof that contains a humanized light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95 in sequence %, 99% or 100% identical to one of the following amino acid sequences:

Humanized B1H10 Light Chain Option 1:

TLSKADY EKHKVYACEVTHQGL SSPVTK SFNRGEC (SEQ ID NO:62)

Humanized B1H10 Light Chain Option 2:

TLSKAD YEKHKVYACEVTHQGL SSPVTK SFNRGEC (SEQ ID NO:63)

Humanized B1H10 Light Chain Option 3:

SKAD YEKHKVYACEVTHQGL SSPVTK SFNRGEC (SEQ ID NO:64)

Heavy chain

In one embodiment, the present invention provides a murine monoclonal anti-B7H4 antibody or antigen binding fragment thereof comprising a heavy chain of at least 50%, 60%, 70%, 80%, 85%, 90%, 95% sequence , 99% or 100% identical to the following amino acid sequence:

EVOLOOSGPELVKPGASVKMSCKASGYTFT DYYMN WVROSHGKSLEWI GRVNPSNGG TNYNOKFKG KATLTVDKSLSTAYMOLSSLTSEDSAVYYCA RRHNYADF WGOGTTLTV SS (SEQ ID NO:65).

The complementarity determining regions (CDRs) from SEQ ID NO:65 are shown in bold and underlined:

CDR1 DYYMN (SEQ ID NO:66);

CDR2 RVNPSNGGTNYNQKFKG (SEQ ID NO:67); And

CDR3 RHNYADF (SEQ ID NO:68).

In another embodiment of the present invention, a nucleic acid encoding a heavy chain is provided (SEQ ID NO:65). An exemplary heavy chain encoding nucleic acid (SEQ ID NO:65) is:

GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAA GATGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACTACATGAACTGGGTGAG GCAGAGTCATGGAAAGAGCCTTGAGTGGATTGGACGTGTTAATCCTAGCAATGGTG GTACTAACTACAACCAGAAATTCAAGGGCAAGGCCACATTGACAGTAGACAAATCC CTCAGCAAGCCTACAT GCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTAT TACTGTGCAAGACGACATAACTACGCAGACTTCTGGGGCCAAGGCACCACTCTCAC AGTCTCCTCA (SEQ ID NO:69).

Humanized heavy chain B1H10

In one embodiment, the present invention provides a monoclonal anti-B7H4 antibody or antigen binding fragment thereof that contains a humanized variant of a heavy chain variable domain that is at least 50%, 60%, 70%, 80%, 85%, 90 in sequence %, 95%, 99% or 100% identical to one of the following amino acid sequences:

Humanized B1H10VH1:

EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWIGRVNP SN GG TNYAQKFQGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVTV SS (SEQ ID NO:70)

Humanized B1H10VH2:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPAN GGTNYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTV TVSS (SEQ ID NO:71)

Humanized B1H10VH3:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSSG GTNYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVT VSS (SEQ ID NO:72)

Humanized B1H10VH4:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSNG GTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVT VSS (SEQ ID NO:73)

Humanized B1H10VH5:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSSG GTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVT VSS (SEQ ID NO:74)

In one embodiment, the present invention provides an anti-B7H4 monoclonal antibody or antigen-binding fragment thereof that contains a humanized heavy chain variant that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to one of the following amino acid sequences:

Humanized B1H10 Heavy Chain Option 1:

EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWIGRVNPSNGG TNYAQKFQGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:75)

Humanized B1H10 Heavy Chain Option 2:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPAN GGTNYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSWTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:76)

Humanized B1H10 Heavy Chain Option 3:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSSG GTNYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSWTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:77)

Humanized B1H10 Heavy Chain Option 4:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSNG

GTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVT

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSNG GTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSWTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:78)

Humanized B1H10 Heavy Chain Variant 5:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSSG GTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSWTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:79)

In one embodiment of the present invention, there is provided an anti-B7H4 monoclonal antibody or antigen binding fragment thereof that comprises a humanized mutant heavy chain variant that is at least 50%, 60%, 70%, 80%, 85%, 90% identical in sequence. , 95%, 99% or 100% identical to one of the following amino acid sequences:

Humanized mutant heavy chain B1H10 variant 1:

EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWIGRVNPSNGG TNYAQKFQGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPDVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:80)

Humanized mutant heavy chain B1H10 variant 2:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPAN GGTNYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSWTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP

(SEQ ID NO:81)

Humanized mutant heavy chain B1H10 variant 3:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSSG GTNYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSWTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:82)

Humanized mutant heavy chain B1H10 variant 4:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSNG GTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSWTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:83)

Humanized mutant heavy chain B1H10 variant 5:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRVNPSSG GTNYAQKFQGRVTLTVDTSKSTAYMELSSLRSEDTAVYYCARRHNYADFWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSWTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:84)

In one embodiment, the present invention provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, which comprises a light chain having complementarity determining regions according to the sequences of SEQ ID NO:53, 54 or 55.

In one embodiment, the present invention provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, comprising a heavy chain having complementarity determining regions according to the sequences of SEQ ID NO:66, 67 or 68.

In one embodiment of the present invention there is provided an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, which comprises a light chain having complementarity determining regions (CDRs) according to the sequences of SEQ ID NO:53, 54 or 55, and a heavy chain containing (CDR) according to the sequences of SEQ ID NO:66, 67 or 68.

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, comprising a light chain of at least 50%, 60%, 70%, 80%, 85% sequence , 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO:52, and a heavy chain which is at least 50%, 60%, 70%, 80%, 85%, 90% identical in sequence, 95%, 99% or 100% identical to the sequence of SEQ ID NO:65.

In another embodiment, the present invention provides an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, comprising a light chain variable domain whose sequence is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% identical to any of SEQ ID NO:58, 59, 60, or 61, and a heavy chain variable domain that is at least 50%, 60% identical in sequence, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to any of SEQ ID NO:70, 71, 72, 73 or 74.

In one embodiment, the present invention provides an anti-B7H4 antibody or antigen binding fragment thereof comprising a light chain having an amino acid sequence corresponding to any of SEQ ID NO: 62, 63, or 64 and a heavy chain having an amino acid sequence corresponding to any of SEQ ID NO: 62, 63, or 64. ID NO:75, 76, 77, 78 or 79.

In another embodiment, the present invention provides an anti-B7H4 antibody or antigen binding fragment thereof comprising a light chain having an amino acid sequence corresponding to any of SEQ ID NO:62, 63 or 64 and a heavy chain having an amino acid sequence corresponding to any of SEQ ID NO:62, 63 or 64 NO:80, 81, 82, 83 or 84.

In one embodiment, the present invention provides an anti-B7H4 antibody or antigen binding fragment thereof comprising two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of the sequences SEQ ID NO:62, 63 or 64, or a variant thereof which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identical to the sequences of SEQ ID NO:62, 63 or 64, and two heavy chains include a polypeptide selected from the group consisting of the sequences of SEQ ID NO: 75, 76, 77, 78 or 79, or a variant thereof, the sequence of which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more identical to SEQ ID NO:75, 76, 77, 78 or 79.

In another embodiment, the present invention provides an anti-B7H4 antibody or antigen binding fragment thereof comprising two light chains and two heavy chains, wherein the two light chains comprise a polypeptide selected from the group consisting of the sequences SEQ ID NO:62, 63 or 64, or a variant thereof which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identical to the sequences of SEQ ID NO:62, 63 or 64, and two heavy chains include a polypeptide selected from the group consisting of the sequences of SEQ ID NO:80, 81, 82, 83 or 84, or a variant thereof, the sequence of which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more identical to SEQ ID NO:80, 81, 82, 83 or 84.

B2E6 sequences

In one embodiment of the present invention, the mouse monoclonal anti-B7H4 antibody is produced by the hybridoma clone B2E6 and contains two light chains and two heavy chains.

Light chain

In one embodiment of the present invention, there is provided a murine monoclonal anti-B7H4 antibody or antigen binding fragment thereof that contains a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95 in sequence %, 99% or 100% identical to the following amino acid sequence:

ETYMTOSHKIMSTSVGDRYTIT CKASODVRTAVAW YOOKPGO SPKLLIS S ASY OYT GV

PDRFTGSGSGTDFTFTIS SLOAEDLAVYY CHOYYNTPLTF GAGTKLELR (SEQ ID NO:85).

The complementarity determining regions (CDRs) from SEQ ID NO:85 are shown in bold and underlined:

CDR1 KASQDVRTAVA (SEQ ID NO:4);

CDR2 SASYQYT (SEQ ID NO:86); And

CDR3 HQYYNTPLT (SEQ ID NO:87).

In another embodiment, the present invention provides a nucleic acid that encodes a light chain (SEQ ID NO:85).

An exemplary light chain encoding nucleic acid (SEQ ID NO:85) is:

GAAACTGTGATGACCCAGTCTCACAAAATCATGTCCACTTCAGTAGGAGACAGGGT CACCATCACCTGCAAGGCCAGTCAGGATGTGAGAACTGCTGTGGCCTGGTATCAAC AGAAACCAGGACAATCTCCTAAATTACTAATTTCCTCGGCATCCTACCAATACACTG GAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCA GCAGTTTGCA GGCTGAAGACCTGGCAGTTTATTACTGTCATCAGTATTATAATACTC CCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAGA (SEQ ID NO:88).

ii. Heavy chain

In one embodiment of the present invention, there is provided a murine monoclonal anti-B7H4 antibody or antigen binding fragment thereof that contains a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95 in sequence %, 99% or 100% identical to the following amino acid sequence:

EVOLOOSGTVLARPGASVKMSCKASGYTFT SYWMH WVKORPGOGLEWI GAIYPGKS DTTYNOKFEG KAKLTAVTSDSTAYMDLSSLTNEDSAVYYCTS SVRNAMDY WGOGTS VTVSS (SEQ ID NO:89).

The complementarity determining regions (CDRs) from SEQ ID NO:89 are shown in bold and underlined:

CDR1 SYWMH (SEQ ID NO:9);

CDR2 AIYPGKSDTTYNQKFEG (SEQ ID NO:90); And

CDR3 SVRNAMDY (SEQ ID NO:91).

In another embodiment, the present invention provides a nucleic acid that encodes a heavy chain (SEQ ID NO:89). An exemplary nucleic acid encoding a heavy chain (SEQ ID NO:89) is:

GAGGTTCAGCTCCAGCAGTCTGGGACTGT GCTGGCAAGGCCTGGGGCTTC AGTGAA GATGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTAAA ACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAAAAAGTG ATACTACCTACAACCAGAAGTTCGAGGGCAAGGCCAACTGACTGCAGTCACATCT GACAGCACAG CCTACATGGATTCTCAGTAGCCTGACAAATGAGGACTCTGCGGTCTAT TACTGTACATCTTCGGTTCGGAATGCTATGGACTACTGGGGTCAAGGAACCTCAGTC ACCGTCTCCTCA (SEQ ID NO:92).

In one embodiment, the present invention provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, which comprises a light chain having complementarity determining regions according to the sequences of SEQ ID Nos: 4, 86 and 87.

In one embodiment, the present invention provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, comprising a heavy chain having complementarity determining regions according to the sequences of SEQ ID NOs: 9, 90 and 91.

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, which comprises a light chain having complementarity determining regions according to the sequences of SEQ ID NOs: 4, 86, and 87, and a heavy chain having complementarity determining regions according to the sequences of SEQ ID NO:9, 90, and 91.

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, the sequence of which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO:85, and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99 identical in sequence % or 100% identical to the sequence of SEQ ID NO:89.

B4B3 sequences

In one embodiment of the present invention, the mouse monoclonal anti-B7H4 antibody is produced by the B4B3 hybridoma clone and contains two light chains and two heavy chains.

i. Light chain

In one embodiment of the present invention, there is provided a murine monoclonal anti-B7H4 antibody or antigen binding fragment thereof that contains a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95 in sequence %, 99% or 100% identical to the following amino acid sequence:

DVLMTOTPLSLPVSLGGOASISC RSSOIIVHSNGNTYLEW YLOKPGOSPKLLI YKVSNR FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FOGSHVPWT FGGGTKLEIK (SEQ ID NO:93)

The complementarity determining regions (CDRs) from SEQ ID NO:93 are shown in bold and underlined:

CDR1 RSSQIIVHSNGNTYLE (SEQ ID NO:94);

CDR2 KVSNRFS (SEQ ID NO:95); And

CDR3 FQGSHVPWT (SEQ ID NO:96).

In another embodiment, the present invention provides a nucleic acid that encodes a light chain (SEQ ID NO:93).

An exemplary light chain encoding nucleic acid (SEQ ID NO:93) is:

GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGGTCAAGCCT CCATCTCTTGCAGATCTAGTCAGATCATTGTACATAGTAATGGAAACACCTATTTAG AATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCA ACCGATTTTCTGGGGTCCCAGACAGTTCAGTGGCAGTGGATCAGGGACAGATTTCA CACTCAAGAT CAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAG GTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAAGCTGGAAATCAAA (SEQ ID NO: 97).

ii. Heavy chain

In one embodiment of the present invention, there is provided a murine monoclonal anti-B7H4 antibody or antigen binding fragment thereof that contains a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95 in sequence %, 99% or 100% identical to the following amino acid sequence:

OVOLOOPGAELVKPGASVKLSCKASGYTFI SYWMHW VKORPGOGLEWI GEIDPSDSY TYYNOKFKGK ATLTVDKSSSTAYMOL SSLTSEP S AVYYCARRKT WDW YFD VW GAG TTVTVSS (SEQ ID NO:98).

The complementarity determining regions (CDRs) from SEQ ID NO:98 are shown in bold and underlined:

CDR1 SYWMH (SEQ ID NO:9);

CDR2 EIDPSDSYTYYNQKFKG (SEQ ID NO:99); And

CDR3 RKTWDWYFDV (SEQ ID NO:100).

In another embodiment, the present invention provides a nucleic acid that encodes a heavy chain (SEQ ID NO:98). An exemplary nucleic acid encoding a heavy chain (SEQ ID NO:98) is:

CAGGTCCAGCTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAA GCTGTCCTGCAAGGCTTCTGGATACACCTTCATTAGCTACTGGATGCACTGGGTGAA GCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGAGATTGATCCTTCTGATAGTT ATACTTACTACAATCAAAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAATCCT CCAGCACAGCCTACATG CAACTCAGCAGCTGACATCTGAGGACTCTGCGGTCTATT ACTGTGCAAGAAGGAAAACCTGGGACTGGTACTTCGATGTCTGGGGCGCAGGGACC ACGGTCACCGTCTCCTCA (SEQ ID NO:101).

In one embodiment, the present invention provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, which comprises a light chain having complementarity determining regions according to the sequences of SEQ ID NOs:94, 95 and 96.

In one embodiment, the present invention provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, which comprises a heavy chain having complementarity determining regions according to the sequences of SEQ ID NOs: 9, 99 and 100.

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or antigen binding fragment, which contains a light chain having complementarity determining regions according to the sequences SEQ ID NO:94, 95 and 96, and a heavy chain a chain having complementarity determining regions according to the sequences of SEQ ID NO:9, 99 and 100.

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, which contains a light chain that is at least 50%, 60%, 70%, 80%, 85 in sequence %, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO:93, and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90% identical in sequence , 95%, 99% or 100% identical to the sequence of SEQ ID NO:98.

B4E11 sequences

In one embodiment of the present invention, the mouse monoclonal anti-B7H4 antibody is produced by the hybridoma clone B4E11 and contains two light chains and two heavy chains.

i. Light chain

In one embodiment of the present invention, there is provided a murine monoclonal anti-B7H4 antibody or antigen binding fragment thereof that contains a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95 in sequence %, 99% or 100% identical to the following amino acid sequence:

DIVMT OSHKFMSTS VGDRYTIT CKASOD VSTA VAWY OOKPGO SPKLLIS SAS YRYT GV PDRFTGSGSGTDFTFTISSVOAEDLAVYYC OOHYSTPT FGGGTKLEIR (SEQ ID NO:102).

The complementarity determining regions (CDRs) from SEQ ID NO:102 are shown in bold and underlined:

CDR1 KASQDVSTAVA (SEQ ID NO:103);

CDR2 SASYRYT (SEQ ID NO:104); And

CDR3 QQHYSTPT (SEQ ID NO:105).

In another embodiment, the present invention provides a nucleic acid that encodes a light chain (SEQ ID NO:102).

An exemplary light chain encoding nucleic acid (SEQ ID NO:102) is:

GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTC ACTATCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGTATCAACAG AAACCAGGACAGTCTCCTAAACTACTGATTTCCTCGGCATCCTACCGGTACACTGGA GTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGC

AGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCG ACGTTCGGT GGAGGC ACCAAGCTGGAAAT CAGA (SEQ ID NO:106).

ii. Heavy chain

In one embodiment of the present invention, there is provided a murine monoclonal anti-B7H4 antibody or antigen binding fragment thereof that contains a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95 in sequence %, 99% or 100% identical to the following amino acid sequence:

EVOLOOSGTVLARPGASVKMSCKASGYTFT SYWMH WVKERPGOGLEWI GAIYPGDSD TRYNOKFKG RAKLTAVTSANTAYMELSSSLTNDDSAVFYCTC TTAGVLDY WGOGTSV

TVSS (SEQ ID NO:107).

The complementarity determining regions (CDRs) from SEQ ID NO:107 are shown in bold and underlined:

CDR1 SYWMH (SEQ ID NO:9);

CDR2 AIYPGDSDTRYNQKFKG (SEQ ID NO:108); And

CDR3 TTAGVLDY (SEQ ID NO:109).

In another embodiment, the present invention provides a nucleic acid that encodes a heavy chain (SEQ ID NO:107). An exemplary nucleic acid encoding a heavy chain (SEQ ID NO:107) is:

GAGGTTC AGCTCC AGCAGTCTGGGACTGT GCTGGC AAGGCCTGGGGCTTC AGTGAA GATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTACTGGATGCACTGGGTAAA AGAGAGGCCTGGAC AGGGTCTGGAAT GGATT GGCGCT ATTTATCCTGGAGAT AGTG ATACTAGGTATAATCAGAAGTT CAAGGGC AGGGCC AAACTGACTGC AGTCACATCT GCCAACACTGCCTACATGGAGCTCAGCAGCCTGACAAATGATGACTCTGCGGTCTTC TACTGTACATGTACTACGGCTGGTGTTTTGGACTACTGGGGTCAAGGAACCTCAGTC ACCGTCTCCTCA (SEQ ID NO:110).

In one embodiment, the present invention provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, which comprises a light chain having complementarity determining regions according to the sequences of SEQ ID NOs: 103, 104 and 105.

In one embodiment, the present invention provides an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, which comprises a heavy chain having complementarity determining regions according to the sequences of SEQ ID NOs: 9, 108 and 109.

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, which comprises a light chain having complementarity determining regions according to the sequences of SEQ ID NOs: 103, 104 and 105, and also a heavy chain having complementarity determining regions according to the sequences of SEQ ID NO:9, 108 and 109.

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, which contains a light chain that is at least 50%, 60%, 70%, 80%, 85 in sequence %, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO:102, and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90% identical in sequence , 95%, 99% or 100% identical to the sequence of SEQ ID NO:107.

B6C8 sequences

In one embodiment of the present invention, the mouse monoclonal anti-B7H4 antibody is produced by the B6C8 hybridoma clone and contains two light chains and two heavy chains.

Light chain

In one embodiment of the present invention, there is provided a murine monoclonal anti-B7H4 antibody or antigen binding fragment thereof that contains a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95 in sequence %, 99% or 100% identical to the following amino acid sequence:

DVVMTOPLSLPVSLGDOASISC TSSOSIVHGNGNTYLEW YLOKPGOSPKLLI YKVSNR

FS GVPDRF SSGSGSGTDF TLKI SR E AEDLGV YY CFO GSHVPYTF GGGTKLEIK

(SEQ ID NO:111).

The complementarity determining regions (CDRs) from SEQ ID NO:111 are shown in bold and underlined:

CDR1 TSSQSIVHGNGNTYLE (SEQ ID NO:112);

CDR2 KVSNRFS (SEQ ID NO:95); And

CDR3 FQGSHVPYT (SEQ ID NO:113).

In another embodiment, the present invention provides a nucleic acid that encodes a light chain (SEQ ID NO:111).

An exemplary light chain encoding nucleic acid (SEQ ID NO:111) is:

GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAGGCC TCCATCTCTTGCACATCTAGTCAGAGCATTGTACATGGTAATGGAAACACCTATTTA GAATGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCC AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTC ACACTCA AGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAA GGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

(SEQ ID NO:114).

ii. Heavy chain

In one embodiment of the present invention, there is provided a murine monoclonal anti-B7H4 antibody or antigen binding fragment thereof that contains a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95 in sequence %, 99% or 100% identical to the following amino acid sequence:

OVOLOOPGAELVKPGASVKLSCKASGYSFT SYWMN WVKORPGRGLEWIG RIHPSDSE

THYNOKFKSK ATLTVDKSSSTAYIOL SSLTSEP SAVYFCARY GLFYGNDGYAMDH W.G.

