JP2014240384A - Polypeptides and polynucleotides, and uses thereof as a drug target for producing drugs and biologics - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel target molecule for production of immune and non-immune based therapeutics and for disease diagnosis.SOLUTION: The invention provides therapeutic antibodies against VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigens, which are predicted co-stimulatory family members and which are differentially expressed in cancers including, lung cancer, ovarian cancer, and colon cancer, and diagnostic and therapeutic usages. The use of these antibodies for modulating B7 costimulation and related therapies such as the treatment of autoimmunity are also provided. This invention further relates to the discovery of extracellular domains of VSIG1 and its variants, FXYD3 and its variants, ILDR1 and its variants, LOC253012 and its variants, AI216611 and its variants, and C1ORF32 and its variants which are suitable targets for immunotherapy, cancer therapy, and drug development.

Description

(Related application)
The claims of the present invention are all filed on Sep. 4, 2007, US Provisional Application Nos. 60 / 969,865, 60 / 969,799, 60 / 969,780, 60/969. , 806, 60 / 969,769, and 60 / 969,788, which are incorporated by reference in their entirety.

(Technical field)
The present invention relates to specific proteins that are specifically expressed in specific tissues and their use as therapeutic and diagnostic targets. More specifically, the present invention relates to VSIG1 protein and variants thereof, FXYD3 and variants thereof, ILDR1 and variants thereof that are specifically expressed in specific cancers and are therefore suitable for immunotherapy, cancer treatment and drug development. The present invention relates to variants, LOC253012 and variants thereof, AI216611 and variants thereof, and C1ORF32 and variants thereof. Further, the present invention relates to immunotherapy, cancer treatment of VSIG1 protein and variants thereof, FXYD3 and variants thereof, ILDR1 and variants thereof, LOC253012 and variants thereof, AI216611 and variants thereof, and C1ORF32 and variants thereof. And an invention of an extracellular region suitable for drug development.

  In addition, because some of the proteins of the present invention appear to be involved in immune co-stimulation because of their B7-like structure, the present invention is useful as an immunomodulator and in immunotherapy, particularly cancer and autoimmunity. It further relates to the use of these proteins or agents that modulate (in agonists and antagonists) these proteins to treat immune related diseases such as diseases. Even more specifically, the present invention relates to prophylactic and therapeutic antibodies, and therapeutic and diagnostic methods using the proteins of the present invention or soluble or secreted portions thereof, particularly the same antibodies and antibody fragments that bind specifically to the extracellular region. About.

  Tumor antigens are ideally positioned as biomarkers and drug targets and play an important role in the development of new strategies for active and passive immunotherapeutic agents used in combination with monotherapy or conventional cancer therapy. Tumor antigens are only expressed in tumor cells and not in normal tissues, tumor specific antigens (TSA), or tumor associated antigens (TAA) that are overexpressed in tumor cells and expressed at low levels in normal tissues It is classified into either.

  TAA and TSA have proven effective as targets for passive (antibody) and active immunotherapy using strategies that break immune tolerance and stimulate the immune system. Antigenic epitopes targeted by these therapeutic approaches are overexpressed in tumor cells compared to non-tumor cells and are targeted by antibodies that prevent functional activity, inhibit cell proliferation, and induce cell death .

  An increasing number of tumor-associated antigens have been tested for monoclonal antibodies or used as cancer treatments. The identification of new tumor antigens expressed in human malignant tumors and the analysis of their molecular properties are actively performed in the field of tumor immunology. Several approaches have been taken to identify tumor-associated antigens as potential immunotherapy targets, including high-throughput bioinformatics approaches based on genomics and proteomics. By identifying new TAAs or TSAs, the range of tumor antigens targeted for immune recognition is expanded and new target molecules for the development of therapeutic agents for passive immunotherapy, such as unmodified or conjugated monoclonal antibodies, Provided. Such new antigens may also indicate the direction of active or adoptive immunotherapy towards more effective therapeutic vaccines.

  Cancer vaccines involve the administration of tumor antigens, as well as breaching immune tolerance and inducing an activated T cell response against the tumor. Vaccine therapy includes whole cell therapy where naked DNA, peptides, recombinant proteins and the patient's own tumor cells are used as a source of vaccine. By identifying specific tumor antigens, dendritic cells are incorporated into related proteins or peptides, or vector DNA or RNA is transfected, and vaccines are more frequently performed by dendritic cell therapy.

  The main uses of anti-TAA antibodies for the treatment of cancer are therapy using naked antibodies, therapy using drug-conjugated antibodies, and fusion therapy with cellular immunity. Since its discovery, antibodies have been expected to carry toxic agents such as drugs, toxins, and radioisotopes specifically to the affected site without affecting non-targeted normal tissue. Was assumed. Indeed, antibodies and especially antibody fragments can function as carriers for cytotoxic substances such as radioisotopes, drugs and toxins. Immunotherapy using such immunoconjugates is more effective than therapy using naked antibodies.

  In contrast to the huge success of hematological malignancies of naked antibodies (such as Rituxan and Campath) and conjugated antibodies (such as Bexar and Zevalin), solid tumor immunity Only slight success was achieved with the therapy. One of the main limitations in the successful use of immunotherapy for solid tumors is the large molecular size of intact immunoglobulins, which results in long blood half-lives but poor tumor penetration and uptake . In fact, only a very small amount (only 0.01%) of the administered antibody reaches the tumor. In addition to its size, the antibody encounters other obstacles before reaching the target antigen expressed on the cell surface of the solid tumor. Disorders include inadequate blood flow in large tumors, vascular endothelium permeability, increased tumor interstitial fluid pressure, and heterologous antibody expression.

  With the advent of antibody engineering, low molecular weight antibody fragments have been created that show improved tumor penetration. Such antibody fragments are often conjugated to cytotoxic molecules and are designed to selectively carry those antibodies to cancer cells. However, even with immunoconjugated antibody fragments, solid tumors remain a challenge.

  A new wave of optimization strategies involves the use of biomodifiers that regulate damage from solid tumors. That is, in combination with antibodies or conjugated antibody fragments, various agents improve tumor blood flow, improve vascular permeability, regulate stromal cells and extracellular matrix components, and reduce tumor interstitial fluid pressure. It has been used to lower, increase target antigen expression and improve the penetration and retention of therapeutic agents.

  Immunotherapy using antibodies represents a great opportunity to combine with standard therapies such as chemotherapy, and with a wide variety of biological agents, creating a synergistic effect. Indeed, unconjugated mAbs are more effective when used in combination with other therapeutic agents, including other antibodies.

  Another component of the immune system response to immunotherapy is the activation of cellular responses, particularly T cell responses and cytotoxic T cells (CTL). The efficiency of the immune system in mediating tumor regression depends on the induction of antibody-specific T cell responses through physiological immune surveillance, vaccine sensitization, or T cell adoptive transfer. Various tumor-associated antigens have been identified and immunotherapy methods have been tried, but objective clinical responses are rare. Reasons for this include current immunotherapy approaches that produce effective T cell responses, the presence of regulatory cells that interfere with T cell responses, and the inactivation of cytotoxic T cells through the expression of negative costimulatory molecules. Other evasion mechanisms of tumor progression are included. Effective immunotherapy against cancer requires the use of appropriate tumor-specific antigens, optimization of interactions between antigenic peptides, APCs and T cells, and simultaneous blockade of negative regulatory mechanisms that impair the effectiveness of immunotherapy Will.

  T cell activation plays a central role in advancing protective and pathogenic immune responses and requires the completion of a carefully organized series of specific steps that can be preempted or interrupted by many important events And Naive T cells must receive two independent signals from antigen presenting cells (APCs) in order to be productively activated. First, signal 1 is antigen specific and occurs when the T cell antigen receptor meets the appropriate antigen-MHC complex on APC. A signal independent of the second antigen (signal 2) is transmitted through a T cell costimulatory molecule involving a ligand expressed in APC. Without a costimulatory signal, T cell activation is weakened and interrupted. This results in antigen-specific unresponsiveness (known as T cell anergy) or T cell apoptotic death.

  A costimulatory signal can be stimulatory (positive costimulation) or inhibitory (negative costimulation or co-inhibition). Positive costimulation is necessary for optimal activation of naïve T cells, and negative costimulation is necessary for the acquisition of immune tolerance against itself and for cessation of effector T cell function. In addition, costimulatory signals, particularly positive costimulatory signals, are involved in the regulation of B cell activity. For example, B cell activation and germinal center B cell survival require T cell-derived signals in addition to stimulation with antigen.