QGTSVTVSS (SEQ ID NO:115).

The complementarity determining regions (CDRs) from SEQ ID NO:115 are shown in bold and underlined:

CDR1 SYWMN (SEQ ID NO:116);

CDR2 RIHPSDSETHYNQKFKS (SEQ ID NO:117); And

CDR3 YGLFYGNDGYAMDH (SEQ ID NO:118)

In another embodiment, the present invention provides a nucleic acid that encodes a heavy chain (SEQ ID NO:115). An exemplary nucleic acid encoding a heavy chain (SEQ ID NO:115) is:

CAGGTCC AACTGC AGCAGCCTGGGGCTGAACTGGT GAAGCCTGGGGCTTC AGTGAA GCTGTCCTGCAAGGCTTCTGGCTACTCTTTCACCAGCTACTGGATGAACTGGGTGAA GCAGAGGCCTGGACGAGGCCTCGAGT GGATTGGAAGGATTC ATCCTTCTGAT AGTG AAACTCACTACAATCAAAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAATCC TC CAGCACAGCCTACATCCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTAT TTTTGTGCAAGATACGGGCTCTTCTATGGTAACGACGGATATGCTATGGACCACTGG GGTCAAGGA ACCTCAG (SEQ ID NO:119).

In one embodiment of the present invention there is provided an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, which comprises a light chain having complementarity determining regions according to the sequences of SEQ ID NOs:97, 116 and 117.

In one embodiment, the present invention provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, which comprises a heavy chain having complementarity determining regions according to the sequences of SEQ ID NOs: 118, 119 and 120.

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody or antigen binding fragment, which comprises a light chain having complementarity determining regions according to the sequences SEQ ID NO:97, 114 and 115, and a heavy chain , having regions that determine complementarity, in accordance with the sequences of SEQ ID NO: 118, 119 and 120.

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, which contains a light chain that is at least 50%, 60%, 70%, 80%, 85 in sequence %, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO:l13, and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90% identical in sequence , 95%, 99% or 100% identical to the sequence of SEQ ID NO:117.

i. B9H1 sequences

In one embodiment of the present invention, the mouse monoclonal anti-B7H4 antibody is produced by the B9H1 hybridoma clone and contains two light chains and two heavy chains.

Light chain

In one embodiment of the present invention, there is provided a murine monoclonal anti-B7H4 antibody or antigen binding fragment thereof that contains a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95 in sequence %, 99% or 100% identical to the following amino acid sequence:

DIOMTOTT SSL SASLGDRVTISC RASODISF YLNW YOOKPDGT VKLLIY YTSRLHS GVPS

RF SGSSGSGTDYSLTISNLEOEDIATYE COOGNTLPWTF GGGTKLEIK (SEQ ID NO:120).

The complementarity determining regions (CDRs) from SEQ ID NO:120 are shown in bold and underlined:

CDR1 RASQDISFYLN (SEQ ID NO:121);

CDR2 YTSRLHS (SEQ ID NO:54); And

CDR3 QQGNTLPWT (SEQ ID NO 55).

In another embodiment, the present invention provides a nucleic acid that encodes a light chain (SEQ ID NO:120).

An exemplary light chain encoding nucleic acid (SEQ ID NO:120) is:

GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTC ACCATCAGTTGCAGGGCAAGTCAGGACATTAGCTTTTATTTAAACTGGTATCAGCAG AAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGA GTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATCTCTCACCATTAGC AACCTGGAGCAAGA AGATATTGCCACTTACTTTTGCCAACAGGGTAATACACTTCCG TGGACGTTCGGTGGAGGCACCAAAGCTGGAAATCAAA (SEQ ID NO:122).

Heavy chain

In one embodiment of the present invention, there is provided a murine monoclonal anti-B7H4 antibody or antigen binding fragment thereof that contains a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95 in sequence %, 99% or 100% identical to the following amino acid sequence:

EVOLOOSGPELVKPGASVKMSCKASGYTFT DYYMN WVKOSHGKSLEWI GRVNPSNG

GTS YNOKFKGK ATLTVDKSL SAAYMOLN SLTSEP SAVYYC ARRHNYPD YW GOGTTL

TVSS (SEQ ID NO:123).

The complementarity determining regions (CDRs) from SEQ ID NO:123 are shown in bold and underlined:

CDR1 DYYMN (SEQ ID NO:66);

CDR2 RVNPSNGGTSYNQKFKG (SEQ ID NO:124); And

CDR3 RHNYPDY (SEQ ID NO:125).

In another embodiment, the present invention provides a nucleic acid that encodes a heavy chain (SEQ ID NO:125). An exemplary nucleic acid encoding a heavy chain (SEQ ID NO:125) is:

C TCAGCGCAGCCTATATGCAGCTCAACAGCCTGACATCTGAGGACTCTGCGGTCTAT TACTGTGCAAGAAGGCATAACTACCCTGACTACTGGGGCCAAGGCACCACTCTCAC AGTCTCCTCA (SEQ ID NO:126).

In one embodiment, the present invention provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, which comprises a light chain having complementarity determining regions according to the sequences of SEQ ID NOs: 54, 55 and 121.

In one embodiment, the present invention provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, which comprises a heavy chain having complementarity determining regions according to the sequences of SEQ ID NOs: 66, 124 and 125.

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, which comprises a light chain having complementarity determining regions according to the sequences of SEQ ID NOs:54, 55 and 121, and a heavy chain having complementarity determining regions according to the sequences of SEQ ID NO:66, 124 and 125.

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, which contains a light chain that is at least 50%, 60%, 70%, 80%, 85 in sequence %, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO:120, and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90% identical in sequence , 95%, 99% or 100% identical to the sequence of SEQ ID NO:123.

B10D7 sequences

In one embodiment of the present invention, the mouse monoclonal anti-B7H4 antibody is produced by the B10D7 hybridoma clone and contains two light chains and two heavy chains.

Light chain

In one embodiment of the present invention, there is provided a murine monoclonal anti-B7H4 antibody or antigen binding fragment thereof that contains a light chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95 in sequence %, 99% or 100% identical to the following amino acid sequence:

EIOMTO SP SSMS ASLGDRITITCOATODIVKSLNW YOOKPGKPP SFLIYYTAOLAEGVP S RFSGSGSGSDYSLTI SNLESEDF ADYY CLOF YEFPPTF GGGTKLEIK (SEQ ID NO:13).

The complementarity determining regions (CDRs) from SEQ ID NO:13 are shown in bold and underlined:

CDR1 QATQDIVKSLN (SEQ ID NO:14);

CDR2 YTAQLAE (SEQ ID NO:15); And

CDR3 LQFYEFPPT (SEQ ID NO:16).

In another embodiment, the present invention provides a nucleic acid that encodes a light chain (SEQ ID NO:13).

An exemplary light chain encoding nucleic acid (SEQ ID NO:13) is:

GAAATCCAGATGACCCAGTCTCCATCCTCTATGTCTGCATCTCTGGGAGACAGAATA ACCATCACTTGCCAGGCAACTCAAGACATTGTTAAGAGTTTAAACTGGTATCAACAA AAACCAGGGAAACCCCCTTCATTCCTGATCTATTATACAGCTCAACTGGCAGAAGGG GTCCCGTCAAGGTTCAGTGGCAGTGGGTCTGGGTCAGACTATTCTCTGACAATCAGC AACCTGGAGTCTGAAGA TTTTGCAGACTATTACTGTCTACAGTTTTATGAGTTTCCTC CGACGTTCGGTGGAGGCACCAAGCTGGAAATCAA (SEQ ID NO:127).

Heavy chain

In one embodiment of the present invention, there is provided a murine monoclonal anti-B7H4 antibody or antigen binding fragment thereof that contains a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95 in sequence %, 99% or 100% identical to the following amino acid sequence:

EVOLOOSGTVLARPGASVKMSCKASGYPFT SYWMHW VKORPGOGLEWI GAIYPGNSD TRYNPNFKG KANLTAVTSATTAYMELSSSLTNEESAVYYCTS TWTHYFDY WGOGTTLT

VSS (SEQ ID NO:128).

The complementarity determining regions (CDRs) from SEQ ID NO:128 are shown in bold and underlined:

CDR1 SYWMH (SEQ ID NO:9);

CDR2 AIYPGNSDTRYNPNFKG (SEQ ID NO:129); And

CDR3 TWTHYFDY (SEQ ID NO:130).

In another embodiment, the present invention provides a nucleic acid that encodes a heavy chain (SEQ ID NO:128). An exemplary nucleic acid encoding a heavy chain (SEQ ID NO:128) is:

GAGGTTC AGCTCC AGCAGTCTGGGACTGT GCTGGC AAGGCCTGGGGCTTC AGTGAA GATGTCCTGCAAGGCTTCTGGCTACCCCTTTACCAGCTACTGGATGCACTGGGTAAA GCAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAAATAGTG ATACTAGGTACAACCCGAATTTCAAGGGCAAGGCCAACCTGACTGCAGTCACATCT GC CACCACTGCCTACATGGAGCTCAGCAGCCTGACAAATGAGGAATCTGCGGTCTA TTACTGTACAAGTACCTGGACCCACTACTTTGACTACTGGGGCCAAGGCACCACTCT CACAGTCTCCTCA (SEQ ID NO:131).

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, which comprises a light chain having complementarity determining regions according to the sequences of SEQ ID NOs: 14, 15 and 16.

In one embodiment, the present invention provides an anti-B7H4 antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, which comprises a heavy chain having complementarity determining regions according to the sequences of SEQ ID NOs: 9, 129 and 130.

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, which comprises a light chain having complementarity determining regions according to the sequences of SEQ ID NOs: 14, 15 and 16, and a heavy chain having complementarity determining regions according to the sequences of SEQ ID NO:9, 129 and 130.

In one embodiment of the present invention there is provided an anti-B7H4 antibody, in a preferred embodiment a monoclonal antibody, or an antigen binding fragment thereof, which contains a light chain that is at least 50%, 60%, 70%, 80%, 85 in sequence %, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO:13, and a heavy chain that is at least 50%, 60%, 70%, 80%, 85%, 90% identical in sequence , 95%, 99% or 100% identical to the sequence of SEQ ID NO:128.

2. Antibody compositions

The described B7-H4 binding molecules may be antibodies or antigen binding fragments thereof. The described antibodies and antigen-binding fragments thereof include native immunoglobulin (ie, intact antibody) of any class, fragments thereof, and synthetic proteins containing at least the antigen-binding variable domain of the antibody. In some embodiments of the present invention, the described molecule comprises both an antibody light chain and at least an antibody heavy chain variable domain. In other embodiments of the present invention, such molecules may further include one or more CHi, hinge, CH2, CH3 and CH4 regions of the heavy chain (especially CHi and hinge regions, or CHi, hinge and CH2 regions, or CHi, hinge, CH2 and CH3 regions ). The antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGi, IgG2, IgG3 and IgG4. In some embodiments of the present invention, the constant domain is a complement-fixing constant domain, where it is desired that the antibody exhibit cytotoxic activity, and is typically of the IgGi class. In other embodiments of the present invention, where such cytotoxic activity is not desired, the constant domain may be of the IgG2 or IgG4 class. The antibody may include sequences from more than one class or isotype, and the selection of specific constant domains to optimize desired effector functions is a matter of common skill in the art.

These variable domains differ in sequence among antibodies and are used to specifically bind each specific antibody to its specific antigen. However, variability is typically not distributed evenly across antibody variable domains. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in both the light chain and heavy chain variable domains. The most conserved parts of variable domains are called framework regions (FR). The variable domains of the native heavy and light chains consist of four FR regions (generally adopting a folded beta sheet configuration) connected by three CDR regions that form loops connecting, and in some cases forming, part of the folded beta sheet structure. CDR regions on each chain are held together in close proximity by FR regions and, together with CDR regions from the other chain, contribute to the formation of the antigen binding site of antibodies.

Antibody fragments with bioactivity have also been described. These fragments (whether attached to other sequences or not) include insertions, deletions, substitutions or other selected modifications of specific regions or specific amino acid residues, provided that the activity of the fragment is not significantly changed or impaired compared to the unmodified an antibody or antibody fragment.

Methods for producing single chain antibodies specific for B7-H4 can also be adapted. Methods for producing single chain antibodies are well known to those skilled in the art. A single-chain antibody can be created by fusing together the heavy and light chain variable domains using a short peptide linker, thereby reconstituting the antigen-binding site on a single molecule. Single-chain variable fragment antibodies (scfvs), in which the C-terminus of one variable domain is linked to the N-terminus of another variable domain via a 15-25 amino acid residue peptide or linker, have been developed without significantly affecting antigen binding or binding specificity. The linker is selected to allow the heavy chain and light chain to be linked together in the correct conformational orientation.

Divalent single chain variable fragments (di-scfv) can be artificially created by joining two scfv fragments. This can be done by producing a single peptide chain with two VH and two VL regions, resulting in tandem scfv fragments. Scfv fragments can also be created using linker peptides that are too short for two variable regions to fold together (about five amino acids), forcing the scfv fragments to dimerize. This type is known as diatel. Diabodies have been shown to have dissociation constants up to 40 times lower than the corresponding scfv fragments, meaning that they have a much higher affinity for their target. Even shorter linkers (one or two amino acids) lead to the formation of trimers (triabody or triobody). Tetrabodies were also created. They demonstrate even higher affinity for their targets than diabodies.

In one embodiment of the present invention, a monoclonal antibody is provided that is derived from a sufficiently homogeneous population of antibodies (ie, individual antibodies within a population are identical except for possible naturally occurring mutations that may be present in a small subpopulation of antibody molecules). Monoclonal antibodies include “chimeric” antibodies in which part of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain(s) is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, because they exhibit the desired antagonistic activity.

In one embodiment of the present invention, antibodies and antigen-binding fragments thereof are provided that specifically bind to human B7-H4.

In one embodiment, the present invention provides antibodies produced by a hybridoma from the group consisting of B1A1, B1H1, B1H3, B1H10, B2E6, B4B2, B4E11, B6C8, B9H1 and b10d7.

a. Antibodies

The immunomodulatory agent may be an antibody. Antibodies suitable for this purpose are known in the art or can be prepared by one skilled in the art. The nucleic acid and polypeptide sequences for B7-H4 are known in the art, and typical protein sequences are given above. These sequences can be used (as discussed in more detail below) by one skilled in the art to produce an antibody or antigen binding fragment thereof specific for B7-H4. Thus, the antibody or antigen binding fragment may be an agonist or antagonist of the B7-H4 signaling system.

The activity (agonist or antagonist) of an antibody or antigen binding fragment thereof that is specific for B7-H4 can be determined using functional assays known in the art, which are discussed below. Typically, these assays involve determining whether an antibody or antigen-binding fragment thereof increases ( ie, agonist) or reduces ( ie, antagonist) signaling through B7-H4. Since B7-H4 signal transduction leads to suppression of the immune response, B7-H4, which has an agonistic effect, causes a suppressed or weakened immune response. B7-H4 signaling, which has an antagonistic effect, inhibits the weakened immune response, resulting in an overall increase in the immune response.

In some embodiments of the present invention, the described antibodies and antigen-binding fragments thereof immunospecifically bind to B7-H4. In some embodiments of the present invention, the antibody binds to the extracellular domain of B7-H4.

For example, molecules have been proposed that can bind immunospecifically to B7-H4:

(I) located on the surface of a cell (especially a living cell);

(II) located on the surface of a cell (especially a living cell) in endogenous concentrations;

(III) located on the surface of a living cell, and which modulate the binding between B7-H4 and its ligand;

(IV) located on the surface of a living cell, and which reduce or inhibit immunosuppression caused by B7-H4;

(V) located on the surface of a living cell, and which induce or enhance immunosuppression caused by B7-H4;

(VI) located on the surface of a living cell, where the cell is a tumor cell;

(VII) combinations I-IV and VI;

(VIII) combinations I-III and V-IV; And

(IX) located on the surface of living cancer cells of myeloid or lymphoid origin (AML or ALL), and enhance apoptosis and differentiation, which leads to decreased self-renewal of cancer stem cells.

In some embodiments of the present invention, these molecules are capable of inducing antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or cellular apoptosis through other mechanisms in cells expressing B7-H4.

To produce an antibody or antigen-binding fragment thereof that specifically binds to B7-H4, purified proteins, polypeptides, fragments, fusions or epitopes with B7-H4, or expressed polypeptides from their nucleic acid sequences can be used. Antibodies or antigen-binding fragments thereof can be produced by any suitable methods known in the art, such as those described in more detail below.

i. Human and humanized antibodies

Many non-human antibodies (eg, those obtained from mice, rats, or rabbits) are naturally occurring antigens in humans and thus can cause unwanted immune reactions when administered to humans. Therefore, the use of human or humanized antibodies in these methods serves to reduce the likelihood that an antibody administered to a human will cause an undesirable immune response.

Transgenic animals (eg mice) capable of producing the full range of human antibodies upon immunization in the absence of endogenous immunoglobulin production can be used. For example, it has been described that homozygous deletion of the antibody heavy chain attachment site gene (J(H)) in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin genetic information into such germline mutant mice will result in the production of human antibodies upon antigen stimulation.

Additionally, antibodies are generated in other forms and “humanized” for administration to humans. Humanized forms of non-human (eg, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab') ₂ or other antigen-binding antibody sequences) that contain the minimal sequence , derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibodies), in which residues from the complementarity determining regions (CDRs) of the recipient antibody are replaced with residues from the CDR of a non-human species (donor antibodies), such as mouse, rat or rabbit, having desired specificity, affinity and potency. In some cases, human immunoglobulin Fv framework residues are replaced by corresponding non-human residues. Humanized antibodies may also contain residues that are not found in either the recipient antibody or the imported CDR or framework sequences. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to regions of non-human immunoglobulin and all or substantially all of the FR regions are regions of the human immunoglobulin consensus sequence. An optimally humanized antibody will also contain at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin region.

Methods for humanizing non-human antibodies are well known in the art. Typically, a humanized antibody has one or more amino acid residues introduced into it from a source that is not human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from the “import” variable domain region. Antibody humanization technologies typically involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Humanization may actually be accomplished by replacing the CDR regions or rodent CDR region sequences with the corresponding human antibody sequences. Accordingly, a humanized form of a non-human antibody (or fragment thereof) is a chimeric antibody or fragment in which significantly less of the intact human variable domain has been replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are usually human antibodies in which some of the CDR residues and possibly some of the FR residues are replaced by residues from similar locations in rodent antibodies.

The selection of human variable domains, both light and heavy, for use in the creation of humanized antibodies is very important to reduce antigenicity. According to the "best match" method, the variable domain sequence of a rodent antibody is screened from the entire bank of known human variable domain sequences. The human sequence closest to the rodent sequence is then adopted as the human framework (FR) for the humanized antibody. Another method uses a specific structure derived from the consensus sequence of all human antibodies of a particular subset of light or heavy chains. The same framework can be used for several different humanized antibodies.

It is also important that antibodies are humanized while maintaining high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be obtained by analyzing the parent sequences and various conceptual humanized products using three-dimensional models of the parent and humanized sequences. Three-dimensional models of immunoglobulins are widespread and familiar to those skilled in the art. Computer programs are available to illustrate and display the likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. The study of these manifestations makes it possible to analyze the likely role of molecular fragments in the functioning of the candidate immunoglobulin sequence, i.e. analyze fragments of the molecule that affect the ability of a candidate immunoglobulin to bind its antigen. Thus, fragments of the FR molecule can be selected and combined from the consensus and import sequences so as to achieve a desired antibody characteristic, such as increased affinity for the target antigen(s). In general, CDR region residues directly and significantly influence antigen binding.

The antibody may be bound to a substrate or labeled with a detectable moiety, or may be both bound and labeled. Detectable moieties contemplated in these compositions include fluorescent, enzymatic and radioactive markers.

ii. Single chain antibodies

Methods for producing single chain antibodies are well known to those skilled in the art. A single-chain antibody is created by fusing together the heavy and light chain variable domains using a short peptide linker, thereby restoring the antigen-binding site on a single molecule. Single-chain variable fragment antibodies (scfvs), in which the C-terminus of one variable domain is linked to the N-terminus of another variable domain via 15-25 amino acid peptides or linkers, have been generated without significantly affecting antigen binding or binding specificity. The linker is selected to allow the heavy chain and light chain to be linked together in the correct conformational orientation. These Fv fragments do not have the constant regions (Fc) present in the heavy and light chains of the native antibody.

Monovalent antibodies

In vitro methods are also suitable for the production of monovalent antibodies. Cleavage of antibodies to obtain their fragments, in particular Fab fragments, can be performed using conventional techniques known in the art. For example, cleavage can be accomplished using papain. Papain cleavage of antibodies typically results in the formation of two identical antigen-binding fragments, called Fab fragments, each with one antigen-binding site and a residual Fc fragment. Treatment with pepsin produces a fragment called the F(ab') ₂ fragment, which has two antigen-binding sites and is capable of cross-linking antigen.