  Both positive and negative costimulatory signals play an important role in the control of cellular immune responses, and molecules that mediate these signals have proven to be effective targets for immune regulation. Based on this finding, several therapeutic approaches targeting costimulatory molecules have been developed and have been shown to be useful for the prevention and treatment of cancer and autoimmune diseases, as well as rejection of allografts. It is by blocking the activation or cessation of the immune response in subjects with these pathologies, respectively.

  A costimulatory molecule pair usually consists of a ligand expressed on APC and its cognate receptor expressed on T cells. Well-characterized B7 / CD28 and CD40 / CD40L costimulatory molecules are important in early T cell activation. In recent years, several more costimulatory molecules have been identified and belong to the B7 / CD28 or TNF / TNF-R gene family. The effects of costimulatory TNFR family members can often be considered functionally, temporally, and spatially separate from the effects of CD28 family members and the members within the family. Sequential and temporal control of T cell activation / survival signals by different costimulators can function to allow long lasting responses while tightly controlling T cell survival.

  The B7 family consists of structurally related cell surface protein ligands that bind to receptors on lymphocytes that control the immune response. Interaction of a B7-family member with its corresponding costimulatory receptor, usually the CD28-related family, enhances the immune response, while interaction with a co-inhibitory receptor such as CTLA4 attenuates the immune response To do. Members of the B7 family have an extracellular region that is structurally related, has 20-40% amino acid identity, and includes tandem regions associated with variable and constant immunoglobulin regions.

  Currently, seven family members are known. B7.1 (CD80), B7.2 (CD86), B7-H1 (PD-Ll), B7-H2 (ICOS-L), B7-DC (PD-L2), B7-H3, and B7-H4 Yes, each has its own function, but many have overlapping functions. Clearly, individual B7 molecules in the immune system have developed their own unique and indispensable fields. As the specific specific areas of B7 family members have been scrutinized, their diagnostic and therapeutic potential has become more apparent. Many of the B7 superfamily members were initially characterized as T cell costimulatory molecules. However, recently it has become clear that they can also co-inhibit T cell responses. That is, B7 family members can have the opposite effect on the immune response.

  Central to the normal functioning of the immune system is the ability to distinguish between self and non-self. Failure to do so can lead to the development of autoimmune diseases. Most autoimmune diseases involve autoreactive T cells and / or autoantibodies. Thus, agents that have the ability to inhibit or eliminate autoreactive lymphocytes have promising therapeutic potential. Moreover, the use of agents that exhibit such immunosuppressive activity should also be beneficial for inhibiting the normal immune response to alloantigens in transplanted patients. That is, new drugs that can modulate costimulatory signals without compromising the ability of the immune system to defend against pathogens are very advantageous for the treatment and prevention of such pathologies.

  The importance of B7 family members in controlling immune responses to self and alloantigens was demonstrated by the development of immune deficiency and autoimmune disease in mice with mutations in the B7-family gene. Thus, manipulating the signals carried by B7 ligands has shown potential in the treatment of autoimmunity, inflammatory diseases and transplant rejection. This approach relies, at least in part, on the ultimate removal of autologous or alloreactive T cells, which, without costimulation (which induces cell survival genes), are extremely susceptible to induction of apoptosis. This is because it becomes easier.

  Suppressing the immune system to treat chronic diseases is a major goal of immunotherapy. Active and passive immunotherapy have been shown to be effective as effective therapeutic strategies. Passive immunotherapy using monoclonal antibodies or receptor Fc-fusion proteins has been well developed and has shown great clinical success. The approved number of such therapeutic agents is growing and is in clinical trials for the prevention of allograft rejection or the treatment of autoimmune diseases and cancer. Active immunotherapy (i.e., vaccines) is usually at the forefront of efforts to prevent infections that are effective against pathogens that cause acute self-limiting infections and subsequently bring about immunity. However, active immunotherapy is less effective against cancer or chronic infections. These are because they develop strategies to avoid normal immune responses. Among these are negative co-stimulators of the B7 family, such as B7-H1 and B7-H4, which are highly expressed in certain tumors and are localized from immune cell-mediated attacks Allow defense.