Fab fragments generated by antibody cleavage also contain light chain constant domains and the first heavy chain constant domain. Fab' fragments differ from Fab fragments by the addition of several residues at the carboxyl terminus of the heavy chain domain, including one or more cysteine residues from the antibody hinge region. The F(ab') ₂ fragment is a divalent fragment consisting of two Fab' fragments connected by a disulfide bridge at the hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. Antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteine residues between them. Other chemical compounds of antibody fragments are also known.

Hybrid antibodies

The antibody may be a hybrid antibody. In hybrid antibodies, one pair of heavy and light chains is homologous to the pair of such chains found in the antibody opposite one epitope, while the other pair of heavy and light chains is homologous to the pair found in the antibody opposite the other epitope. The result of this is the property of multifunctional valence, i.e. the ability to bind at least two different epitopes simultaneously. Such hybrids can be created by fusion of hybridomas producing the corresponding component antibodies, or by recombinant methods. Of course, such hybrids can also be created using chimeric chains.

Conjugates or fusions of antibody fragments

The target function of an antibody can be used for therapeutic purposes by combining the antibody or fragment thereof with a therapeutic agent. Such coupling of an antibody or fragment (eg, at least a portion of an immunoglobulin constant region (Fc)) with a therapeutic agent can be achieved by creating an immunoconjugate or by creating a fusion protein comprising the antibody or antibody fragment and the therapeutic agent.

Such coupling of an antibody or fragment to a therapeutic agent can be achieved by creating an immunoconjugate or by creating a fusion protein, or by coupling the antibody or fragment to a nucleic acid, such as small interfering RNA (siRNA), containing the antibody or antibody fragment and the therapeutic agent.

In some embodiments of the present invention, the antibody is modified to change its half-life. In some embodiments of the present invention, it is desirable to increase the half-life of the antibody so that it remains in the general circulation or at the treatment site for longer periods of time. For example, it may be desirable to maintain antibody titers in the general circulation or at the site being treated over a long period of time. Antibodies can be artificially generated with Fc variants that extend the half-life of the antibody, for example, using Xtend™ antibody half-life extension technology (Xencor, Monrovia, California, USA). In other embodiments of the present invention, the half-life of the anti-DNA antibody is reduced to reduce possible side effects. The described conjugates can be used to modify a given biological response. A fragment of a drug molecule should not be considered limited to classical chemical therapeutic agents. For example, a fragment of a drug molecule may be a protein or polypeptide that has the desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin a, Pseudomonas aeruginosa exotoxin, or diphtheria toxin.

Type antibodies B7H4

This application describes generic B7H4 antibodies or antigen binding fragments. The antibodies may include one or more heavy chains and one or more light chains B1H1 or B1H10 of the non-human murine B7H4 antibody. In some embodiments of the present invention, the B7H45 antibody includes some or all of the light chain complementarity determining regions (CDRs), the entire light chain variable region, some or all of the heavy chain CDRs, the entire heavy chain variable region, or a combination thereof in any mouse non-human B1H1 or B1H10 antibodies to B7H4 antibody. Typical combinations are described below.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain with an amino acid sequence corresponding to the sequence of SEQ ID NO:24 and a heavy chain with an amino acid sequence corresponding to the sequence of SEQ ID NO:38.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain with an amino acid sequence corresponding to the sequence of SEQ ID NO:24 and a heavy chain with an amino acid sequence corresponding to the sequence of SEQ ID NO:39.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain with an amino acid sequence corresponding to the sequence of SEQ ID NO:24 and a heavy chain with an amino acid sequence corresponding to the sequence of SEQ ID NO:40.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain with an amino acid sequence corresponding to the sequence of SEQ ID NO:24 and a heavy chain with an amino acid sequence corresponding to the sequence of SEQ ID NO:41.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain with an amino acid sequence corresponding to the sequence of SEQ ID NO:24 and a heavy chain with an amino acid sequence corresponding to the sequence of SEQ ID NO:42.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:24 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:43.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain containing the amino acid sequence corresponding to the sequence of SEQ ID NO:24 and a heavy chain containing the amino acid sequence corresponding to the sequence of SEQ ID NO:44.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:24 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:45.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:25 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:38.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:25 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:39.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:25 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:40.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:25 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:41.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:25 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:42.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:25 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:43.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:25 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:44.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:25 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:45.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:26 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:38.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:26 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:39.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:26 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:40.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:26 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:41.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:26 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:42.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:26 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:43.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:26 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:44.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:26 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:45.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:27 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:38.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:27 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:39.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:27 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:40.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:27 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:41.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:27 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:42.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:27 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:43.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:27 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:44.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:27 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:45.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:28 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:38.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:28 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:39.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:28 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:40.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:28 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:41.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:28 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:42.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:28 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:43.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:28 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:44.

An anti-B7H4 antibody or antigen binding fragment thereof having a light chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:28 and a heavy chain having an amino acid sequence corresponding to the sequence of SEQ ID NO:45.

2. Proteins and polypeptides

a. Protein and polypeptide compositions

The immunomodulatory agent may be a protein, a polypeptide, or a fusion protein. For example, the immunomodulatory agent may be an isolated or recombinant protein or polypeptide, a functional fragment, variant thereof, or a B7-H4 fusion protein thereof.

The specified protein or polypeptide, or a functional fragment, variant thereof, or a fusion protein thereof may be an agonist or antagonist. For example, in some embodiments of the present invention, the B7-H4 antagonist is a B7-H4 polypeptide or fragment or fusion protein thereof that binds to a B7-H4 ligand. Said polypeptide may be a soluble fragment, for example the B7-H4 extracellular domain, or a functional fragment thereof, or a fusion protein thereof. In some embodiments of the present invention, a soluble B7-H4 ligand may serve as an agonist, increasing signal transduction through B7-H4.

The activity (ie, agonist or antagonist) of a B7-H4 protein or polypeptide, or any fragment, variant or fusion protein thereof, can be determined using functional assays that are known in the art and include the assays presented below. Typically, the assays involve determining whether a protein, polypeptide, or fragment, variant, or fusion protein thereof increases (ie, agonist) or decreases (ie, antagonist) signaling through the B7-H4 receptor. In some embodiments of the present invention, the analysis includes determining whether the protein, polypeptide, or fragment, variant, or fusion protein thereof increases (ie, agonist) or decreases (ie, antagonist) the immune response associated with B7-H4. Typically, these assays involve determining whether a protein, polypeptide, or fragment, variant, or fusion protein thereof increases (ie, agonist) or decreases (ie, antagonist) signaling through B7-H4. In some embodiments of the present invention, the assay includes determining whether the protein, polypeptide, or fragment, variant, or fusion protein thereof reduces (ie, agonist) or increases (ie, antagonist) the immune response inhibited by B7-H4. In some embodiments of the present invention, the assay includes determining whether the protein, polypeptide, or fragment, variant, or fusion protein (i.e., antagonist) increases apoptosis and differentiation of acute myeloid leukemia cells and acute lymphoblastic leukemia cells, resulting in decreased self-renewal capacity AML and ALL stem cells.

The nucleic acid and polypeptide sequences for B7-H4 are known in the art, and typical protein and peptide sequences are given above. These sequences can be used, as presented in more detail below, by one skilled in the art to produce any B7-H4 protein or polypeptide, or any fragment, variant or fusion protein thereof. In general, proteins, polypeptides, fragments, variants and fusions thereof in B7-H4 are expressed from nucleic acids that include sequences encoding a signal sequence. The signal sequence is typically cleaved from the immature polypeptide to produce a mature polypeptide lacking the signal sequence. The signal sequence can be replaced by the signal sequence of another polypeptide using standard molecular biology techniques to affect the expression levels, secretion, solubility or other properties of the B7-H4 proteins of the polypeptide with or without the described signal sequence. It will be understood that in some cases, the mature protein as it is known or described in the art (ie, the protein sequence without the signal sequence) is a hypothetical mature protein. During normal cellular expression, the signal sequence can be removed by cellular peptidase to produce the mature protein. The sequence of the mature protein can be determined or confirmed using methods known in the art.

i. Fragments

As used herein, fragment B7-H4 refers to any polypeptide species that is at least one amino acid shorter than the full-length protein. Useful moieties include moieties that retain the ability to bind to their natural ligand or ligands. A polypeptide that is a fragment of any full-length B7-H4 typically has at least 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 98 percent , 99 percent, 100 percent, or even more than 100 percent ability to bind its natural ligand, respectively, compared to the full-length protein.

The B7-H4 fragments include non-cellular fragments. Non-cellular polypeptides can be fragments of full-length transmembrane polypeptides that can be separated, secreted or otherwise recovered from the producing cells. Noncellular fragments of polypeptides may include some or all of the extracellular domains of the polypeptide and may not contain some or all of the intracellular and/or transmembrane domains of the full-length protein. In one embodiment of the present invention, the polypeptide fragments include the entire extracellular domain of the full-length protein. In other embodiments of the present invention, the noncellular fragments of the polypeptides include extracellular domain fragments that retain the biological activity of the full-length protein. The extracellular domain may include 1, 2, 3, 4 or 5 contiguous amino acids from the transmembrane domain and/or 1, 2, 3, 4 or 5 contiguous amino acids from the signal sequence. Alternatively, the extracellular domain may have 1, 2, 3, 4, 5 or more amino acids removed from the C-terminus, N-terminus, or both. In some embodiments of the present invention, the extracellular domain is the only functional domain of the fragment (eg, the ligand binding domain).

ii. Options

Options B7-H4 and their fragments are also proposed. In some embodiments of the present invention, such a variant is at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99 percent identical to any of SEQ ID NO:1. Useful options include options that increase biological activity, as indicated in any of the assays described herein, or that increase the half-life or stability of the protein. B7-H4 protein and polypeptides, and fragments, variants and fusion proteins thereof, can be artificially created to enhance biological activity. For example, in some embodiments of the present invention, the B7-H4 polypeptide, protein, or fragment, variant, or fusion thereof has been modified by at least the substitution, deletion, or insertion of one amino acid that enhances its function.

Finally, variant polypeptides can be artificially engineered to have an increased half-life compared to the wild type. These variants are typically modified to resist enzymatic degradation. Typical modifications include modified amino acid residues and modified peptide bonds that are resistant to enzymatic degradation. To achieve this goal, various modifications are known in the art. These options can be modified to match the effect of receptor affinity on the half-life of proteins, polypeptides, fragments or fusions thereof at serum and endosomal pH.

iii. Fusion proteins

Fusion polypeptides comprise a first fusion component comprising all or part of a B7-H4 polypeptide fused to a second polypeptide either directly or through a linker peptide sequence that is fused to the second polypeptide. Additionally, the fusion proteins contain a domain that functions to dimerize or multimerize two or more fusion proteins. The linker peptide/polypeptide domain may be either a separate domain or alternatively may be contained within one of the other domains (the first polypeptide or the second polypeptide) of the fusion protein. Likewise, the domain that functions to dimerize or multimerize the fusion proteins may be either a separate domain or alternatively may be contained in one of the other domains (the first polypeptide, the second polypeptide, or the linker peptide/polypeptide domain) of the fusion protein. In one embodiment of the present invention, the dimerization/multimerization domain and the linker peptide/polypeptide domain are the same.

The fusion proteins described in this application have formula I:

N-R1-R2-R3-C

where "N" represents the N-terminus of the fusion protein, "C" represents the C-terminus of the fusion protein. In some embodiments of the present invention, "Ri" is a B7-H4 polypeptide or protein, or a fragment or variant thereof, "R2" is an additional linker peptide/polypeptide domain, and "R3" is a second polypeptide. Alternatively, R ₃ may be a B7-H4 polypeptide or protein, or a fragment or variant thereof, and Ri may be a second polypeptide. In some embodiments of the present invention, the B7-H4 polypeptide is an extracellular domain or fragment thereof, such as a C2 Ig-like domain, or a region framed by cysteine residues that form a disulfide bond, as discussed above.

Dimerization or multimerization can occur between two or among more fusion proteins through dimerization or multimerization domains. Alternatively, dimerization or multimerization of the fusion proteins can occur by chemical cross-linking. The resulting dimers or multimers may be homodimeric/homomultimeric or heterodimeric/heteromultimeric.

In some embodiments of the present invention, the fusion protein includes the extracellular domain of the B7-H4 antibody or a fragment or variant thereof fused to an Ig Fc region. Recombinant Ig fusion proteins can be produced by fusing the coding region of said extracellular domain of any extracellular domain or fragment or variant thereof with the Fc region of human IgGl, IgG2, IgG3 or IgG4, or mouse IgG2a, or other suitable Ig domain, such as described previously (Chapoval, et al ., Methods Mol. Med., 45:247-255 (2000)).

iv. Polypeptide modifications

Polypeptides and fusion proteins can be modified by functional chemical groups that may be present on polypeptides in the normal cellular environment, such as phosphorylation, methylation, amidation, sulfation, acylation, glycosylation, sumoylation and ubiquitylation. Fusion proteins can also be modified with a label capable of providing a detectable signal, directly or indirectly, including (but not limited to) radioisotopes and fluorescent compounds.

Polypeptides and fusion proteins can also be modified with functional chemical groups that are not typically added to polypeptides in the cellular environment. For example, the disclosed fusion proteins can also be modified by covalently attaching polymer chains, including but not limited to polyethylene glycol (PEG) polymer chains (ie, PEGylation). Conjugation of macromolecules with PEG has recently emerged as an effective strategy for altering the pharmacokinetic (PK) profile of various drugs and thereby increasing their therapeutic potential. PEG conjugation increases the retention of drugs in the bloodstream by protecting against enzymatic degradation, slowing renal filtration, and reducing the production of neutralizing antibodies. In addition, PEG conjugates can be used to provide multimerization of fusion proteins.

Modifications can be introduced into the molecule by reacting the target amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Another modification is protein cyclization.

Examples of chemical derivatives of polypeptides include lysinyl and amino-terminal residues derivatized with succinic anhydrides or other carboxylic acids. The consequence of obtaining derivatives with cyclic carboxylic anhydride is the reversal of the charge of the lysinyl residues. Other useful reagents for derivatization of amine-containing moieties include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, o-methylisourea. 2,4-pentanedione, and a transaminase-catalyzed reaction with glyoxylate. Carboxyl side groups, aspartyl or glutamyl, can be selectively modified by reaction with carbodiimides (R-N=C=N-R'), such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or 1-ethyl- 3-(4-azonia-4,4-dimethylpentyl) carbodiimide. In addition, the aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonia. The fusion proteins may also include one or more D-amino acids that are replaced by one or more L-amino acids.

v. Modified binding capacity

The binding capacity of proteins, polypeptides, their fragments, variants and fusions corresponds to the dose and schedule of administration. In one embodiment of the present invention, the described proteins, polypeptides, fragments, variants and fusions thereof have a property of binding to the B7-H4 ligand that exhibits a longer duration or a higher percentage of occupancy of the binding site (e.g., on the ligand) compared to other receptor molecules, who contact him. In other embodiments of the present invention, the described proteins, polypeptides, fragments, variants, and fusions thereof have reduced binding affinity for the B7-H4 ligand compared to the wild type protein.

In some embodiments of the present invention, the proteins, polypeptides, fragments, variants, and fusions thereof have a relatively high affinity for the B7-H4 ligand and therefore may have a relatively slow dissociation rate. In other embodiments of the present invention, proteins, polypeptides, fragments, variants, and fusions thereof are administered periodically over several days, weeks, or months to dampen immune responses, which are allowed to recover before the next administration is made, which may serve to alter the immune response. without turning the immune response on or off completely, and to avoid long-term side effects.

3. Isolated Nucleic Acid Molecules

This application describes isolated nucleic acid sequences encoding proteins, polypeptides, fragments, variants and fusions thereof. As used herein, “isolated nucleic acid” refers to a nucleic acid that is separated from other nucleic acid molecules present in the mammalian genome, including nucleic acids that are typically located at the ends of one or both sides of the nucleic acid in the mammalian genome. The term “isolated” as used herein with respect to nucleic acids also includes combination with any non-naturally occurring nucleic acid sequence, since such non-naturally occurring sequences do not occur in nature and do not have immediately contiguous sequences in the natural genome.

The isolated nucleic acid may be, for example, a DNA molecule, provided that one of the nucleic acid sequences typically found immediately at the ends of the DNA molecule in the natural genome is deleted or missing. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid or complementary DNA (cDNA), or a fragment of genomic DNA obtained by polymerase chain reaction (PCR) or restriction endonuclease treatment), as well as recombinant DNA that is included in a vector, an autonomously replicating plasmid, a virus (for example, a retrovirus, lentivirus, adenovirus or herpes virus) or in the genomic DNA of a prokaryote or eukaryote. In addition, the isolated nucleic acid may include an artificially generated nucleic acid, such as a recombinant DNA molecule, that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds or millions of other nucleic acids, for example in a cDNA bank or genomic bank, or on a gel plate containing restriction fragments of genomic DNA, should not be considered an isolated nucleic acid.

Nucleic acids encoding proteins, polypeptides, fragments, variants and fusions thereof can be optimized for expression in a selected expression host. Codons can be replaced by alternative codons encoding the same amino acid to account for differences in codon usage between the mammal from which the nucleic acid sequence is derived and the expression host.

Thus, nucleic acids can be synthesized using the expression host's preferred codons.

Nucleic acids may be in sense or antisense orientation, or may be complementary to a reference sequence encoding a B7-H4 polypeptide or protein. Nucleic acids can be analogues of DNA, RNA or nucleic acids. Nucleic acid analogues can be modified in a base residue, a sugar residue, or a phosphate backbone. Such modification may improve, for example, the stability, hybridization or solubility of the nucleic acid. Modifications to the base residue may include deoxyuridine for deoxythymidine and 5-methyl-2'-deoxycytidine or 5-bromo-2'-deoxycytidine for deoxycytidine. Modifications to the sugar residue may include modification of the 2'-hydroxyl of the ribose sugar to form 2'-O-methyl or 2'-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids in which each basic residue is linked to a six-membered morpholine ring, or peptide nucleic acids in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev . 7: 187-195; and the work of Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5-23. In addition, the deoxyphosphate backbone can be replaced, for example, by a phosphorothioate or phosphorodithioate backbone, a phosphoramidite backbone, or an alkyl phosphotriester backbone.

Nucleic acids encoding polypeptides can be administered to subjects in need. Nucleic acid delivery involves the introduction of “foreign” nucleic acids into a cell and, ultimately, into a living animal. Compositions and methods for delivering nucleic acids to a subject are known in the art (see Understanding Gene Therapy, Lemoine, N. R., ed., BIOS Scientific Publishers, Oxford, 2008).

4. Vectors and host cells

Vectors encoding proteins, polypeptides, fragments, variants and fusions thereof are also proposed. Nucleic acids, such as those described above, can be inserted into vectors for expression in cells. As used herein, a "vector" is a replicon, such as a plasmid, phage, virus, or cosmid, into which another segment of DNA can be inserted to cause replication of the inserted segment. The vectors may be expression vectors. An "expression vector" is a vector that includes one or more expression control sequences, and an "expression control sequence" is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.

The nucleic acids in the vectors may be operably linked to one or more expression control sequences. As used herein, the term "operably linked" means included in a genetic construct such that the expression control sequences effectively control the expression of the coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription termination sites. A promoter is an expression-driving sequence consisting of a region of a DNA molecule typically within 100 nucleotides upstream of the point at which transcription begins (usually near the initiation site of RNA polymerase II). To bring a coding sequence under the control of a promoter, it is necessary to locate the translation initiation site of the polypeptide's translational reading frame downstream of the promoter at a distance of one to approximately fifty nucleotides. Enhancers provide specificity of expression in terms of timing, location, and level. Unlike promoters, enhancers can function at different distances from the site of transcription. The enhancer may also be located downstream of the transcription initiation site. A coding sequence is "operably linked" and is "under the control" of the sequences that control expression in the cell when RNA polymerase is able to transcribe the coding sequence into messenger RNA (mRNA), which can then be translated into the protein encoded by the coding sequence.

Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses, vaccinia viruses, adenoviruses and adeno-associated viruses. A variety of vectors and expression systems are commercially available from companies such as Novagen (Madison, WI, USA), Clontech (Palo Alto, CA, USA), Stratagene (La Jolla, CA, USA), and Invitrogen Life Technologies (Carlsbad). , California, USA).