  The efficiency of the immune system in mediating tumor regression depends on eliciting an antigen-specific T cell response through physiological immunological surveillance, vaccine sensitization, or following T cell adoptive transfer. Although a variety of tumor-associated antigens have been identified and many immunotherapy methods have been tested, objective clinical responses are rare. This is because current immunotherapy approaches are unable to produce an efficient T cell response, there are regulatory cells that inhibit the T cell response, and cytotoxicity through the expression of negative costimulatory molecules. Other evasion mechanisms for tumor progression are included, such as inactivation of sex T cells. Effective immunotherapy for cancer requires the use of appropriate tumor-specific antigens, optimization of interactions between antigenic peptides, APCs and T cells, and simultaneous blockade of negative regulatory mechanisms that interfere with immunotherapeutic effects. Seem.

  B7 family costimulators play an important role in the activation and inhibition of anti-tumor immune responses. New drugs targeting these molecules could find important applications in modulating immune responses and improving cancer immunotherapy. Such an agent could be administered with a tumor specific antigen as an adjuvant that serves to enhance the immune response to the antigen in the patient. In addition, such agents could be useful for other types of cancer immunotherapy, such as adoptive immunotherapy, which leads to an expansion of the tumor-specific T cell population that attacks and destroys tumor cells. . Agents that can enhance such anti-tumor responses have great therapeutic potential and may be beneficial in efforts to overcome the obstacles of tumor immunotherapy.

  Passive tumor immunotherapy takes advantage of the immune system's superior selectivity and lytic capacity. Targeting tumor-specific antigens to treat malignant tumor diseases with minimal damage to normal tissue. Several approaches have been used to identify new tumor-associated antigens as potential immunotherapy targets. The identification of new tumor-specific antigens broadens the range of antigen targets available for immune recognition and provides new target molecules for the development of therapeutic agents for passive immunotherapy, such as unmodified or conjugated monoclonal antibodies. Such new antigens may indicate a direction towards more effective therapeutic vaccines for active or adoptive immunotherapy.

  The clinical development of costimulatory blockade has come to fruition with the approval of CTLA4Ig (Abatacept) for rheumatoid arthritis. This soluble fusion protein acts as a competitive inhibitor of the B7 / CD28 costimulatory pathway and is also in clinical trials for other immune diseases such as psoriasis and multiple sclerosis, and transplant rejection. Belatacept, a processed CTLA4Ig with increased binding affinity for its ligands B7.1 and B7.2 (CD80 and CD86, respectively), has shown promising results in phase II clinical trials of renal transplantation. Yes. Two fully human anti-CTLA4 monoclonal antibodies, ipilimumab and tremelimumab, cancel CTLA4 / B7 inhibitory interactions and are in clinical (study) phase III for metastatic melanoma and other cancers and HIV infection . Galiximab is a primatized monoclonal antibody that targets CD80 and is in phase II for rheumatoid arthritis, psoriasis, and non-Hodgkin lymphoma.

  It is important to note that strategies that use a single agent to block costimulation are often found to be inadequate. In view of the diversity of various costimulatory molecules, future strategies may involve simultaneous blockade of several selected pathways, or conventional drugs such as immunosuppressants for immune related disorders or cytotoxic drugs for cancer. Combination therapy may be included.

  Despite recent advances in understanding cancer biology and cancer therapy, and better understanding of molecules involved in the immune response, the success rate in cancer therapy and treatment of autoimmune diseases remains low. Thus, there is an unmet need for new therapies that can successfully treat both cancer and autoimmune deficiencies.

  The object of the present invention is to provide novel therapeutic and diagnostic compositions comprising at least one of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 protein, or one of the novel splice variants disclosed herein. It is to be. And these novel VSIG1 splice variants, specifically, ILDR1 splice variant, LOC25303012 splice variant, AI216611s splice variant, C1ORF32 splice variant, and FXYD3 splice variant, and nucleic acid sequences encoding them or fragments thereof In particular, providing a nucleic acid sequence encoding the extracellular region or secreted form of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 protein and / or splice variant.

  Another object of the present invention is to provide agents that specifically bind to VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 protein and / or splice variants, and / or other sites (VSIG1, ILDR1, LOC253012). Utilizing said proteins, splice variants and nucleic acid sequences as novel targets for the development of agents that agonize or antagonize binding to AI216611, C1ORF32, FXYD3 proteins and / or splice variants.