The expression vector may contain a sequence of tags. Tag sequences are typically expressed as a fusion to the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including either the carboxyl terminus or the amino terminus. Examples of useful tags include (but are not limited to) green fluorescent protein (GFP), glutathione-S-transferase (GST), polyhistidine, c-myc, hemagglutinin, Flag™ tag (Kodak, New Haven, CT, USA), maltose-e-binding protein and protein A. In one embodiment of the present invention, a nucleic acid molecule encoding one of the described polypeptides is present in a vector containing nucleic acids that encode one or more Ig heavy chain constant region domains, for example, having an amino acid sequence corresponding to the hinge region, CH2 and CH3 regions of the Cγl chain of human immunoglobulin.

Vectors containing nucleic acids to be expressed can be transferred into host cells. The term "host cell" includes prokaryotic and eukaryotic cells into which the recombinant expression vector can be introduced. As used herein, the terms “transformed” and “transfected” mean the introduction of a nucleic acid molecule (eg, a vector) into a cell using one of a number of technologies. Although these technologies are not limited to any particular technology, a number of them are well known in the art. Prokaryotic cells can be transformed using nucleic acids, for example by electroporation or calcium chloride-mediated transformation. Nucleic acids can be transfected into mammalian cells using technologies including, for example, calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation or microinjection. Host cells (eg, a prokaryotic cell or a eukaryotic cell, such as a CHO cell) can be used, for example, to produce the proteins, polypeptides, fragments, variants, and fusions thereof described herein.

The described vectors can be used to express proteins, polypeptides, fragments, variants and fusions thereof in cells. An exemplary vector includes, but is not limited to, an adenoviral vector. One approach to the problem involves transfer of nucleic acids into primary cells in culture followed by autologous transplantation of the transformed cells ex vivo into the host, either systemically or into a specific organ or tissue. Ex vivo methods may include, for example, the steps of collecting cells from a subject, culturing the cells, transducing them with an expression vector, and maintaining the cells under conditions suitable for expression of the encoded polypeptides. These methods are known in the field of molecular biology. The transduction step can be performed by any of the standard means used for ex vivo gene therapy, including, for example, calcium phosphate, lipofection, electroporation, viral infection and biolistic gene transfer. Alternatively, liposomes or polymer microparticles can be used. Cells that have been successfully transduced can be selected, for example, to express a coding sequence or a drug resistance gene. The cells can then be exposed to a lethal dose of radiation (if desired) and injected or implanted into the subject. In one embodiment of the present invention, expression vectors containing nucleic acids encoding fusion proteins are transfected into cells that are administered to a subject in need thereof.

In vivo nucleic acid therapy can be achieved by direct transfer of functionally active DNA into a somatic tissue or organ of a mammal in vivo . For example, nucleic acids encoding the polypeptides described herein can be administered directly into lymphoid tissues. Alternatively, specific targeting of lymphoid tissue can be achieved using lymphoid tissue-specific transcriptional regulatory elements (TREs), such as B lymphocyte, T lymphocyte, or dendritic cell-specific transcriptional regulatory elements (TREs). Transcriptional regulatory elements (TREs) specific to lymphoid tissue are known in the art.

Nucleic acids can also be administered in vivo via viral mechanisms. Nucleic acid molecules encoding fusion proteins can be packaged into retroviral vectors using packaging cell lines that produce replication-defective retroviruses, as is well known in the art. Other viral vectors can be used, including recombinant adenoviruses and vaccinia virus, which can be made non-replicating. In addition to naked DNA or RNA, or viral vectors, artificially created bacteria can be used as vectors.

Nucleic acids can also be delivered by other carriers, including liposomes, polymeric micro- and nanoparticles, and polycations such as asialoglycoprotein/polylysine.

In addition to in vivo gene transfer mediated by viruses and carriers, physical means well known in the art, including introduction of plasmid DNA and particle bombardment-mediated gene transfer, can be used for direct DNA transfer.

5. Small molecules

The immunomodulatory agent may be a small molecule. Small molecule B7-H4 agonists and antagonists are known in the art or can be identified using conventional screening methods.

In some embodiments of the present invention, screening assays may involve randomly searching large banks of test compounds. Alternatively, these assays can be applied to focus on specific classes of compounds that have the potential to cause B7-H4 modulations. Assays may include determination of B7-H4 signaling activity or B7-H4-mediated inhibitory response. Other tests may include determination of nucleic acid transcription or translation, messenger RNA (mRNA) levels, mRNA stability, mRNA degeneration, transcription rate, and translation rate.

C. Pharmaceutical Compositions

Pharmaceutical compositions are proposed that include the described immunomodulatory agents. Pharmaceutical compositions containing immunomodulatory agents may be administered parenterally (by intramuscular, intraperitoneal, intravenous (IV), or subcutaneous injection), transdermally (either passively, or by iontophoresis or electroporation), or transmucosally (nasally, vaginally, rectally, or sublingually ), or using biodegradable inserts; they can be prepared in dosage forms suitable for each route of administration.

In some in vivo methods, the compositions described herein are administered to a subject in a therapeutically effective amount. As used herein, the term “effective amount” or “therapeutically effective amount” means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of the disease being treated, or to otherwise provide a desired pharmacological and/or physiological effect. The exact dosage will vary depending on a number of factors, such as subject-specific variables (eg, age, immune system status, etc.), disease, and treatment being administered.

For the immunomodulatory agents described, as further research is conducted, information regarding appropriate dosage levels for the treatment of various pathological conditions in different patients will become available, and the correct dosage will be determined by the person skilled in the art, taking into account the therapeutic context, age and general health of the recipient. The dosage chosen depends on the desired therapeutic effect, the route of administration and the duration of the desired treatment. The described immunomodulatory agents are typically administered to mammals at a dose of 0.001 to 20 mg/kg body weight daily. In general, the dosage may be lower for intravenous injection or infusion.

In certain embodiments of the present invention, the immunomodulatory agent is administered locally, for example, by injection directly into the site being treated. Typically, the injection causes an increased local concentration of the immunomodulatory agent composition, which is higher than the concentration that can be achieved with systemic administration. The immunomodulatory agent compositions may be combined with a matrix as described above to help create an increased local concentration of the polypeptide compositions by reducing the passive diffusion of the polypeptides from the site being treated.

1. Dosage forms for parenteral administration

In some embodiments of the present invention, the compositions described herein, including compositions containing peptides and polypeptides, are administered as an aqueous solution by parenteral injection. The dosage form can also be in the form of a suspension or emulsion. In general, the pharmaceutical compositions provided herein include an effective amount of a peptide or polypeptide, and further include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. These compositions may additionally include one (or more) of the following: diluents, sterile water, buffered saline of various buffer compositions (eg, Tris-HCl, acetate, phosphate), various pH and ionic strength of the solution; and impurities such as detergents and solubilizing agents (eg, TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), antioxidants (eg, ascorbic acid, sodium metabisulfite), preservatives (eg, thimerosol, benzyl alcohol) and fillers (eg lactose, mannitol). Examples of non-aqueous solvents or excipients are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin and injectable organic esters such as ethyl oleate. These dosage forms may be lyophilized and redissolved/reconstituted immediately prior to administration. Such dosage form can be sterilized, for example, by filtration through a bacteria-retaining filter, introducing sterilizing agents into the compositions, irradiating the compositions, or heating the compositions.

2. Oral dosage forms

In embodiments of the present invention, there are compositions formulated for oral delivery. Solid oral dosage forms are generally described in Remington Pharmaceutical Sciences, 18th edition. 1990 (Mack Publishing Co. Easton Pa. 18042) in Chapter 89. Solid dosage forms include tablets, capsules, pills, troches or lozenges, wafers, microspheres, powders or granules, or the inclusion of the said material in aerosol preparations of polymeric compounds such such as polylactic acid, polyglycolic acid, etc., or into liposomes. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the disclosed compositions. See, for example, Remington Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042), pages 1435-1712, which are incorporated herein by reference. These compositions may be prepared in liquid form or may be in dried powder form (eg, lyophilized). To prepare these compositions, liposomal or proteinoid encapsulation can be used. Liposomal encapsulation can be used, and liposomes can be derivatized with various polymers (eg, US Pat. No. 5,013,556). Also see Marshall, K. In: Modem Pharmaceutics, edited by G. S. Banker and C. T. Rhodes, Chapter 10, 1979. Typically, a given dosage form will include the peptide (or chemically modified forms thereof) and inert ingredients that protect the peptide in the gastric environment and release biologically active material in the intestine.

These agents can be chemically modified so that oral delivery of such a derivative is effective. In general, the chemical modification in question is the addition of at least one fragment to the component molecule itself, where this fragment allows entry into the bloodstream from the stomach or intestines, or absorption directly by the intestinal mucosa. It is also desirable to increase the overall stability of the component or components and increase the circulation time in the body. A typical chemical modification for pharmaceutical use is PEGylation. Other moieties that may be used include: propylene glycol, ethylene glycol-propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyproline, poly-1,3-dioxolane and poly-1,3,6-thioxocane [see, e.g. , Abuchowski and Davis (1981) "Soluble Polymer-Enzyme Adducts" in Enzymes as Drugs, edited by Hocenberg and Roberts (Wiley-Interscience: New York, NY.) , pp. 367-383; and the book by Newmark, et al. (1982) J. Appl. Biochem (Applied Biochemistry) 4:185-189].

In another embodiment, the present invention provides liquid oral dosage forms, including pharmaceutically acceptable emulsions, solutions, suspensions and syrups, which may contain other components, including inert diluents; adjuvants such as wetting agents, emulsifying and suspending agents; and sweetening, flavoring and flavoring agents.

Controlled-release oral dosage forms may be desirable. The agent may be included in an inert matrix that allows release by either a diffusion or leaching mechanism, such as gum. Slowly decomposing matrices may also be included in the dosage form. Another form of controlled release is based on the Oros therapeutic system (Alza Corp.), i.e. the drug is enclosed in a semi-permeable membrane that allows water to enter and expel the drug through a single small hole through an osmotic effect.

For oral dosage forms, the site of release may be the stomach, small intestine (duodenum, jejunum, or ileum), or colon. In some embodiments of the present invention, the release will prevent harmful effects of the gastric environment either by protecting the agent (or derivative) or by releasing the agent (or derivative) outside the gastric environment, such as in the intestine. To provide complete resistance to the gastric environment, a coating that is impermeable to at least pH 5.0 is required. Examples of the most common inert ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D™, Aquateric™. , cellulose acetate phthalate (CAP), Eudragit L™, Eudragit S™ and Shellac™ (Shellac). These coatings can be used as mixed films.

3. Dosage forms for topical use

The described immunomodulatory agents can be applied topically. For most peptide drugs, topical application is not effective, although it can be effective, especially if applied to the mucous membranes of the lungs, nose, mouth (sublingual, buccal), vagina or rectum.

The compositions can be delivered to the lungs during inhalation and penetrate the epithelium lining the lungs, entering the bloodstream when delivered either as an aerosol or as spray-dried particles having an aerodynamic diameter of less than 5 microns.

A wide range of mechanical devices can be used to deliver drugs to the lungs, including but not limited to nebulizers, metered dose inhalers, and dry powder inhalers; all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are the Ultravent nebulizer (Mallinckrodt Inc., St. Louis, MO, USA); Acorn II nebulizer (Marquest Medical Products, Englewood, CO, USA); Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, NC) and Spinhaler powder inhaler (Fisons Corp., Bedford, MA, USA). Nektar, Alkermes and Mannkind have inhaled insulin powder formulations approved or undergoing clinical trials where this technology can be applied to the dosage forms described in this application.

Mucosal dosage forms are typically spray-dried drug particles that may be contained in a tablet, gel, capsule, suspension or emulsion. Standard pharmaceutical excipients available in any dosage form are used.

Transdermal preparations can also be prepared. Usually these are ointments, lotions, sprays or patches that can be prepared using standard technology. Transdermal dosage forms may require the inclusion of skin penetration enhancers.

4. Controlled Delivery Polymer Matrices

The immunomodulatory agents described herein can also be formulated in controlled release dosage forms. Controlled-release polymer devices can be formulated for sustained systemic release following implantation of a polymer device (rod, barrel, film, disk) or injection (microparticles). The matrix may be in the form of microparticles such as microspheres, where the agent is dispersed within a solid polymer matrix, or microcapsules, in which the core consists of a different material from the polymer shell and the peptide is dispersed or suspended in the core, which may be liquid or solid in nature nature. Microparticles, microspheres and microcapsules are used interchangeably, unless specifically stated in this application. Alternatively, the polymer can be cast as a thin plate or film, ranging in size from nanometers to four centimeters, from a powder obtained by grinding or other standard methods, or even as a gel, such as a hydrogel.

Either non-biodegradable or biodegradable matrices can be used to deliver fusion polypeptides or nucleic acids encoding fusion polypeptides, although biodegradable matrices are preferred in some embodiments of the present invention. Such matrices may be natural or synthetic polymers, although in some embodiments of the present invention synthetic polymers are preferred due to superior degradation and release dynamics. The polymer is selected based on the period over which release is required. In some cases, a linear release may be most beneficial, although in other cases a pulsed release or "massive release" may produce more dramatic results. The polymer can be in the form of a hydrogel (typically absorbing up to 90% of its mass of water) and can be further cross-linked with multivalent ions or polymers.

These matrices can be formed by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art. Biodegradable microspheres can be prepared using any method designed to make microspheres for drug delivery, for example, as described in Mathiowitz and Langer, J. Controlled Release, 5:13-22 (1987); Mathiowitz, et al., Reactive Polymers , 6:275-283 (1987), and Mathiowitz, et al. , J. Appl. Polymer Sci . (Applied Polymer Science), 35:755-774 (1988).

These devices may be formulated for local release to treat the implantation or injection site - which typically provides a dosage that is much less than that for whole body treatment - or for systemic delivery. They can be implanted or injected subcutaneously, into muscle, fatty tissue, or ingested.

Methods of obtaining

A. Methods for producing antibodies

Antibodies can be produced in cell culture, in phage, or in a variety of animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and apes. Therefore, in one embodiment of the present invention, the antibody is a mammalian antibody. Phage technologies can be used to isolate the original antibody or to create variants with altered specificity or avidity characteristics. Such techniques are common and well known in the art. In one embodiment of this invention, the antibody is produced by recombinant methods known in the art. For example, a recombinant antibody can be produced by transfecting a host cell with a vector containing a DNA sequence encoding the antibody. One or more vectors can be used to transfect a DNA sequence expressing at least one VL region and one VH region into a host cell. Typical descriptions of recombinant methods for creating and producing antibodies are presented in Delves, Antibody Production: Essential Techniques (Wiley, 1997); Shephard, et al., Monoclonal Antibodies (Oxford University Press, 2000); Goding, Principles and Practice (Academic Press, 1993); Current Protocols in Immunology (John Wiley & Sons, latest edition).

The disclosed antibodies can be modified by recombinant methods to make the antibody more effective in mediating the desired function. Thus, within the scope of this invention, antibodies can be modified by substitution using recombinant methods. Typically, such substitutions will be conservative substitutions. For example, at least one amino acid in the constant region of an antibody may be replaced by another residue. See, for example, ET.S patent No. 5,624,821, US patent No. 6194551, Application No. WO 9958572; and the work of Angal, et al ., Mol. Immunol.("Molecular Immunology") 30:105-08 (1993). Amino acid modification includes deletions, additions, and substitutions of amino acids. In some cases, such changes are made to reduce undesirable activity, such as complement-dependent cytotoxicity. Antibodies are often labeled by attaching, either covalently or non-covalently, a substance that provides a detectable signal. A wide variety of labels and conjugation technologies are known and widely reported in both the scientific and patent literature. These antibodies can be tested for binding to proteins, polypeptides or B7-H4 fusion proteins. See, for example, Antibody Engineering: A Practical Approach (Oxford University Press, 1996).

For example, useful antibodies with the desired biological activity can be identified using in vitro assays including, but not limited to, assays for proliferation, migration, adhesion, growth on soft agar, angiogenesis, intercellular communication, apoptosis, transport, signal transduction, and in vivo assays such as tumor growth inhibition assays. Antibodies proposed in this application may also be useful in diagnosis. As immobilized or non-neutralizing antibodies, they can be tested for the ability to bind to a specific antigen without inhibiting the receptor binding or biological activity of the antigen. As neutralizing antibodies, these antibodies may be useful in competitive binding assays.

Antibodies that can be used in the described compositions and methods include intact immunoglobulin (ie, intact antibody) of any class, fragments thereof, and synthetic proteins containing at least the antigen-binding variable domain of the antibody. Variable domains vary in sequence among antibodies and are used in the specific binding of each specific antibody to its specific antigen. However, variability is typically not evenly distributed among the variable domains of antibodies. Typically, it is concentrated in three segments called complementarity determining regions (CDRs), or hypervariable regions, in both the light chain and heavy chain variable domains. The most conserved parts of the variable domains are called the framework (FR). The variable domains of the native heavy and light chains consist of four framework regions (FRs) largely adopting a folded beta sheet configuration, connected by three CDR regions that form loops connecting, and in some cases forming part of, the folded beta sheet structure. CDR regions on each chain are held together in close proximity by framework regions (FRs) and, together with CDR regions from the other chain, contribute to the formation of the antigen-binding site of antibodies.

Antibody fragments with bioactivity have also been described. These fragments, whether attached to other sequences or not, include insertions, deletions, substitutions or other selected modifications of specific regions or specific amino acid residues, provided that the activity of the fragment is not significantly changed or impaired compared to the unmodified an antibody or antibody fragment.

Technologies can also be adapted to produce single-chain antibodies specific for an antigenic peptide. Methods for producing single chain antibodies are well known to those skilled in the art. A single chain antibody can be created by fusing together the heavy and light chain variable domains using a short peptide linker, thereby restoring the antigen binding site on a single molecule. Single-chain variable fragment antibodies (scfvs), in which the C-terminus of one variable domain is linked to the N-terminus of another variable domain via 15-25 amino acid peptides or linkers, have been generated without significantly affecting antigen binding or binding specificity. The linker is selected to allow the heavy chain and light chain to link together in the correct conformational orientation.

Divalent single chain variable fragments (di-scfv) can be artificially created by joining two scfvs. This can be achieved by producing a single peptide chain with two VH and two VL regions, resulting in tandem scfvs. scfv fragments can also be designed with linker peptides that are too short for the two variable regions to fold together (about five amino acids), causing the scfv fragments to dimerize. This type is known as diatel. Diabodies have been shown to have dissociation constants up to 40 times lower than the corresponding scfv fragments; this means that they have a much higher affinity for their target. Even shorter linkers (one or two amino acids) lead to the formation of trimers (triabody or triobody). Tetrabodies were also created. They demonstrate even higher affinity for their targets than diabodies.

A monoclonal antibody is obtained from a fairly homogeneous population of antibodies, i.e., individual antibodies within the population are identical, excluding antibodies with possible natural mutations that may be present in a small subpopulation of antibody molecules. Monoclonal antibodies include "chimeric" antibodies in which part of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain(s) is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies if they exhibit the desired antagonistic activity.

Monoclonal antibodies can be produced by any procedure that produces monoclonal antibodies. In the hybridoma method, a mouse or other suitable animal host is typically immunized with an immunizing agent to isolate lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro .

Antibodies can also be produced using recombinant DNA methods. The DNA encoding the antibodies described can be readily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of murine antibodies). Banks of antibodies or active antibody fragments can also be generated and sorted using phage display technologies.

Methods for producing antibodies using protein chemistry are also known in the art. One way to produce antibody-containing proteins is to combine two or more peptides or polypeptides together using protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) chemistry or Boc (tert-butyloxycarbonyl) chemistry. (Applied Biosystems, Inc., Foster City, California, USA). One skilled in the art can readily appreciate that a peptide or polypeptide corresponding to an antibody, for example, can be synthesized using standard chemical reactions. For example, a peptide or polypeptide may be synthesized and not cleaved from the synthesis resin, whereas another antibody fragment may be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group that is operably blocked on the other fragment. These two fragments, through a peptide condensation reaction, can be covalently linked through a peptide bond at their carboxyl and amino termini, respectively, to form an antibody or fragment thereof. Alternatively, the peptide or polypeptide is synthesized independently in vivo as described above. Once isolated, these independent peptides or polypeptides can be combined to form an antibody or antigen-binding fragment thereof through similar peptide condensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segments allows relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides, or entire protein domains. Alternatively, native chemical ligation of synthetic peptides can be used to construct long synthetic peptides or polypeptides from shorter peptide fragments. This method involves a two-step chemical reaction. The first step is the chemoselective reaction of an unprotected synthetic alpha-thioester peptide with another unprotected peptide segment containing a cis-amino-terminal residue to produce a thioester-linked intermediate as the starting covalent product. This intermediate, without changing the reaction conditions, undergoes a spontaneous rapid intramolecular reaction with the formation of a native peptide bond at the site of ligation.

B. Methods for obtaining proteins

The described proteins, polypeptides, fragments, variants and fusions thereof can be prepared using conventional methods known in the art. Isolated fusion proteins can be produced, for example, by chemical synthesis or recombinant production in a host cell. For the recombinant production of a protein, polypeptide, fragment, variant, or fusion thereof, a nucleic acid comprising a nucleotide sequence encoding a protein, polypeptide, fragment, variant, or fusion thereof may be used to transform, transduce, or transfect a bacterial or eukaryotic host cell (e.g., an insect cell , yeast or mammal). In general, nucleic acid constructs include a regulatory sequence operably linked to a nucleotide sequence encoding a protein, polypeptide, fragment, variant, or fusion thereof. Regulatory sequences (also referred to herein as expression control sequences) typically do not encode a gene product, but instead affect the expression of the nucleic acid sequences to which they are operably linked.

Useful prokaryotic and eukaryotic systems for the expression and production of polypeptides are well known in the art, including, for example, Escherichia coli strains such as BL-21, and cultured mammalian cells such as CHO cells.

A number of virus-based expression systems can be used to express fusion proteins in eukaryotic host cells. Viral expression systems are well known in the art and include (but are not limited to) baculovirus vectors, SV40 vectors, retroviral vectors or non-vaccinia viral vectors.

Mammalian cell lines stably expressing proteins, polypeptides, fragments, variants or fusions thereof can be produced using expression vectors with appropriate control elements and a selectable marker. For example, the eukaryotic expression vectors pCR3.1 (Invitrogen Life Technologies) and p91023(B) (Wong et al . (1985) Science 228: 810-815) are suitable for the expression of proteins, polypeptides, fragments, variants or fusions thereof, for example, in Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells and human vascular endothelial cells (HUVEC). Additional useful expression systems include the GS Gene Expression System™, which is available through Lonza Group Ltd.

Following introduction of the expression vector by electroporation, lipofection, co-precipitation of calcium phosphate or calcium chloride, dextran DEAE, or other suitable transfection method, stable cell lines can be selected (eg, by metabolic selection or detection of antibiotic resistance: G418, kanamycin, or hygromycin). Transfected cells can be cultured such that the polypeptide of interest is expressed; this polypeptide can be recovered, for example, from cell culture supernatant or from lysed cells. Alternatively, a protein, polypeptide, fragment, variant, or fusion thereof can be produced by (a) ligating the amplified sequences into a mammalian expression vector such as pcDNA3 (Invitrogen Life Technologies); and (b) in vitro transcription and translation using wheat germ extract or rabbit reticulocyte lysate.

Proteins, polypeptides, fragments, variants or fusions thereof can be isolated using, for example, chromatographic methods such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, DEAE ion exchange chromatography, gel filtration and hydroxylapatite chromatography. In some embodiments of the present invention, proteins, polypeptides, fragments, variants, or fusions thereof can be artificially engineered to contain an additional domain containing an amino acid sequence that allows the polypeptides to be captured on an affinity matrix. For example, an Fc-fusion polypeptide in a cell culture supernatant or cytoplasmic extract can be isolated using a protein A column. Additionally, tags such as c-myc, hemagglutinin, polyhistidine, or Flag™ (Kodak) can be used to facilitate purification of the polypeptides. Such tags can be inserted anywhere in the polypeptide, including at the carboxyl or amino terminus. Other fusions that may be useful include enzymes that aid in polypeptide detection, such as alkaline phosphatase. Immunoaffinity chromatography can also be used to purify polypeptides. It is further possible to artificially engineer fusion proteins to contain a secretory signal (if the secretory signal is not already present) that causes the cells in which it is produced to secrete the proteins, polypeptides, fragments, variants, or fusions thereof. Secreted proteins, polypeptides, fragments, variants and fusions thereof can then be freely isolated from the cellular environment.

C. Methods for producing individual nucleic acid molecules

Individual nucleic acid molecules can be obtained using standard technologies, including, without limitation, well-known molecular cloning and chemical nucleic acid synthesis technologies. For example, polymerase chain reaction (PCR) technologies can be used to obtain isolated nucleic acid encoding a variant polypeptide. PCR is a technology in which enzymatic amplification of target nucleic acids occurs. In general, sequence information from the ends of the region of interest or beyond can be used to design oligonucleotide primers that are identical in sequence to the opposite strands of the template that is being amplified. PCR can be used to amplify specific DNA sequences as well as RNA, including sequences of total genomic DNA or total cellular RNA. Primers are typically 14 to 40 nucleotides in length, but can vary from 10 nucleotides to hundreds of nucleotides. General PCR technologies are described, for example, in the book PCR Primer: A Laboratory Manual , ed. Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. When using RNA as a template source, reverse transcriptase can be used to synthesize a strand of complementary DNA (cDNA). Ligase chain reaction, strand displacement amplification, self-sustaining sequence replication, or nucleic acid sequence-based amplification can also be used to obtain individual nucleic acids. See, for example, Lewis (1992) Genetic Engineering News 12:1; Guatelli et al . (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878; and Weiss (1991) Science 254: 1292-1293.

Individual nucleic acids can be chemically synthesized either as a single nucleic acid molecule or as a series of oligonucleotides (eg, using phosphoramidite technology for automated DNA synthesis in the 3' to 5' direction). For example, one or more pairs of long oligonucleotides (eg, >100 nucleotides) containing the desired sequence can be synthesized, where each pair contains a short complementarity segment (eg, about 15 nucleotides) such that when the pair of oligonucleotides hybridizes, a duplex is formed. DNA polymerase can be used to expand oligonucleotides, resulting in one double-stranded nucleic acid molecule per oligonucleotide pair, which can then be ligated into a vector. Individual nucleic acids can also be obtained by mutagenesis. Nucleic acids encoding proteins can be mutated using standard techniques, including oligonucleotide-directed mutagenesis and/or site-directed mutagenesis using PCR. See Short Protocols in Molecular Biology . Chapter 8, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al. , 1992.

IV. Antibody tests and screening

Antibody screening tests include:

1. Analysis of the binding affinity of B7-H4-Fc to ligands in comparison with B7-H4.

2. Functional assays to confirm that B7-H4-Fc interferes with signal transduction in cells expressing B7-H4. These assays can use reporter cells or primary B7-H4+ cells, which are another option.

B7-H4-deficient (“gene knockout”) or wild-type mice can be used to generate high-affinity monoclonal antibodies (mAbs) to B7-H4 using proprietary immunization methods. NZB/WF1 mice, which are prone to autoimmune diseases, can be used to generate monoclonal antibodies to overcome “tolerance.”

1. Phase I Screening: Binding of monoclonal antibodies (mAbs) to cell lines transfected to express B7-H4 on the cell surface. In addition, monoclonal antibodies must have the ability to bind endogenously expressed B7-H4 on the surface of primary human cell subpopulations. These monoclonal antibodies should be highly specific for B7-H4. Antibody screening can be done using an enzyme-linked immunosorbent assay (ELISA) with purified B7-H4 protein to detect anti-B7-H4 antibodies.

2. Phase II screening: B7-H4-specific monoclonal antibodies must block the binding of B7-H4 to its ligands and/or target cells.

3. Phase III screening: Functional assays to confirm that anti-B7-H4 monoclonal antibodies or a combination of monoclonal antibodies modulate B7-H4 signaling. These assays will use cell lines that express endogenous B7-H4, or primary cells such as human monocytes, macrophages, and dendritic cell subsets, to assess function in the presence of anti-B7-H4 monoclonal antibodies. In addition, reporter cell lines can be used to determine whether signaling cascades such as NFkB (nfkb reporter) or NFAT (NFAT reporter) are altered after culture with monoclonal antibodies to B7-H4.

4. Phase IV screening: Functional assays to determine the ability of anti-B7-H4 monoclonal antibodies to induce, through other mechanisms, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or cell apoptosis in B7-H4-expressing cell lines. Specifically, monoclonal antibodies to B7-H4 will be tested for depletion ability by one of these methods against leukemia cell lines known to express B7-H4 on the cell surface. Monoclonal antibodies to B7-H4 can also be artificially generated to deplete cells expressing B7-H4 and tested by known methods, as described later herein.

5. Phase V Screening: Functional assays to determine the ability of anti-B7-H4 monoclonal antibodies to deliver or induce a negative signal (agonist) through B7-H4 into cells expressing B7-H4 to inhibit cellular function. Cell lines endogenously expressing B7-H4 or transfectant cell lines will be assessed for changes in phenotype and survival after culture with anti-B7-H4 monoclonal antibodies. In other assays, reporter cell lines will be used to identify in anti-B7-H4 monoclonal antibodies modulatory positive signaling cascades such as NF-kB (NF-kB reporter) or other known cellular signaling reporters. Induction of apoptosis in cell lines will also be assessed.

Phase II and III assays can be used to predict the concentrations of anti-B7-H4 monoclonal antibodies required to block physiological levels of ligands in vivo .

V. Method of application

B7-H4 antagonists or agonists may be used to modulate immune responses in subjects requiring such treatment.

Typical applications are discussed in detail below.

A. Stimulation of the immune response

1.Therapeutic strategies

Methods of inducing or enhancing an immune response in a subject are provided. Typically, these methods involve administering to a subject an effective amount of an immunomodulatory agent or cells that have been primarily exposed to an immunomodulatory antigen ex vivo . The immune response may be, for example, a primary immune response to an antigen or an enhancement of effector cell function, such as increasing antigen-specific T cell proliferation, increasing T cell cytokine production, promoting differentiation, or a combination thereof. In some embodiments of this invention, the agent may enhance the development of naïve T cells into Thl, Thl7, Th22, or other cells that secrete or cause other cells to secrete inflammatory molecules, including but not limited to IL-lp, TNF-alpha , TGF-beta, IFN-gamma, IL-17, IL-6, IL-23, IL-22, IL-21 and matrix metalloproteinases (MMPs). In some embodiments of the present invention, the agent may reduce or inhibit the activity of regulatory T cells (Tregs), reduce the production of cytokines such as IL-10 from Tregs, reduce the differentiation of Tregs, reduce the number of Tregs, reduce the ratio of Tregs in the immune cell population, or reduce Treg survival. The immunomodulatory agent may be administered to a subject in need thereof in an effective amount to overcome T cell exhaustion and/or T cell anergy. Overcoming T cell exhaustion or T cell anergy can be determined by measuring T cell function by known methods.

These methods can be used in vivo or ex vivo as therapeutic methods for stimulating an immune response. Thus, in some embodiments of the present invention, the agent or a nucleic acid encoding the agent is administered directly to the subject. In some embodiments of the present invention, the agent or a nucleic acid encoding the agent is contacted with cells (eg, immune cells) ex vivo and the treated cells are administered to the subject (eg, adoptive transfer). In general, the immunomodulatory agents described can be used to treat a subject having or predisposed to any disease or disorder to which the subject's immune system mounts an immune response. These agents may cause a stronger immune response. The described compositions are useful for stimulating or enhancing immune responses involving T cells.

Immunomodulatory agents used to enhance the immune response are typically agents that reduce B7-H4 expression, ligand binding, cross-linking, negative signaling, or a combination thereof. For example, the agent may be a B7-H4 antagonist, such as an antagonist of an anti-B7-H4 antibody, or an antigen-binding fragment thereof. Such an agent may also be a B7-H4 polypeptide, for example a soluble polypeptide, or a fusion protein thereof, which may serve as a decoy receptor for one or more B7-H4 ligands or receptors.

Blockade of B7-H4, for example by an anti-B7-H4 antibody blocking function, may be an alternative or complementary agent to soluble B7-H4 polypeptides and fusion proteins. For example, in some embodiments of the present invention, the B7-H4 blockade is combined with a decoy receptor, such as soluble B7-H4 or a fusion protein thereof. This combination treatment (eg, B7-H4-Fc and B7-H4 blockade) may be complementary.

In some embodiments of the present invention, therapy that stimulates an immune response (eg, in the treatment of cancer or infections) includes depleting B7-H4+ cells.

The creation and identification of depleting monoclonal antibodies to B7-H4 can be performed in accordance with known methods of artificial generation and screening, including the methods that are presented in this application. See, for example, Reff, et al. , Blood (“Blood”). Vol.83, No. 2, 1994: pp. 435-445, which describes the production of an anti-CD20 chimeric antibody that binds to human Clq and mediates complement-dependent cell lysis (CDCL) in the presence of human complement and antibody-dependent cellular cytotoxicity (ADCC) using human effector cells. Rituximab destroys B cells and is therefore used to treat diseases that are characterized by hyperactivity, dysfunction or excessive numbers of B cells. Other β-cell depleting antibodies include ocrelizumab and ofatumumab. In another example, anti-CD3 antibodies can preferentially target and deplete activated effector T cells while sparing CD4 ⁺ Foxp3 ⁺ regulatory cells. Antibodies transiently deplete T cells, although they exhibit little or no complement-dependent and antibody-dependent cellular cytotoxicity. Redirected cell lysis has been shown to be due to the ability to cross-link CD3 molecules expressed by two different cells (cytotoxic CD8+ T cells on the one hand and other target T cells on the other), but T cell depletion is primarily a result of AICD (reviewed in You Front Immunol 2015;6:242).

An agent that inhibits or blocks the B7-H4-attenuated immune response may be administered to a subject in need of enhancing the immune response in an amount effective to enhance antigen uptake by antigen presenting cells (APCs).

2. Subjects being treated

A. Cancer treatment

The described compositions and methods can be used for the treatment of cancer. Typically, the agents are used to stimulate or enhance the immune response to cancer in a subject by administering to the subject an amount of an immunomodulatory agent that reduces B7-H4 expression, ligand binding, cross-linking, negative signaling, or a combination thereof. During their development, cancer cells acquire a characteristic set of functional capabilities, although through different mechanisms. Such possibilities include cell loss of apoptosis, self-sufficiency of growth signals, insensitivity to anti-growth signals, tissue invasion/metastasis, limitless replication potential, and robust angiogenesis. The term "cancer cell" should cover both precancerous and malignant cancer cells. In some embodiments of the present invention, cancer refers to a benign tumor that remains localized. In other embodiments of the present invention, cancer refers to a malignant tumor that has invaded and destroyed adjacent structures of the body, and has spread to distant sites. In other embodiments, cancer is associated with a specific cancer antigen (e.g., pancarcinoma antigen (KS 1/4), ovarian carcinoma antigen (CA125), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), CD 19, CD20, HER2 /neu, etc.).

The methods and compositions described herein are useful for the treatment or prevention of various types of cancer or other abnormal proliferative diseases, including but not limited to the following: carcinoma, including cancer of the bladder, breast, colon, kidney, liver, lung , ovaries, pancreas, stomach, cervix, thyroid and skin; including squamous cell carcinoma; lymphoid hemoblastoses, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; myeloid hemoblastoses, including acute and chronic myeloid leukemia and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; other tumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscaroma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid cancer and teratocarcinoma.

Cancers caused by aberrations in apoptosis can also be treated with the described methods and compositions. Such cancers may include, but are not limited to, follicular lymphomas, p53-mutant carcinomas, hormone-dependent breast, prostate, and ovarian tumors, as well as precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndromes. In specific embodiments of the present invention, these methods and compositions treat or prevent malignant or dysplastic changes (such as metaplasias and dysplasias) or hyperproliferative disorders in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus. In other specific embodiments of the present invention, these methods and compositions treat or prevent sarcoma, melanoma, or leukemia.

The described compositions and methods are particularly useful for treating cancers associated with cells expressing abnormally high levels of B7-H4.

Specific cancers and related disorders that can be treated or prevented by the methods and compositions described herein include, but are not limited to, leukemias, including but not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, leukemia and myelodysplastic syndrome, chronic leukemia, including (but not limited to), chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas, including (but not limited to) Hodgkin's lymphoma or non-Hodgkin's lymphoma (eg, diffuse large B-cell lymphoma (DLBCL) not expressing anaplastic lymphoma kinase (ALK) (ALK-negative DLBCL); diffuse large B-cell lymphoma (DLBCL), anaplastic lymphoma kinase (ALK)-expressing (ALK-positive DLBCL), anaplastic large cell lymphoma (ALCL), anaplastic lymphoma kinase (ALK)-expressing (AKL-positive ALCL), acute myeloid lymphoma (AML)); multiple myelomas, including (but not limited to) smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytomas; Waldenström's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; Bone and connective tissue sarcomas, including (but not limited to) bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, bone fibrosarcoma, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma , liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors, including (but not limited to) glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, non-glial tumors, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer, including (but not limited to), adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease and inflammatory breast cancer ; adrenal cancer, including (but not limited to) pheochromocytoma and adrenocortical carcinoma; thyroid cancer, including (but not limited to) papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer, including (but not limited to) insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor and carcinoid or islet cell tumor; pituitary cancer, including (but not limited to) Cushing's disease, prolactin-secreting tumor, acromegaly and diabetes insipidus; eye cancer, including (but not limited to) ocular melanoma such as iris melanoma, choroidal melanoma, ciliary body melanoma and retinoblastoma; vaginal cancer, including (but not limited to) squamous cell carcinoma, adenocarcinoma and melanoma; vulvar cancer, including (but not limited to) squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma and Paget's disease; cervical cancer, including (but not limited to) squamous cell carcinoma and adenocarcinoma; uterine cancer, including (but not limited to) endometrial carcinoma and uterine sarcoma; ovarian cancer, including (but not limited to) epithelial ovarian carcinoma, borderline tumor, germ cell tumor and stromal tumor; esophageal cancer, including (but not limited to) squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma and oat cell (small cell) carcinoma; gastric cancer, including (but not limited to) adenocarcinoma with the formation of necrotic ulcers (polypoid), ulcerating, superficial spreading, diffuse spreading, malignant lymphoma, liposarcoma, fibrosarcoma and carcinosarcoma; colon cancer; rectal cancer; liver cancer, including (but not limited to) hepatocellular carcinoma and hepatoblastoma, gallbladder cancer, including (but not limited to) adenocarcinoma; cholangiocarcinomas, including (but not limited to) papillary, nodular and diffuse; lung cancer, including (but not limited to) non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell lung cancer and small cell lung cancer; testicular cancer, including (but not limited to) germinoma, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratomucarcinoma, choriocarcinoma (yolk sac tumor); prostate cancer, including (but not limited to) adenocarcinoma, leiomyosarcoma and rhabdomyosarcoma; penile cancer; oral cancer, including (but not limited to) squamous cell carcinoma; basal cancer; salivary gland cancer, including (but not limited to) adenocarcinoma, mucoepidermoid carcinoma and adenoid cystic carcinoma; pharyngeal cancer, including (but not limited to) squamous cell carcinoma and verrucous carcinoma; skin cancer, including (but not limited to) basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancer, including (but not limited to) renal cell carcinoma, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell carcinoma (renal pelvis and/or ureter); Wilms tumor; Bladder cancer, including (but not limited to) transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma and papillary adenocarcinoma carcinoma (for a review of such diseases, see Fishman et al ., 1985, Medicine, 2d ed., J. B. Lippincott Co., Philadelphia and Murphy et al. , 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery. cancer diagnosis, treatment and recovery"), Viking Penguin, Penguin Books USA, Inc., United States of America).

b. Treatment of infections

The described compositions and methods can be used to treat infections and infectious diseases. Typically, these agents are used to stimulate or enhance an immune response to an agent causing an infection in a subject by administering to the subject an amount of an immunomodulatory agent that reduces B7-H4 expression, ligand binding, cross-linking, negative signaling, or a combination thereof. This method may relieve one or more symptoms of the infection.

Neutrophils are a major component of the host's innate defense against infection and also contribute to the development of autoimmune diseases and chronic inflammation. During infection, neutrophils quickly migrate to sites of inflammation, are activated and trigger a cascade of protective mechanisms, including phagocytosis, destruction and degeneration of microorganisms by antimicrobial and proteolytic proteins, as well as the generation of reactive oxygen species. Neutrophils are also involved in tissue destruction, remodeling, wound healing, and modulation of other inflammatory and adaptive immune components. Due to the short lifespan of neutrophils, their numbers must be constantly replenished during infection and inflammation by increasing their production from myeloid progenitor cells in the bone marrow. B7-H4 inhibits in vitro the growth of bone marrow-derived neutrophil progenitor cells, indicating an inhibitory function of B7-H4 in neutrophil formation (Zhu, G. et al. , Blood, 113: 1759-1767 ( 2009)).

In one embodiment, the present invention provides a method of treating an infection in a patient by administering an agent that inhibits or blocks an immune response weakened by B7-H4 in an amount effective to produce neutrophils and/or enhance innate immunity.

The infection or disease may be caused by a bacterium, virus, protozoan, helminth, or other microbial pathogen that penetrates intracellularly and is attacked, in particular, by cytotoxic T lymphocytes.

The infection or infectious disease can be acute or chronic. An acute infection is usually an infection of short duration. During acute microbial infection, immune cells begin to express immunomodulatory receptors. Accordingly, in some embodiments of the present invention, the method includes enhancing a stimulating immune response to an acute infection.

The infection may be caused by, for example, but not limited to , Candida albicans, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria meningitidis, Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, or Mycobacterium.