  Yet another object of the present invention is to provide an agent that modulates (activates or antagonizes) at least one of the biological activities associated with VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3. Examples of such agents include antibodies, small molecules, peptides, ribozymes, antisense molecules, and siRNA. These molecules can directly bind or modulate the activity elicited by VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 protein or VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 DNA or part thereof or variants thereof Or an activity associated with VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3, such as by modulating the binding of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 to a competing receptor or endogenous ligand, or VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FX D3 as well as modulate indirectly the binding of the to some and variants

  In a more specific embodiment, the present invention diagnoses that agonize or antagonize binding of other sites to VSIG1 protein and / or modulate (agonize or antagonize) at least one VSIG1-related biological activity. V-set and immunoglobulin region containing 1 (SEQ ID NO: 11), which is a known protein that can be used as a marker and / or therapeutic agent (RefSequencementidentifierNP _872413, synonyms: RP5-889N15.1, 1700062D20Rik, GPA34, MGC44287, dJ889N15.) A novel splice variant, or a polynucleotide encoding the same.

  According to one more specific embodiment, the novel splice variant is a nucleic acid having the nucleic acid sequence described in any one of AI581519_T10 (SEQ ID NO: 9), AI581519_T11 (SEQ ID NO: 10) or a sequence homologous thereto. An isolated polynucleotide comprising. According to another embodiment, the isolated polynucleotide is at least 95% homologous to any one of AI581519_T10 (SEQ ID NO: 9), AI581519_T11 (SEQ ID NO: 10).

  According to yet another more specific embodiment, the novel splice variant has an amino acid sequence set forth in any one of AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), or a sequence homologous thereto. An isolated protein or polypeptide. According to another embodiment, the isolated polypeptide is at least 95% homologous to any one of AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16).

  Another specific object of the invention is a molecule and isolated polypeptide comprising a soluble extracellular region (ECD) of VSIG1 protein and fragments thereof, and a nucleic acid sequence encoding said soluble extracellular region, and its Fragments and conjugates and their use as therapeutic agents, including their use in immunotherapy (which promotes or inhibits immune co-stimulation).

  In a more specific embodiment, the present invention relates to residues 23-234 of the VSIG1 protein sequence contained in the sequence of AI581519_P3 (SEQ ID NO: 11), corresponding to the amino acid sequence shown in SEQ ID NO: 138, or SEQ ID NO: 139 Residues 23-270 of the VSIG1 protein sequence included in the sequence of AI581519_P4 (SEQ ID NO: 12) corresponding to the amino acid sequence shown, or the sequence of AI581519_P5 (SEQ ID NO: 13) corresponding to the amino acid sequence shown in SEQ ID NO: 140 Residues 23-296 of the VSIG1 protein sequence contained in VSIG1, or residues 23-193 of the VSIG1 protein sequence contained in the sequence of AI581519_P7 (SEQ ID NO: 14) corresponding to the amino acid sequence shown in SEQ ID NO: 141, or SEQ ID NO: 142 Residue 23-203 of the VSIG1 protein sequence contained in the sequence of AI581519_P9 (SEQ ID NO: 15) corresponding to the amino acid sequence, or included in the sequence of AI581519_P10 (SEQ ID NO: 16) corresponding to the amino acid sequence shown in SEQ ID NO: 143 Extracellular region corresponding to residues 26-293 of the VSIG1 protein sequence contained in the sequence of AI581519_P5 (SEQ ID NO: 13) corresponding to residues 23-231 of the VSIG1 protein sequence or the amino acid sequence shown in SEQ ID NO: 302 Or a variant thereof having at least 80% sequence identity, more preferably at least 90% sequence identity, more preferably 95, 96, 97, 98 or 99% sequence identity thereto. Contains VSIG1 protein It supplies another part.

  According to another more specific embodiment, the present invention activates or antagonizes the binding of other sites to ILDR1 protein and / or modulates at least one ILDR1-related biological activity (actuation). Also known as immunoglobulin-like region-containing receptor 1 (SEQ ID NO: 21) (RefSequencementidentifierNP_787120, ILDR1alpha, ILDR1beta, ILDR1), a known protein that can be utilized as a diagnostic marker and / or therapeutic agent (or antagonizes) A novel splice variant, or a polynucleotide encoding the same.

  In one specific embodiment, the novel splice variant is an isolated polynucleotide comprising a nucleic acid sequence set forth in AA424839_1_T7 (SEQ ID NO: 20) or a nucleic acid having a sequence homologous thereto. According to another embodiment, the isolated polynucleotide has at least 95, 96, 97, 98 or 99% homology to AA424839_1_T7 (SEQ ID NO: 20).