In some embodiments of the present invention, the described compositions are used to treat chronic infections, such as infections in which T cell exhaustion or T cell anergy has occurred, resulting in the infection remaining in the host for an extended period of time.

Typical infections that are treated are chronic infections caused by hepatitis virus, human immunodeficiency virus (HIV), human T-lymphotrophic virus (HTLV), herpes virus, Epstein-Barr virus or human papillomavirus.

Since cure of viral infections occurs primarily through the action of T cells, increasing T cell activity would be therapeutically beneficial in situations where the animal or human requires more rapid or complete clearance of the infectious viral agent from the body. Thus, the disclosed compositions can be administered for the treatment of local or systemic viral infections, including but not limited to immunodeficiency (e.g., HIV), papilloma (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., human influenza A virus), colds (eg human rhinovirus) and other viral infections such as HTLV, hepatitis virus, respiratory syncytial virus, vaccinia virus and rabies virus. These molecules can be administered topically to treat viral skin diseases such as herpes lesions or herpes zoster, or genital warts. These molecules can also be administered systemically to treat systemic viral diseases, including but not limited to AIDS, influenza, the common cold or encephalitis.

Representative infections that may be treated include, but are not limited to, those caused by microorganisms including, but not limited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type B (HIB), Hyphomicrobium, Legionella, Leptspirosis, Listeria, Meningococcus A, B and c, Methanobacterium, Micrococcus, Mycoplasma, Myxococcus, Neisseria, Nitrobacter , Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus and Treponema, Vibrio, Yersinia, Cryptococcus neoformans, Histoplasma capsulatum, C andida albicans, Candida tropicalis , Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis and Schistosoma mansoni.

Other microorganisms that can be treated using the compositions and methods described include bacteria such as Klebsiella, Serratia, Pasteurella ; pathogens associated with cholera, tetanus, botulism, anthrax, plague and Lyme disease; or fungal or parasitic pathogens such as Candida (albicans, krusei, glabrata, tropicalis, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrix (schenkii), Blastomyces (dermatitidis), Paracoccidioides (brasiliensis), Coccidioides (immitis ) and Histoplasma (capsulatuma), Entamoeba, histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondi, etc., Sporothrix , Blastomyces, Paracoccidioides, Coccidioides, Histoplasma, Entamoeba, Histolytica, Balantidium, Naegleria, Acanthamoeba, Giardia, Cryptosporidium, Pneumocystis, Plasmodium, Babesia or Trypanosoma , etc.

B. Inhibition of the immune response

1. Therapeutic strategies

Methods are provided for reducing or inhibiting the immune response in a subject. Typically, these methods involve administering to a subject an effective amount of an immunomodulatory agent or cells that have been primarily exposed to the immunomodulatory agent ex vivo . The immune response may be, for example, a primary immune response to an antigen or an increase in cell effector function, such as an increase in antigen-specific T cell proliferation, an increase in T cell cytokine production, promotion of differentiation, or a combination thereof. Thus, in some embodiments of the present invention, the agent reduces T cell proliferation, T cell cytokine production, T cell differentiation, or a combination of these mechanisms. In some embodiments of the present invention, the agent may attenuate the development of naïve T cells into Thl, Thl7, Th22, or other cells that secrete or cause other cells to secrete inflammatory molecules, including but not limited to IL-1p, TNF-alpha, TGF-beta, IFN-gamma, IL-17, IL-6, IL-23, IL-22, IL-21 and matrix metalloproteinases (MMPs). In some embodiments of the present invention, the agent may increase or stimulate the activity of regulatory T cells (Tregs), increase the production of cytokines such as IL-10 from Tregs, increase the differentiation of Tregs, increase the number of Tregs, increase the ratio of Tregs in the immune cell population, or increase Treg survival.

These methods can be used in vivo or ex vivo as treatments that inhibit the immune response. Thus, in some embodiments of the present invention, the agent or the nucleic acid encoding the agent is administered directly to the subject. In some embodiments of the present invention, the agent or a nucleic acid encoding the agent is contacted with cells (eg, immune cells) ex vivo and the treated cells are administered to the subject (eg, adoptive transfer). In general, the described immunomodulatory agents can be used to treat a subject having or predisposed to any disease or disorder to which the subject's immune system mounts an overactive or inappropriate immune response. These agents may produce a less potent immune response. The compositions described are useful for attenuating or inhibiting immune responses involving T cells.

Immunomodulatory agents used to attenuate the immune response are typically agents that enhance B7-H4 expression, ligand binding, cross-linking, negative signaling, or a combination thereof. For example, the agent may be a B7-H4 agonist, such as an anti-B7-H4 agonist antibody or antigen binding fragment thereof.

a. Inflammatory reactions

The described compositions and methods can be used to treat inflammation. Typically, these agents are used to reduce or inhibit the immune response in a subject by administering an amount of an immunomodulatory agent that enhances B7-H4 expression, ligand binding, cross-linking, negative signaling, or a combination thereof. This method can weaken or intensify the symptoms of inflammation. Inflammation can be acute, chronic or chronic recurrent.

In some embodiments of the present invention, immunomodulatory agents slow down the functioning of the immune system. For example, the agent can be used to regulate a hyperinflammatory response that causes damage to healthy tissue. Accordingly, in some embodiments of the present invention, these agents are administered to a subject who is having a hyperinflammatory response. In such cases, it may be beneficial for the subject to regulate excessive immune responses.

b. Inflammatory and autoimmune diseases/disorders

Agents that enhance B7-H4 expression, ligand binding, cross-linking, negative signaling, or a combination thereof may also be used to treat inflammatory or autoimmune diseases and disorders. Representative inflammatory or autoimmune diseases/disorders include (but are not limited to) rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome ( alps), autoimmune thrombocytopenic purpura (ATP), Behçet's disease, bullous pemphigoid, cardiomyopathy, dermatitis in celiac sprue, chronic fatigue and immune dysfunction syndrome (CFIDS syndrome), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, Crest syndrome, disease Crohn's disease, Dego's disease, dermatomyositis, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia - fibromyositis, Grave's disease, Guillain-Barre disease, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin dependent diabetes ( type I), juvenile arthritis, Meniere's disease, mixed connective tissue diseases, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis , Raynaud's disease, Reiter's syndrome, rheumatism, sarcoidosis, scleroderma, Sjögren's syndrome, stiff muscle syndrome, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo and Wegener's granulomatosis.

In some embodiments of the present invention, the inflammation or autoimmune disease is caused by a pathogen or results from an infection.

VI. Combined treatments

The immunomodulatory agents described may be administered to a subject in need thereof, as monotherapy or in combination with one or more additional therapeutic agents. In some embodiments of the present invention, the immunomodulatory agent and the additional therapeutic agent are administered separately but simultaneously. The immunomodulatory agent and the additional therapeutic agent may also be administered in the same composition. In other embodiments of the present invention, the immunomodulatory agent and the second therapeutic agent are administered separately and at different times, but within the same treatment regimen.

The subject may be administered the first therapeutic agent 1, 2, 3, 4, 5, 6 or more hours, or 1, 2, 3, 4, 5, 6, 7 or more days before the administration of the second therapeutic agent. In some embodiments of the present invention, the subject may be administered one or more doses of the first agent 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, or 48 days before the first administration of the second agent. The immunomodulatory agent may be the first or second therapeutic agent.

An immunomodulatory agent and an additional therapeutic agent may be administered as part of the treatment regimen. For example, if the first therapeutic agent can be administered to a subject every fourth day, then the second therapeutic agent can be administered on the first, second, third or fourth day, or a combination thereof. The first therapeutic agent or the second therapeutic agent may be re-administered throughout the treatment regimen.

Exemplary molecules include (but are not limited to) cytokines, chemotherapeutic agents, radionuclides, other immunotherapies, enzymes, antibiotics, antivirals (especially protease inhibitors, as monotherapy or in combination with nucleosides for the treatment of HIV or hepatitis B or C), antiparasitics (against helminths, protozoa), growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and their biologically active fragments (including humanized, single-chain and chimeric antibodies), antigens and vaccines (including adjuvants), peptide drugs, anti-inflammatory, ligands molecules that bind to Toll-like receptors (including, but not limited to, CpG oligonucleotides) to activate the innate immune system, molecules that mobilize and optimize the adaptive immune system, other molecules that activate or enhance the activity of cytotoxic T lymphocytes, natural killer and helper T cells, as well as other molecules that deactivate or reduce the activity of suppressor or regulatory T cells.

Additional therapeutic agents are selected depending on the condition, disorder or disease being treated. For example, an immunomodulatory agent may be administered in conjunction with one or more additional agents that act to enhance or stimulate the immune response, or attenuate or inhibit the immune response.

A. Strengthening immune responses

1. Antimicrobials

For example, the B7-H4 immunomodulatory agent may be used to serve a prophylactic or prophylactic role in the treatment and prevention of disease as described above, as well as in the treatment regimen for severe injuries such as a major burn, open bone fracture, traumatic amputation of a limb, or other wounds. Thus, B7-H4 immunomodulatory agents can be administered to a subject in combination with an antimicrobial agent, such as an antibiotic, antifungal, antiviral, antiparasitic, or essential oil.

In some embodiments of the present invention, the subject is administered a B7-H4 immunomodulatory agent and/or an antimicrobial agent upon admission to the hospital to prevent further bacterial, fungal, or viral complications. This antibiotic can target pathogenic microorganisms, and the immunomodulatory agent B7-H4 can stimulate the immune system to produce an enhanced response to treat or prevent further infection or infectious disease.

2. Chemotherapeutic agents

B7-H4 immunomodulatory agents can be combined with one or more chemotherapeutic agents and pro-apoptotic agents. Representative chemotherapeutic agents include, but are not limited to, amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, chrysantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pen tostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine or a combination thereof. Representative pro-apoptotic agents include, but are not limited to, fludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2), and combinations thereof.

3. Other immunomodulators

a. PD-1 antagonists

In some embodiments of the present invention, the B7-H4 immunomodulatory agents are co-administered with a PD-1 antagonist. PD-1 (programmed death) is a member of the CD28 family of receptors that mediates a negative immune response when induced on T cells. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that reduces T cell proliferation and/or the strength and/or duration of the T cell response. Suitable PD-1 antagonists are described in US Pat. Nos. 8,114,845, 8,609,089, and 8,709,416, which are specifically incorporated herein by reference in their entirety, and include compounds or agents that either bind to and block a PD-1 ligand to interfere with or inhibit ligand binding to the PD-1 receptor, or bind directly to the PD-1 receptor and block it without inducing transduction of an inhibitory signal through the PD-1 receptor.

In some embodiments of the present invention, a PD-1 receptor antagonist binds directly to the PD-1 receptor without initiating inhibitory signal transduction, and also binds to a PD-1 receptor ligand to reduce or inhibit the ligand's signal transduction through the PD-1 receptor. By reducing the number and/or amount of ligands that bind to the PD-1 receptor and trigger inhibitory signal transduction, fewer cells are attenuated by the negative signal provided by PD-1 signal transduction and a stronger immune response can be achieved.

PD-1 signaling is thought to be driven by binding to a PD-1 ligand (such as B7-H1 or B7-DC) in close proximity to a major histocompatibility complex (MHC) peptide antigen (see, for example, Freeman, Proc . Natl Acad Sci US A, 105:10275-10276 (2008). Therefore, proteins, antibodies or small molecules that prevent co-ligation of PD-1 and TCR on the T cell membrane are also useful PD-1 antagonists.

In some embodiments of the present invention, PD-1 receptor antagonists are small molecule antagonists or antibodies that reduce or interfere with PD-1 receptor signal transduction by binding to PD-1 ligands or to PD-1 itself, especially when after such binding does not occur when PD-1 co-ligates with the TCR, thereby causing no inhibitory signal transduction through the PD-1 receptor. Other PD-1 antagonists provided by the methods of this invention include antibodies that bind to PD-1 or PD-1 ligands, and other antibodies.

Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following publications:

PCT/IL03/00425 (Hardy et al ., WO/2003/099196)

PCT/JP2006/309606 (Korman et al ., WO/2006/121168)

PCT/US2008/008925 (Li et al ., WO/2009/014708)

PCT/JP03/08420 (Honjo et al ., WO/2004/004771)

PCT/JP04/00549 (Honjo et al ., WO/2004/072286)

PCT/IB2003/006304 (Collins et al ., WO/2004/056875)

PCT/US2007/088851 (Ahmed et al ., WO/2008/083174)

PCT/US2006/026046 (Korman et al ., WO/2007/005874)

PCT/US2008/084923 (Terrett et al ., WO/2009/073533)

Berger et al. , Clin. Cancer Res., 14:30443051 (2008).

A specific example of an anti-PD-1 antibody is the antibody described in Kosak, US 20070166281 (published July 19, 2007) in paragraph 42), a human anti-PD-1 antibody, which in some embodiments of the present invention is administered at a dose of 3 mg/kg .

Exemplary anti-B7-Hl antibodies include, but are not limited to, those described in the following publications:

PCT/US06/022423 (WO/2006/133396, published December 14, 2006)

PCT/US07/088851 (WO/2008/083174, published July 10, 2008)

US 2006/0110383 (published May 25, 2006)

A specific example of an anti-B7-Hl antibody is the antibody described in (WO/2007/005874, published January 11, 2007)), human anti-B7-Hl antibody.

Additional anti-PD-1 and anti-B7-Hl antibodies described in 2014/0044738, which are incorporated herein by reference in their entirety.

For anti-B7-DC antibodies: see 7,411,051, 7,052,694, 7,390,888 and US Patent Application No. 2006/0099203.

Other exemplary PD-1 receptor antagonists include, but are not limited to, B7-DC polypeptides, including homologs and variants thereof, as well as active fragments of any of the above, and fusion proteins that include any of them. In some embodiments of the present invention, the fusion protein includes a soluble portion of B7-DC linked to the Fc portion of an antibody, such as human IgG, and does not include all or part of the transmembrane portion of human B7-DC.

The PD-1 antagonist may also be a mammalian B7-H1 fragment, such as a mouse or a primate such as a human, where the fragment binds to and blocks PD-1 but does not result in transduction of an inhibitory signal through PD-1. These fragments may also be part of a fusion protein, such as an Ig fusion protein.

Other useful PD-1 polypeptide antagonists include antagonists that bind to PD-1 receptor ligands. Ligands include PD-1 receptor protein or soluble fragments thereof, which can bind to PD-1 ligands, such as B7-H1 or B7-DC, and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction. B7-H1 has also been shown to bind the B7.1 protein (Butte et al., Immunity , Vol. 27, pp. 111-122, (2007)). Such fragments also include the soluble portion of the PD-1 ECD protein, which includes mutations such as the A99L mutation, which increases binding to natural ligands (Molnar et al., PNAS, 105: 10483-10488 (2008)). also useful are B7-1 or its soluble fragments, which can bind to the B7-H1 ligand and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction.

PD-1 and B7-H1 antisense nucleic acids, both DNA and RNA, as well as small interfering RNA (siRNA) molecules can also be PD-1 antagonists. Such antisense molecules prevent the expression of PD-1 on T cells, as well as the production of T cell ligands such as B7-H1, PD-L1 and/or PD-L2. For example, small interfering RNA (siRNA) (e.g., about 21 nucleotides in length that is specific for the gene encoding PD-1, or encoding a PD-1 ligand, and the oligonucleotides of which are readily available commercially) complexes with carriers such as as polyethylenimine (see Cubillos-Ruiz et al. , J. Clin. Invest. 119(8): 2231-2244 (2009), is readily taken up by cells that express PD-1, as well as by PD-1 ligands, and reduces expression these receptors and ligands to produce a decrease in inhibitory signal transduction in T cells, thereby activating the T cells.

b. CTLA4 antagonists

Other molecules useful in mediating the effects of T cells in the immune response are also being considered as additional therapeutic agents. In some embodiments of the present invention, such a molecule is a CTLA4 antagonist, such as an antagonistic anti-CTLA4 antibody. An example of an anti-CTLA4 antibody provided for use in the methods of this invention includes the antibody described in PCT/US2006/043690 (Fischkoff et al ., wo/2007/056539).

Dosages for anti-PD-1, anti-B7-Hl and anti-CTLA4 antibodies are known in the art and may range from, for example, 0.1 to 100 mg/kg, or shorter ranges: 1 to 50 mg/kg or 10 to 20 mg/kg. An appropriate dose for a human may be from 5 to 15 mg/kg, where a dose of 10 mg/kg antibody (eg, human anti-PD-1 antibody) is a particular embodiment of the invention.

Specific examples of an anti-CTLA4 antibody useful in the methods of this invention are ipilimumab, a human anti-CTLA4 antibody administered at a dose of, for example, about 10 mg/kg, and Tremelimumab, a human anti-CTLA4 antibody administered at a dose of, for example, about 15 mg/kg. See also Sammartino, et al. , Clinical Kidney Journal, 3(2): 135-137 (2010), published online December 2009.

In other embodiments of the present invention, the antagonist is a small molecule. A number of small organic compounds have been shown to bind to the B7-1 ligand to prevent binding to CTLA4 (Erbe et al ., J. Biol. Chem ., 277:7363-7368 (2002)). Such small organic compounds can be administered alone or together with an anti-CTLA4 antibody to reduce T cell inhibitory signal transduction.

4. Potentiating agents

In some embodiments of the present invention, the additional therapeutic agents include a potentiating agent. The potentiating agent acts to increase the effectiveness of the regulator that activates the immune response, possibly through more than one mechanism, although the exact mechanism of action is not essential to the general practical application of the present invention.

In some embodiments of this invention, the potentiating agent is cyclophosphamide. Cyclophosphamide (CTX, Cytoxan® or Neosar®) is an oxazaphosphorine drug whose analogues include ifosfamide (IFO, Ifex), perfosfamide, trofosfamide (trofosfamide; Ixoten) and their pharmaceutically acceptable salts, solvates, prodrugs and metabolites (US Patent Application 2007 /0202077, which is fully included). Ifosfamide (MITOXANA®) is a structural analogue of cyclophosphamide, and its mechanism of action is considered identical or largely similar to that of cyclophosphamide. Perfosfamide (4-hydroperoxycyclophosphamide) and trophosfamide are also alkylating agents that are structurally related to cyclophosphamide. For example, perfosfamide alkylates DNA, thereby inhibiting DNA replication and RNA and protein synthesis. New oxazaphosphorine derivatives have been developed and evaluated to improve selectivity and response with reduced host toxicity (Liang J., Huang M., Duan W., Yu XQ, Zhou S. Design of new oxazaphosphorine anticancer drugs. Curr Pharm Des 2007;13(9):963-78. Review). These include mafosfamide (NSC 345842), glufosfamide (D19575, beta-D-glucosyl isophosphoramide mustard), S-(-)-bromophosphamide (CBM-11), NSC 612567 (aldophosphamide perhydrothiazine) and NSC 613060 (aldophosphamide thiazolidine). Mafosfamide is an oxazaphosphorine analogue that is a chemically stable salt of 4-thioethanesulfonic acid 4-hydroxy-CPA. Glufosfamide is an IFO derivative in which isophosphoramide mustard, an alkylating metabolite of IFO, is glycosidically linked to a beta-D-glucose molecule. Additional cyclophosphamide analogs are described in US Pat. No. 5,190,929, entitled “Cyclophosphamide Analogs Useful as Antineoplastic Agents,” which is incorporated herein by reference in its entirety.

Although CTX itself is nontoxic, some of its metabolites are cytotoxic alkylating agents that induce DNA cross-linking and, at higher doses, strand breaks. Many cells are resistant to CTX because they express high levels of the detoxifying enzyme aldehyde dehydrogenase (ALDH). CTX targets proliferating lymphocytes because lymphocytes (but not hematopoietic stem cells) express only low levels of ALDH and dividing cells are quite sensitive to DNA alkylating agents.

Low doses of CTX (<200 mg/kg) can have immunostimulatory effects, including stimulation of antitumor immune responses in humans and in mouse models of cancer (Brode & Cooke CritRev. Immunol. 28:109-126 (2008)). Such low doses are subtherapeutic and do not have direct antitumor activity. In contrast, high doses of CTX suppress the antitumor response. The role of CTX in potentiating the antitumor immune response may be explained by several mechanisms: (a) depletion of CD4+CD25+FoxP3+Tregs (and especially proliferating regulatory T cells (Tregs), which may be particularly suppressive); (b) depletion of B lymphocytes; (c) induction of nitric oxide (NO), leading to suppression of tumor cell growth; (d) mobilization and increase in the formation of CD1 lb+Gr-l+ BMSCs. These primary effects have numerous secondary effects; for example, after depleting regulatory T cells, macrophages produce more IFN-γ and less IL-10. CTX has also been shown to induce type I IFN expression and promote homeostatic lymphocyte proliferation.