  According to yet another specific embodiment, the novel splice variant has the amino acid sequence set forth in AA424839_1_P11 (SEQ ID NO: 24) or a sequence homologous thereto, ie, at least 80 or 90% sequence identity thereto An isolated protein or polypeptide. According to another related embodiment, the isolated polypeptide is at least 95, 96, 97, 98 or 99% homologous to AA424839_1_P11 (SEQ ID NO: 24).

  Another embodiment of the invention relates to molecules and isolated polypeptides comprising a soluble extracellular region (ECD) of ILDR1 protein and fragments thereof, as well as nucleic acid sequences encoding said soluble extracellular region, and fragments and inclusions thereof. As well as providing its use as a therapeutic agent, including its use in immunotherapy (which promotes or inhibits immune co-stimulation).

  According to a further embodiment, the present invention is represented by residues 24-162 of sequences AA424839_P3 (SEQ ID NO: 22) and AA424839_P5 (SEQ ID NO: 11), or SEQ ID NO: 76, corresponding to the amino acid sequence set forth in SEQ ID NO: 75 Residues 24-45 of AA424839_P7 (SEQ ID NO: 23) corresponding to the amino acid sequence, or residues 24-105 of AA424839_1_P11 (SEQ ID NO: 24) corresponding to the amino acid sequence shown in SEQ ID NO: 296, or SEQ ID NO: 301 A different portion of the extracellular region corresponding to residues 50-160 of AA424839_1_P3 (SEQ ID NO: 22) corresponding to the amino acid sequence shown, or at least 80% sequence identity thereto, more preferably at least 90% sequence identity Sex, even better Properly supplies a different part of ILDR1 protein comprising a variant thereof having it 95, 96, 97, 98 or 99% sequence identity.

  Another embodiment of the invention is an isolated having or encoding an extracellular region of an ILDR1 protein that may be added directly or indirectly to a non-ILDR1 protein or nucleic acid sequence, such as a soluble immunoglobulin region or fragment. Or to provide purified soluble protein or nucleic acid sequence.

  According to certain embodiments, the present invention provides diagnostics that agonize or antagonize the binding of other sites to LOC2530102 protein or modulate (activate or antagonize) at least one of the LOC253012-related biological activities. Novel splice variant of protein LOC253012 isoform 1 (SEQ ID NO: 35) (RefSeq accession number NP_001034461) based on known hypotheses, which can be utilized as a marker and / or therapeutic agent, or encodes it A polynucleotide is provided.

  According to one embodiment, the novel LOC253012 splice variants are H68654_1_T8 (SEQ ID NO: 28), H68654_1_T15 (SEQ ID NO: 29), H68654_1_T16 (SEQ ID NO: 30), H68654_1_T17 (SEQ ID NO: 31), H68654_1_T18 (SEQ ID NO: 32), An isolated polynucleotide comprising a nucleic acid sequence described in any one of H68654_1_T19 (SEQ ID NO: 33) or H68654_1_T20 (SEQ ID NO: 34), or a nucleic acid having a sequence homologous thereto. According to another embodiment, the isolated polynucleotide is H68654_1_T8 (SEQ ID NO: 28), H68654_1_T15 (SEQ ID NO: 29), H68654_1_T16 (SEQ ID NO: 30), H68654_1_T17 (SEQ ID NO: 31), H68654_1_T18 (SEQ ID NO: 32), H68654_1_T19 (SEQ ID NO: 33) or H68654_1_T20 (SEQ ID NO: 34) is at least 95% homologous.

  According to yet another embodiment, the novel LOC253012 splice variant is described in any one of H68654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40). An isolated protein or polypeptide having the amino acid sequence of or a sequence homologous thereto. According to another embodiment, the isolated polypeptide is any one of H68654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40). There is at least 95, 96, 97, 98 or 99% homology.

  Another object of the present invention is to provide molecules and isolated polypeptides comprising the soluble extracellular region (ECD) of LOC2530112 protein and fragments thereof, as well as nucleic acid sequences encoding said soluble extracellular region, and fragments and inclusions thereof. And its use as a therapeutic agent, including its use in immunotherapy (which promotes or inhibits immune co-stimulation).