Treg depletion is most often cited as the mechanism by which CTX potentiates the antitumor immune response. This conclusion is based in part on the results of adoptive transfer experiments. In the AB1-HA tumor model, treatment with CTX on day 9 yielded a 75% cure rate. Transfer of purified Tregs on day 12 almost completely inhibited the CTX response (van der Most et al . Cancer Immunol. Immunother. 58:1219–1228 (2009). A similar result was observed in the HHD2 tumor model: adoptive transfer of CD4+CD25+ Tregs after pretreatment with CTX abolished the therapeutic response to the vaccine (Taieb, J. J. Immunol. 176:2722-2729 (2006)).

Numerous human clinical trials have shown that, at low doses, CTX is a safe, well-tolerated, and effective agent in stimulating antitumor immune responses (Bas, & Mastrangelo Cancer Immunol. Immunother. 47:1-12 (1998)).

The optimal dose of CTX to potentiate the antitumor immune response is one that reduces the total number of T cells by reducing the number of regulatory T cells below normal levels, being subtherapeutic (see Machiels et al. Cancer Res. 61: 3689-3697 (). 2001)).

In human clinical trials where CTX was used as an immunopotentiating agent, a dose of 300 mg/ ^m2 was typically used. For the average male (height 183 cm (6 ft), weight 78 kg (170 lb) with a body surface area of 1.98 m ² ), a dose of 300 mg/m ² is 8 mg/kg, or 624 mg of total protein. In mouse models of cancer, efficacy was seen at doses ranging from 15 to 150 mg/kg, which represents 0.45 to 4.5 mg of total protein in a 30 g mouse (Machiels et al. Cancer Res. 61:3689-3697 (2001 ), Hengst et al. Cancer Res. 41:2163-2167 (1981), Hengst Cancer Res. 40:2135-2141 (1980)).

For larger mammals, such as a primate, eg human patient, such mg/m ² doses may be used, but unit doses administered over a period of time may also be used. Such unit doses may be administered daily for a specified period of time, for example up to 3 days, or up to 5 days, or up to 7 days, or up to 10 days, or up to 15 days, or up to 20 days, or up to 25 days; all intervals are specifically provided for by the present invention. The same scheme can be applied to other potentiating agents described in this application.

In other embodiments of the present invention, the potentiating agent is an agent that reduces the activity and/or number of regulatory T lymphocytes (T-reg), such as Sunitinib (SUTENT®), anti-TGF-β, or Imatinib (GLEEVAC®). The described treatment regimen may also include the administration of an adjuvant.

Useful potentiating agents also include mitosis inhibitors such as paclitaxol, aromatase inhibitors (eg letrozole) and angiogenesis inhibitors (VEGF inhibitors, eg Avastin, VEGF-Trap) (see, for example, Li et al. Vascular endothelial growth factor blockade reduces intratumoral regulatory T cells and enhances the efficacy of a GM-CSF-secreting cancer immunotherapy. Clin Cancer Res. 2006 Nov 15 ; 12(22):6808-16.)) anthracyclines, oxaliplatin, doxorubicin, TLR4 antagonists and IL-18 antagonists.

B. Weakening of the immune response

1. Immunosuppressants

In some embodiments of the present invention, the immune response or inflammatory/autoimmune disease/disorder is treated by administering to the subject an immunomodulatory agent B7-H4 and a second agent that is an immunosuppressant. Immunosuppressive agents include, but are not limited to, antibodies to other lymphocyte surface markers (eg, CD40, alpha-4 integrin) or cytokine fusion proteins (eg, CTLA-4-Ig (Orencia®), TNFR-Ig (Enbrel ®)), TNF-alpha blockers such as Enbrel, Remicade, Cimzia and Humira, cyclophosphamide (CTX) (i.e. Endoxan®, Cytoxan®, Neosar®, Proxitox®, Revimmune™), methotrexate (MTX) (t i.e. Rheumatrex®, Trexall®), belimumab (i.e. Benlysta®) or other immunosuppressive agents (e.g. cyclosporine A, FK506 class compounds, rapamycin compounds or steroids), drugs with antiproliferative effects, cytostatics or other compounds, which may contribute to immunosuppression.

The therapeutic agent may be a CTLA-4 fusion protein, such as CTLA-4-Ig (abatacept). CTLA-4-Ig fusion proteins compete with the co-stimulatory receptor CD28 on T cells for binding to CD80/CD86 (B7-1/B7-2) on antigen presenting cells and thus function to inhibit T cell activation. In another embodiment of the present invention, the therapeutic agent is a CTLA-4-Ig fusion protein known as belatacept. Belatacept contains two amino acid substitutions (L104E and A29Y) that markedly increase its avidity for CD86 in vivo. In another embodiment of the present invention, the therapeutic agent is Maxy-4.

In another embodiment of the present invention, the therapeutic agent is cyclophosphamide (CTX). Cyclophosphamide (international nonproprietary name for Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune™), also known as cytophosphamide, is a nitrogen mustard alkylating agent from the oxazophorin group. It is used to treat various types of cancer and some autoimmune diseases. Cyclophosphamide (CTX) is the main drug used for diffuse proliferative glomerulonephritis in patients with renal lupus.

This therapeutic agent may be administered in an effective amount to reduce the level of anti-double-stranded DNA (anti-dsDNA) autoantibodies in the blood or serum and/or to reduce proteinuria in a patient in need thereof.

In another embodiment of the present invention, the therapeutic agent increases the amount of adenosine in the blood serum, see, for example, WO 08/147482. For example, the second therapeutic agent may be CD73-Ig, recombinant CD73, or another agent (eg, a cytokine or monoclonal antibody or small molecule) that increases CD73 expression, see, for example, WO 04/084933. In another embodiment of the present invention, the therapeutic agent is interferon-beta.

The therapeutic agent may be a small molecule that inhibits or reduces differentiation, proliferation, activity and/or cytokine production and/or secretion by Thl, Thl7, Th22 cells and/or other cells that secrete or cause other cells to secrete inflammatory molecules, including but not limited to but not limited to) IL-1p, TNF-alpha, TGF-beta, IFN-gamma, IL-18, IL-17, IL-6, IL-23, IL-22, IL-21 and matrix metalloproteinases (MMPs). In another embodiment of the present invention, the therapeutic agent is a small molecule that interacts with T regulatory cells (T-reg), increases T-reg activity, promotes or enhances the secretion of IL-10 by T regulatory cells, increases the number of T-regs , increases T-reg suppressive capacity or a combination of the above.

In some embodiments of the present invention, the composition increases T-reg activity or production. Exemplary agents that enhance T-reg activity include, but are not limited to, the glucocorticoid fluticasone, salmeteroal, antibodies to IL-12, IFN-γ and IL-4, vitamin D3 and dexamethasone, and combinations thereof.

In some embodiments of the present invention, the therapeutic agent is an antibody, such as a function-blocking antibody, to a proinflammatory molecule such as IL-6, IL-23, IL-22, or IL-21.

As used herein, the term “rapamycin compound” includes the neutral tricyclic compound rapamycin, rapamycin derivatives, rapamycin analogs, and other macrolide compounds that are believed to have the same mechanism of action as rapamycin (eg, inhibition of cytokine function). The term "rapamycin compounds" includes compounds having structural similarity to rapamycin, for example compounds with a similar macrocyclic structure, which have been modified to increase their therapeutic effectiveness. Exemplary rapamycin compounds are known in the art (see, for example, W095122972, WO 95116691, WO 95104738, US Patent Nos. 6015809; 5989591; US Patent Nos. 5567709; 5559112; 5530006; 5484790; 5385908; 5202332; 5162333; 5780462; 5120727).

The term "FK506 class compounds" includes fk506 and derivatives, as well as analogues of FK506, for example, compounds with structural similarity to FK506, for example, compounds with a similar macrocyclic structure, which have been modified to increase their therapeutic effectiveness. Examples of compounds of the FK506 class include, for example, those described in WO 00101385. In some embodiments of the present invention, the term “rapamycin compound” as used herein does not include compounds of the fk506 class.

2. Anti-inflammatory

Other suitable therapeutic agents include, but are not limited to, anti-inflammatory agents. The anti-inflammatory agent may be non-steroidal, steroidal, or a combination thereof. In one embodiment, the present invention provides oral compositions containing from about 1% (of the total weight of the composition) to about 5% (of the total weight of the composition), most often about 2.5% (of the total weight of the composition), or anti-inflammatory agent. Representative examples of non-steroidal anti-inflammatory agents include, but are not limited to, oxicams such as piroxicam, isoxicam, tenoxicam, sudoxicam; salicylates such as aspirin, disalcid, benorylate, trilisate, safaprin, solprin, diflunisal and fendosal; acetic acid derivatives such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acematacin, fentiazac, zomepirac, clindanac, oxepinac, felbin and ketorolac; fenamates such as mefenamic, meclofenamic, flufenamic, niflumic and tolfenamic acids; and propionic acid derivatives such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indopropfen, pyrprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen and tiaprofen; pyrazoles such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone and trimethasone. Mixtures of these nonsteroidal anti-inflammatory agents may also be used.

Representative examples of steroidal anti-inflammatory drugs include, without any limitation, corticosteroids such as hydrocortisone, hydroxyl-triamcinolone, alpha-methyldexamethasone, dexamethasone phosphate, beclomethasone dipropionates, clobetasol valerate, desonide, deoxymethasone, deoxycorticosterone acetate, dexamethasone, dichlorizone, diflorazone di acetate, diflucortolone valerate , fluadrenolone, fluclorolone acetonide, fludrocortisone, flumethasone pivalate, fluocinolone acetonide, fluocinonide, flucortine butyl esters, fluocortolone, fluprednidene (fluprednilidene) acetate, flurandrenolone, chalcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylpred nisolone, triamcinolone acetonide, cortisone, cortodoxon, flucetonide, fludrocortisone, difluorozone diacetate, fluradrenolone, flucrocortisone, diflorazone diacetate, fluradrenolone acetonide, medrisone, amcinafel, amcinafide, betamethasone and its other esters, chlorprednisone, chlorprednisone acetate, clocortelone, cleskinolone, dichlorizone, diflurprednate, flucloronide, flunisol id, fluorometholone, fluperolone, fluprednisolone, hydrocortisone valerate , hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone and mixtures thereof.

VII. Sets

The disclosed B7-H4 immunomodulatory agents may be packaged in a hermetically sealed container, such as an ampoule or sachet, with the quantity indicated. Such an agent may be supplied as a dry, sterilized, lyophilized powder or an anhydrous concentrate in a sealed container and may be reconstituted, for example, with water or saline to the appropriate concentration for administration to a subject. For example, the agent may be supplied as a dry, sterile, lyophilized powder in a sealed container in a single dose of at least 5 mg, or at least 10 mg, at least 15 mg, at least 25 mg, at least at least 35 mg, at least 45 mg, at least 50 mg or at least 75 mg. The lyophilized agent can be stored at 2 to 8°C in the original container and is usually administered within 12 hours, or within 6 hours, or within 5 hours, or within 3 hours, or within 1 hour after reconstitution.

In an alternative embodiment of this invention, the agent may be supplied in liquid form in a sealed container with the amount and concentration indicated. In some embodiments of this invention, the liquid form of the agent may be provided in a sealed container comprising at least 1 mg/ml or at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml. ml, at least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 50 mg/ml, at least 100 mg/ml, at least 150 mg/ml, at least 200 mg/ml agent.

Also available are pharmaceutical packages and kits comprising one or more containers filled with an agent. In addition, one or more other prophylactic or therapeutic agents useful for treating the disease may also be included in the pharmaceutical package or kit. The pharmaceutical package or kit may also include one or more containers filled with one or more ingredients of the described pharmaceutical compositions. In addition, such container(s) may be accompanied by a notice in a form prescribed by the government agency regulating the manufacture, use, or sale of drugs or biological products that reflects that agency's approval of the manufacture, use, or sale for human administration.

Kits designed for the methods described above are also available. Embodiments of the invention typically include one or more B7-H4 immunomodulatory agents. In specific embodiments of the present invention, the kit also includes one or more other prophylactic or therapeutic agents useful for treating cancer in one or more containers. In other embodiments of the present invention, the kit also includes one or more anti-inflammatory agents useful for treating inflammatory and autoimmune diseases in one or more containers.

Unless otherwise defined, all technical and scientific terms as used in this application have the same meanings as commonly understood by one skilled in the art to which the invention is described. Publications cited in this application and the materials to which they are cited are expressly incorporated herein by reference.

Those skilled in the art will identify, or be able to determine, by no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to fall within the scope of the following claims.

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LIST OF SEQUENCES

<110> NextCure, Inc.

Langerman, Soloman

Liu, Linda

Flies, Dallas B. (Flies, Dallas B)

<120> Antibodies B7-H4 and methods of their use

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Trp Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr Lys Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Thr Val Ser Ser

115

<210> 9

<211> 5

<212> Protein

<213> House mouse (Mus musculus)

<400> 9

Ser Tyr Trp Met His

15

<210> 10

<211> 18

<212> Protein

<213> House mouse (Mus musculus)

<400> 10

Ala Ile Tyr Pro Gly Asn Ser Asp Thr Lys Tyr Asn Gln Lys Phe Lys

1 5 10 15

Asp Lys

<210> 11

<211> 8

<212> Protein

<213> House mouse (Mus musculus)

<400> 11

Thr Val Arg Asn Val Met Asp Tyr

15

<210> 12

<211> 351

<212> DNA

<213> House mouse (Mus musculus)

<400> 12

gaggttcagc tccagcagtc tgggactgtg ctggcaaggc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggcta cacctttacc agctactgga tgcactggat aaaacagagg 120

cctggacagg gtctggaatg gattggcgct atttatcctg gaaatagtga tactaaatac 180

aaccagaagt tcaaggacaa ggccaaactg actgcagtca catctgccag cactgcctac 240

atggagctca gcagcctgac aaatgaggac tctgcggtct attactgtac atctacggta 300

cggaatgtta tggactactg gggtcaagga acctcagtca ccgtctcctc a 351

<210> 13

<211> 107

<212> Protein

<213> House mouse (Mus musculus)

<400> 13

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Ala Ser Leu Gly

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Ser Phe Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Ser

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 14

<211> 11

<212> Protein

<213> House mouse (Mus musculus)

<400> 14

Gln Ala Thr Gln Asp Ile Val Lys Ser Leu Asn

1 5 10

<210> 15

<211> 7

<212> Protein

<213> House mouse (Mus musculus)

<400> 15

Tyr Thr Ala Gln Leu Ala Glu

15

<210> 16

<211> 9

<212> Protein

<213> House mouse (Mus musculus)

<400> 16

Leu Gln Phe Tyr Glu Phe Pro Pro Thr

15

<210> 17

<211> 321

<212> DNA

<213> House mouse (Mus musculus)

<400> 17

gaaatccaga tgacccagtc tccatcctct atgtctgcat ctctgggaga cagaataacc 60

atcacttgcc aggcaactca agacattgtt aagagtttaa actggtatca acaaaaacca 120

gggaaacccc cttcattcct gatctattat acagctcaac tggcagaagg ggtcccatca 180

aggttcagtg gcagtgggtc tgggtcagac tattctctga caatcagcaa cctggagtct 240

gaagattttg cagactatta ctgtctacag ttttatgagt ttcctccgac gttcggtgga 300

ggcaccaagc tggaaatcaa a 321

<210> 18

<211> 107

<212> Protein

<213> House mouse (Mus musculus)

<400> 18

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 19

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 19

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Phe Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 20

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 20

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Phe Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 21

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 21

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 22

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 22

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Phe Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 23

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 23

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Phe Ala Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 24

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 24

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Phe Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly 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> 25

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 25

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Phe Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly 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> 26

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 26

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly 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> 27

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 27

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Phe Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly 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> 28

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 28

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Phe Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly 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> 29

<211> 117

<212> Protein

<213> House mouse (Mus musculus)

<400> 29

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Val Ser Gly Tyr Pro Phe Thr Ser Tyr

20 25 30

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

35 40 45

Gly Ala Ile Tyr Pro Gly Lys Ser Asp Thr Glu Tyr Asn Pro Asn Phe

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ser Thr Trp Thr His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 30

<211> 17

<212> Protein

<213> House mouse (Mus musculus)

<400> 30

Ala Ile Tyr Pro Gly Lys Ser Asp Thr Glu Tyr Asn Pro Asn Phe Lys

1 5 10 15

Gly

<210> 31

<211> 351

<212> DNA

<213> House mouse (Mus musculus)

<400> 31

gaggttcagc tccagcagtc tgggactgtt ctggcaaggc ctggggcttc agtgaagatg 60

tcctgcaagg tttctggcta cccctttacc agctactgga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggcgct atttatcctg gaaaaagtga cactgaatac 180

aacccgaact tcaagggcaa ggccaaactg actgcagtca catctgccac cactgcctac 240

atggagctca gcagcctgac aaatgaggac tctgcggtct attactgtac aagtacctgg 300

acccactact ttgactactg gggccaaggc accactctca cagtctcctc a 351

<210> 32

<211> 330

<212> Protein

<213> House mouse (Mus musculus)

<400> 32

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

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

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

50 55 60

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

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

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

100 105 110

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

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

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

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

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

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 33

<211> 330

<212> Protein

<213> House mouse (Mus musculus)

<400> 33

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

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

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

50 55 60

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

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

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

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

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

195 200 205

Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

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

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 34

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 34

Glu 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 Val Ser Gly Tyr Pro Phe Thr Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Gln Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Ser Thr Trp Thr His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 35

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 35

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

50 55 60

Gln Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Ser Thr Trp Thr His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 36

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 36

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

50 55 60

Gln Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Ser Thr Trp Thr His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 37

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 37

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 Val Ser Gly Tyr Pro Phe Thr Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Gln Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Ser Thr Trp Thr His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 38

<211> 447

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 38

Glu 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 Val Ser Gly Tyr Pro Phe Thr Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Gln Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Ser Thr Trp Thr His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

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 Asp 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> 39

<211> 447

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 39

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

50 55 60

Gln Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Ser Thr Trp Thr His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

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 Asp 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> 40

<211> 447

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 40

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

50 55 60

Gln Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Ser Thr Trp Thr His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

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 Asp 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> 41

<211> 447

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<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 Val Ser Gly Tyr Pro Phe Thr Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Gln Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Ser Thr Trp Thr His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

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 Asp 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> 42

<211> 447

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 42

Glu 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 Val Ser Gly Tyr Pro Phe Thr Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Gln Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Ser Thr Trp Thr His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

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 Asp 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 Asp 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 Leu Pro Glu 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> 43

<211> 447

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 43

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

50 55 60

Gln Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Ser Thr Trp Thr His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

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 Asp 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 Asp 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 Leu Pro Glu 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> 44

<211> 447

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

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

50 55 60

Gln Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Ser Thr Trp Thr His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

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 Asp 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 Asp 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 Leu Pro Glu 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> 45

<211> 447

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 45

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 Val Ser Gly Tyr Pro Phe Thr Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Gln Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Ser Thr Trp Thr His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

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 Asp 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 Asp 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 Leu Pro Glu 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> 46

<211> 107

<212> Protein

<213> House mouse (Mus musculus)

<400> 46

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Ala Ser Leu Gly

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Ser Phe Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Asn Leu Asp Ser

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 47

<211> 8

<212> Protein

<213> House mouse (Mus musculus)

<400> 47

Tyr Tyr Thr Thr Gln Leu Ala Glu

15

<210> 48

<211> 321

<212> DNA

<213> House mouse (Mus musculus)

<400> 48

gaaatccaga tgacccagtc tccatcctct atgtctgcat ctctgggaga cacaataacc 60

atcacttgcc aggcaactca agacattgtt aagagtttaa actggtatca acaaaaacca 120

gggaaacccc cttcattcct gatctattat acaactcaac tggcagaagg ggtcccatca 180

aggttcagtg gcagtgggtc tgggtcagac tattctctga caatcagcaa cctggactct 240

gaagattttg cagactatta ctgtctacag ttttatgagt ttcctccgac gttcggtgga 300

ggcaccaagc tggaaatcaa a 321

<210> 49

<211> 117

<212> Protein

<213> House mouse (Mus musculus)

<400> 49

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Ala Ile Tyr Pro Gly Lys Ser Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ser Thr Trp Thr His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 50

<211> 17

<212> Protein

<213> House mouse (Mus musculus)

<400> 50

Ala Ile Tyr Pro Gly Lys Ser Asp Thr Glu Tyr Asn Pro Asn Phe Lys

1 5 10 15

Gly

<210> 51

<211> 351

<212> DNA

<213> House mouse (Mus musculus)

<400> 51

gaggttcagc tccagcagtc tgggactgtt ctggcaaggc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggcta caccttttcc agctactgga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggcgct atttatcctg gaaaaagtga tactagctac 180

aaccagaagt tccagggcaa ggccaaactg actgcagtca catctgccag cactgccttc 240

atggagctca ccagcctgac aaatgaggac tctgcggtct attactgtac aagtacctgg 300

acccactact ttgactactg gggccaaggc accactctca cagtctcctc a 351

<210> 52

<211> 107

<212> Protein

<213> House mouse (Mus musculus)

<400> 52

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Phe Lys

100 105

<210> 53

<211> 11

<212> Protein

<213> House mouse (Mus musculus)

<400> 53

Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 54

<211> 7

<212> Protein

<213> House mouse (Mus musculus)

<400> 54

Tyr Thr Ser Arg Leu His Ser

15

<210> 55

<211> 9

<212> Protein

<213> House mouse (Mus musculus)

<400> 55

Gln Gln Gly Asn Thr Leu Pro Trp Thr

15

<210> 56

<211> 321

<212> DNA

<213> House mouse (Mus musculus)

<400> 56

gatatccaga tgacacaaac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagc aattatttaa actggtatca gcagaaacca 120

gatggaacta ttaaactcct gatctattac acatcaagat tacattcagg agtcccatca 180

aggttcagtg gcagtgggtc tggatcagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtggac gttcggtgga 300

ggcaccaagc tggaattcaa a 321

<210> 57

<211> 214

<212> Protein

<213> House mouse (Mus musculus)

<400> 57

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 Gln Asp Ile Ser Asn Tyr

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp

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

<210> 58

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 58

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 Gln Asp Ile Ser Asn Tyr

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 59

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 59

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 Gln Asp Ile Ser Asn Tyr

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 60

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 60

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 Gln Asp Ile Ser Asn Tyr

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Gln Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 61

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 61

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 Gln Asp Ile Ser Asn Tyr

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 62

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 62

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 Gln Asp Ile Ser Asn Tyr

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp

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

<210> 63

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<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 Gln Asp Ile Ser Asn Tyr

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Gln Thr Leu Pro Trp

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

<210> 64

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 64

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 Gln Asp Ile Ser Asn Tyr

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

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

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

<210> 65

<211> 116

<212> Protein

<213> House mouse (Mus musculus)

<400> 65

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

1 5 10 15

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

20 25 30

Tyr Met Asn Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Arg Val Asn Pro Ser Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Ser Ser

115

<210> 66

<211> 5

<212> Protein

<213> House mouse (Mus musculus)

<400> 66

Asp Tyr Tyr Met Asn

15

<210> 67

<211> 17

<212> Protein

<213> House mouse (Mus musculus)

<400> 67

Arg Val Asn Pro Ser Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 68

<211> 7

<212> Protein

<213> House mouse (Mus musculus)

<400> 68

Arg His Asn Tyr Ala Asp Phe

15

<210> 69

<211> 348

<212> DNA

<213> House mouse (Mus musculus)

<400> 69

gaggtccagc tgcaacagtc tggacctgag ctggtgaagc ctggggcttc agtgaagatg 60

tcctgtaagg cttctggata cacattcact gactactaca tgaactgggt gaggcagagt 120

catggaaaga gccttgagtg gattggacgt gttaatccta gcaatggtgg tactaactac 180

aaccagaaat tcaagggcaa ggccacattg acagtagaca aatccctcag cacagcctac 240

atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc aagacgacat 300

aactacgcag acttctgggg ccaaggcacc actctcacag tctcctca 348

<210> 70

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 70

Glu 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 Asp Tyr

20 25 30

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

35 40 45

Gly Arg Val Asn Pro Ser Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg His Asn Tyr Ala Asp Phe Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 71

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 71

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 Asp Tyr

20 25 30

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

35 40 45

Gly Arg Val Asn Pro Ala Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Arg His Asn Tyr Ala Asp Phe Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 72

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 72

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 Asp Tyr

20 25 30

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

35 40 45

Gly Arg Val Asn Pro Ser Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Arg His Asn Tyr Ala Asp Phe Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 73

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 73

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 Asp Tyr

20 25 30

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

35 40 45

Gly Arg Val Asn Pro Ser Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg His Asn Tyr Ala Asp Phe Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 74

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<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 Thr Phe Thr Asp Tyr

20 25 30

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

35 40 45

Gly Arg Val Asn Pro Ser Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg His Asn Tyr Ala Asp Phe Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 75

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 75

Glu 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 Asp Tyr

20 25 30

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

35 40 45

Gly Arg Val Asn Pro Ser Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg His Asn Tyr Ala Asp Phe Trp Gly Gln Gly Thr Thr 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 Asp 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> 76

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<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 Thr Phe Thr Asp Tyr

20 25 30

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

35 40 45

Gly Arg Val Asn Pro Ala Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Arg His Asn Tyr Ala Asp Phe Trp Gly Gln Gly Thr Thr 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 Asp 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> 77

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 77

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 Asp Tyr

20 25 30

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

35 40 45

Gly Arg Val Asn Pro Ser Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Arg His Asn Tyr Ala Asp Phe Trp Gly Gln Gly Thr Thr 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 Asp 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> 78

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 78

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 Asp Tyr

20 25 30

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

35 40 45

Gly Arg Val Asn Pro Ser Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg His Asn Tyr Ala Asp Phe Trp Gly Gln Gly Thr Thr 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 Asp 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> 79

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 79

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 Asp Tyr

20 25 30

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

35 40 45

Gly Arg Val Asn Pro Ser Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg His Asn Tyr Ala Asp Phe Trp Gly Gln Gly Thr Thr 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 Asp 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> 80

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 80

Glu 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 Asp Tyr

20 25 30

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

35 40 45

Gly Arg Val Asn Pro Ser Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg His Asn Tyr Ala Asp Phe Trp Gly Gln Gly Thr Thr 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 Asp 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 Asp 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 Leu Pro Glu 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> 81

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 81

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 Asp Tyr

20 25 30

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

35 40 45

Gly Arg Val Asn Pro Ala Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Arg His Asn Tyr Ala Asp Phe Trp Gly Gln Gly Thr Thr 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 Asp 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 Asp 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 Leu Pro Glu 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> 82

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<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 Thr Phe Thr Asp Tyr

20 25 30

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

35 40 45

Gly Arg Val Asn Pro Ser Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Arg His Asn Tyr Ala Asp Phe Trp Gly Gln Gly Thr Thr 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 Asp 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 Asp 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 Leu Pro Glu 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> 83

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 83

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 Asp Tyr

20 25 30

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

35 40 45

Gly Arg Val Asn Pro Ser Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg His Asn Tyr Ala Asp Phe Trp Gly Gln Gly Thr Thr 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 Asp 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 Asp 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 Leu Pro Glu 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> 84

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 84

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 Asp Tyr

20 25 30

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

35 40 45

Gly Arg Val Asn Pro Ser Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg His Asn Tyr Ala Asp Phe Trp Gly Gln Gly Thr Thr 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 Asp 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 Asp 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 Leu Pro Glu 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> 85

<211> 107

<212> Protein

<213> House mouse (Mus musculus)

<400> 85

Glu Thr Val Met Thr Gln Ser His Lys Ile Met Ser Thr Ser Val Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ser Ser Ala Ser Tyr Gln 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 Leu Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys His Gln Tyr Tyr Asn Thr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Arg

100 105

<210> 86

<211> 7

<212> Protein

<213> House mouse (Mus musculus)

<400> 86

Ser Ala Ser Tyr Gln Tyr Thr

15

<210> 87

<211> 9

<212> Protein

<213> House mouse (Mus musculus)

<400> 87

His Gln Tyr Tyr Asn Thr Pro Leu Thr

15

<210> 88

<211> 321

<212> DNA

<213> House mouse (Mus musculus)

<400> 88

gaaactgtga tgacccagtc tcacaaaatc atgtccactt cagtaggaga cagggtcacc 60

atcacctgca aggccagtca ggatgtgaga actgctgtgg cctggtatca acagaaacca 120

ggacaatctc ctaaattact aatttcctcg gcatcctacc aatacactgg agtccctgat 180

cgcttcactg gcagtggatc tgggacggat ttcactttca ccatcagcag tttgcaggct 240

gaagacctgg cagtttatta ctgtcatcag tattataata ctccgctcac gttcggtgct 300

gggaccaagc tggagctgag a 321

<210> 89

<211> 117

<212> Protein

<213> House mouse (Mus musculus)

<400> 89

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Ala Ile Tyr Pro Gly Lys Ser Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Thr Val Ser Ser

115

<210> 90

<211> 17

<212> Protein

<213> House mouse (Mus musculus)

<400> 90

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

1 5 10 15

Gly

<210> 91

<211> 8

<212> Protein

<213> House mouse (Mus musculus)

<400> 91

Ser Val Arg Asn Ala Met Asp Tyr

15

<210> 92

<211> 351

<212> DNA

<213> House mouse (Mus musculus)

<400> 92

gaggttcagc tccagcagtc tgggactgtg ctggcaaggc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggcgct atttatcctg gaaaaagtga tactacctac 180

aaccagaagt tcgagggcaa ggccaaactg actgcagtca catctgacag cacagcctac 240

atggatctca gtagcctgac aaatgaggac tctgcggtct attactgtac atcttcggtt 300

cggaatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc a 351

<210> 93

<211> 112

<212> Protein

<213> House mouse (Mus musculus)

<400> 93

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

1 5 10 15

Gly Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ile Ile Val His Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 94

<211> 16

<212> Protein

<213> House mouse (Mus musculus)

<400> 94

Arg Ser Ser Gln Ile Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 95

<211> 7

<212> Protein

<213> House mouse (Mus musculus)

<400> 95

Lys Val Ser Asn Arg Phe Ser

15

<210> 96

<211> 9

<212> Protein

<213> House mouse (Mus musculus)

<400> 96

Phe Gln Gly Ser His Val Pro Trp Thr

15

<210> 97

<211> 336

<212> DNA

<213> House mouse (Mus musculus)

<400> 97

gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggagg tcaagcctcc 60

atctcttgca gatctagtca gatcattgta catagtaatg gaaacaccta tttagaatgg 120

tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttccg 300

tggacgttcg gtggaggcac caagctggaa atcaaa 336

<210> 98

<211> 119

<212> Protein

<213> House mouse (Mus musculus)

<400> 98

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Tyr Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg Lys Thr Trp Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 99

<211> 17

<212> Protein

<213> House mouse (Mus musculus)

<400> 99

Glu Ile Asp Pro Ser Asp Ser Tyr Thr Tyr Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 100

<211> 10

<212> Protein

<213> House mouse (Mus musculus)

<400> 100

Arg Lys Thr Trp Asp Trp Tyr Phe Asp Val

1 5 10

<210> 101

<211> 357

<212> DNA

<213> House mouse (Mus musculus)

<400> 101

caggtccagc tgcagcagcc tggggctgaa ctggtgaagc ctggggcttc agtgaagctg 60

tcctgcaagg cttctggata caccttcatt agctactgga tgcactgggt gaagcagagg 120

cctggacaag gccttgagtg gatcggagag attgatcctt ctgatagtta tacttactac 180

aatcaaaagt tcaagggcaa ggccacattg actgtagaca aatcctccag cacagcctac 240

atgcaactca gcagcctgac atctgaggac tctgcggtct attactgtgc aagaaggaaa 300

acctgggact ggtacttcga tgtctggggc gcagggacca cggtcaccgt ctcctca 357

<210> 102

<211> 106

<212> Protein

<213> House mouse (Mus musculus)

<400> 102

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

1 5 10 15

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

20 25 30

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

35 40 45

Ser Ser Ala Ser Tyr 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 Ala

65 70 75 80

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

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Arg

100 105

<210> 103

<211> 11

<212> Protein

<213> House mouse (Mus musculus)

<400> 103

Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210> 104

<211> 7

<212> Protein

<213> House mouse (Mus musculus)

<400> 104

Ser Ala Ser Tyr Arg Tyr Thr

15

<210> 105

<211> 8

<212> Protein

<213> House mouse (Mus musculus)

<400> 105

Gln Gln His Tyr Ser Thr Pro Thr

15

<210> 106

<211> 318

<212> DNA

<213> House mouse (Mus musculus)

<400> 106

gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcact 60

atcacctgca aggccagtca ggatgtgagt actgctgtag cctggtatca acagaaacca 120

ggacagtctc ctaaactact gatttcctcg gcatcctacc ggtacactgg agtccctgat 180

cgcttcactg gcagtggatc tgggacggat ttcactttca ccatcagcag tgtgcaggct 240

gaagacctgg cagtttatta ctgtcagcaa cattatagta ctccgacgtt cggtggaggc 300

accaagctgg aaatcaga 318

<210> 107

<211> 116

<212> Protein

<213> House mouse (Mus musculus)

<400> 107

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Gly Arg Ala Lys Leu Thr Ala Val Thr Ser Ala Asn Thr Ala Tyr Met

65 70 75 80

Glu Leu Ser Ser Leu Thr Asn Asp Asp Ser Ala Val Phe Tyr Cys Thr

85 90 95

Cys Thr Thr Ala Gly Val Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val

100 105 110

Thr Val Ser Ser

115

<210> 108

<211> 17

<212> Protein

<213> House mouse (Mus musculus)

<400> 108

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

1 5 10 15

Gly

<210> 109

<211> 8

<212> Protein

<213> House mouse (Mus musculus)

<400> 109

Thr Thr Ala Gly Val Leu Asp Tyr

15

<210> 110

<211> 351

<212> DNA

<213> House mouse (Mus musculus)

<400> 110

gaggttcagc tccagcagtc tgggactgtg ctggcaaggc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggcta cacctttacc agctactgga tgcactgggt aaaagagagg 120

cctggacagg gtctggaatg gattggcgct atttatcctg gagatagtga tactaggtat 180

aatcagaagt tcaagggcag ggccaaactg actgcagtca catctgccaa cactgcctac 240

atggagctca gcagcctgac aaatgatgac tctgcggtct tctactgtac atgtactacg 300

gctggtgttt tggactactg gggtcaagga acctcagtca ccgtctcctc a 351

<210> 111

<211> 112

<212> Protein

<213> House mouse (Mus musculus)

<400> 111

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Thr Ser Ser Gln Ser Ile Val His Gly

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 112

<211> 16

<212> Protein

<213> House mouse (Mus musculus)

<400> 112

Thr Ser Ser Gln Ser Ile Val His Gly Asn Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 113

<211> 9

<212> Protein

<213> House mouse (Mus musculus)

<400> 113

Phe Gln Gly Ser His Val Pro Tyr Thr

15

<210> 114

<211> 336

<212> DNA

<213> House mouse (Mus musculus)

<400> 114

gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaggcctcc 60

atctcttgca catctagtca gagcattgta catggtaatg gaaacaccta tttagaatgg 120

tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttccg 300

tacacgttcg gaggggggac caagctggaa ataaaa 336

<210> 115

<211> 123

<212> Protein

<213> House mouse (Mus musculus)

<400> 115

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

1 5 10 15

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

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 His Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Tyr Gly Leu Phe Tyr Gly Asn Asp Gly Tyr Ala Met Asp His

100 105 110

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 116

<211> 5

<212> Protein

<213> House mouse (Mus musculus)

<400> 116

Ser Tyr Trp Met Asn

15

<210> 117

<211> 17

<212> Protein

<213> House mouse (Mus musculus)

<400> 117

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

1 5 10 15

Ser

<210> 118

<211> 14

<212> Protein

<213> House mouse (Mus musculus)

<400> 118

Tyr Gly Leu Phe Tyr Gly Asn Asp Gly Tyr Ala Met Asp His

1 5 10

<210> 119

<211> 355

<212> DNA

<213> House mouse (Mus musculus)

<400> 119

caggtccaac tgcagcagcc tggggctgaa ctggtgaagc ctggggcttc agtgaagctg 60

tcctgcaagg cttctggcta ctctttcacc agctactgga tgaactgggt gaagcagagg 120

cctggacgag gcctcgagtg gattggaagg attcatcctt ctgatagtga aactcactac 180

aatcaaaagt tcaagagcaa ggccacactg actgtagaca aatcctccag cacagcctac 240

atccaactca gcagcctgac atctgaggac tctgcggtct atttttgtgc aagatacggg 300

ctcttctatg gtaacgacgg atatgctatg gaccactggg gtcaaggaac ctcag 355

<210> 120

<211> 107

<212> Protein

<213> House mouse (Mus musculus)

<400> 120

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

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Phe Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 121

<211> 11

<212> Protein

<213> House mouse (Mus musculus)

<400> 121

Arg Ala Ser Gln Asp Ile Ser Phe Tyr Leu Asn

1 5 10

<210> 122

<211> 321

<212> DNA

<213> House mouse (Mus musculus)

<400> 122

gatatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagc ttttatttaa actggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctactac acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacac ttccgtggac gttcggtgga 300

ggcaccaagc tggaaatcaa a 321

<210> 123

<211> 116

<212> Protein

<213> House mouse (Mus musculus)

<400> 123

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

1 5 10 15

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

20 25 30

Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Arg Val Asn Pro Ser Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg His Asn Tyr Pro Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210> 124

<211> 17

<212> Protein

<213> House mouse (Mus musculus)

<400> 124

Arg Val Asn Pro Ser Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 125

<211> 7

<212> Protein

<213> House mouse (Mus musculus)

<400> 125

Arg His Asn Tyr Pro Asp Tyr

15

<210> 126

<211> 348

<212> DNA

<213> House mouse (Mus musculus)

<400> 126

gaggtccagc tgcaacagtc tggacctgaa ctggtgaagc ctggggcttc agtgaagatg 60

tcctgtaagg cttctggata cacattcact gactactaca tgaactgggt gaagcagagt 120

catggaaaga gccttgagtg gattggacgt gttaatccta gcaatggtgg tactagctac 180

aaccagaagt tcaagggcaa ggccacattg acagtagaca aatccctcag cgcagcctat 240

atgcagctca acagcctgac atctgaggac tctgcggtct attactgtgc aagaaggcat 300

aactaccctg actactgggg ccaaggcacc actctcacag tctcctca 348

<210> 127

<211> 320

<212> DNA

<213> House mouse (Mus musculus)

<400> 127

gaaatccaga tgacccagtc tccatcctct atgtctgcat ctctgggaga cagaataacc 60

atcacttgcc aggcaactca agacattgtt aagagtttaa actggtatca acaaaaacca 120

gggaaacccc cttcattcct gatctattat acagctcaac tggcagaagg ggtcccgtca 180

aggttcagtg gcagtgggtc tgggtcagac tattctctga caatcagcaa cctggagtct 240

gaagattttg cagactatta ctgtctacag ttttatgagt ttcctccgac gttcggtgga 300

ggcaccaagc tggaaatcaa 320

<210> 128

<211> 117

<212> Protein

<213> House mouse (Mus musculus)

<400> 128

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ser Thr Trp Thr His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 129

<211> 17

<212> Protein

<213> House mouse (Mus musculus)

<400> 129

Ala Ile Tyr Pro Gly Asn Ser Asp Thr Arg Tyr Asn Pro Asn Phe Lys

1 5 10 15

Gly

<210> 130

<211> 8

<212> Protein

<213> House mouse (Mus musculus)

<400> 130

Thr Trp Thr His Tyr Phe Asp Tyr

15

<210> 131

<211> 351

<212> DNA

<213> House mouse (Mus musculus)

<400> 131

gaggttcagc tccagcagtc tgggactgtg ctggcaaggc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggcta cccctttacc agctactgga tgcactgggt aaagcagagg 120

cctggacagg gtctggaatg gattggcgct atttatcctg gaaatagtga tactaggtac 180

aacccgaatt tcaagggcaa ggccaacctg actgcagtca catctgccac cactgcctac 240

atggagctca gcagcctgac aaatgaggaa tctgcggtct attactgtac aagtacctgg 300

acccactact ttgactactg gggccaaggc accactctca cagtctcctc a 351

<---

Claims (13)

1. Anti-B7-H4 antibody or antigen-binding fragment thereof, containing: (a) a light chain with the amino acid sequence of SEQ ID NO:63; And (b) a heavy chain having the amino acid sequence of SEQ ID NO:81. 2. A pharmaceutical composition that increases the immune response, containing an anti-B7H4 antibody or an antigen-binding fragment thereof according to claim 1 and a pharmaceutically acceptable carrier. 3. Nucleic acid encoding an antibody or its antigen-binding fragment according to claim 1. 4. An expression vector containing the nucleic acid according to claim 3. 5. A host cell for producing an antibody according to claim 1, containing a nucleic acid according to claim 3 or a vector according to claim 4. 6. The use of an anti-B7H4 antibody or an antigen-binding fragment thereof according to claim 1 or a pharmaceutical composition according to claim 2 in a method of increasing the immune response in a patient, comprising administering to said patient said antibody, antigen-binding fragment or pharmaceutical composition. 7. Use according to claim 6, wherein the method increases the immune response and the patient suffers from cancer. 8. Use according to claim 6, wherein the cancer is ovarian, lung or gastrointestinal cancer. 9. Use according to claim 6, wherein the method increases the immune response and the patient suffers from an infectious disease. 10. Use according to claim 6, wherein the method enhances the immune response and the pharmaceutical composition is administered to the patient simultaneously with the vaccine or a component thereof. 11. Use according to any one of claims 6 to 10, wherein the method includes administering a second therapeutic agent to the patient.