  According to a further embodiment, the present invention relates to residues 38-349 of the sequence H68654_1_P2 (SEQ ID NO: 35), which corresponds to the amino acid sequence shown in SEQ ID NO: 144, or the amino acid sequence shown in SEQ ID NO: 145 H68654_1_P5 (SEQ ID NO: 36), H68654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40) residues 19-337, or the amino acid sequence shown in SEQ ID NO: 300 A different portion of the extracellular region corresponding to residues 1-335 of the sequence H68654_1_P5 (SEQ ID NO: 36), or at least 80% sequence identity thereto, more preferably at least 90% sequence identity , More preferably supplies a different part of the LOC253012 protein comprising a variant thereof having it 95, 96, 97, 98 or 99% sequence identity.

  Another object of the invention is an isolated or encoding or having or encoding an extracellular region of a LOC253012 protein that may be added directly or indirectly to a non-LOC253012 protein or nucleic acid sequence, such as a soluble immunoglobulin region or fragment. To provide a purified soluble protein or nucleic acid sequence.

  According to certain embodiments, the invention activates or antagonizes the binding of other sites to AI216611 protein, or modulates (activates or antagonizes) at least one AI216611-related bioactivity. A novel splice variant of AI216611, or a polynucleotide encoding it, that can be utilized as a diagnostic marker and / or therapeutic agent.

  According to one embodiment, the novel AI216611 splice variant is an isolated polynucleotide comprising a nucleic acid sequence having the nucleic acid sequence set forth in AI216611_T1 (SEQ ID NO: 42) or a nucleic acid sequence homologous thereto. According to another embodiment, the isolated polynucleotide has at least 95, 96, 97, 98 or 99% homology to AI216611_T1 (SEQ ID NO: 42).

  According to yet another embodiment, the novel AI216611 splice variant is an isolated protein or polypeptide having the amino acid sequence set forth in AI216611_P1 (SEQ ID NO: 44) or a sequence homologous thereto. According to another embodiment, the isolated polypeptide is AI216611_P1 (SEQ ID NO: 44). Have at least 95, 96, 97, 98 or 99% homology.

  Another object of the present invention is to provide molecules and isolated polypeptides comprising the soluble extracellular region (ECD) of AI216611 protein and fragments thereof, as well as nucleic acid sequences encoding said soluble extracellular region, and fragments and inclusions thereof. And its use as a therapeutic agent, including its use in immunotherapy (which promotes or inhibits immune co-stimulation). According to a further embodiment of the invention, residues 29-147 of sequence AI216611_P0 (SEQ ID NO: 43) or AI216611_P1 (SEQ ID NO: 44), corresponding to the amino acid sequence set forth in SEQ ID NO: 146, or the amino acid set forth in SEQ ID NO: 298 A different portion of the extracellular region corresponding to residues 1-1145 of sequence AI216611_P0 (SEQ ID NO: 43), corresponding to the sequence, or at least 80% sequence identity thereto, more preferably at least 90% sequence identity thereto, More preferably there is another part of the AI216611 protein which includes variants thereof with 95, 96, 97, 98 or 99% sequence identity.

  Another object of the invention is an isolation having or encoding an extracellular region of AI216611 protein, which may be added directly or indirectly to a non-AI216611 protein or nucleic acid sequence, such as a soluble immunoglobulin region or fragment. Or providing a purified soluble protein or nucleic acid sequence.

  Another object of the invention is plasmids and recombinant viral vectors expressing AI216611, secreted or soluble forms thereof and / or E216 of AI216611 protein and variants thereof or polypeptide conjugates containing any of the foregoing And the like, as well as containing host cells.

  According to certain embodiments, the present invention provides a diagnostic marker that agonizes or antagonizes binding of other sites to the C1ORF32 protein or modulates (activates or antagonizes) at least one of the C1ORF32 related biological activities. And / or protein LOC387597 (SEQ ID NO: 47) (RefSeq accession identifier NP_955383, synonyms: NP_955383; LISCH-like, C1ORF32, based on known hypotheses that may be used as therapeutic agents A novel splice variant of RP4-782G3.2, dJ782G3.1) or a polynucleotide encoding the same is provided.

  According to certain embodiments, the novel LOC387597 splice variant is an isolated nucleic acid sequence comprising a nucleic acid sequence according to any one of H19011_1_T8 (SEQ ID NO: 45), H19011_1_T9 (SEQ ID NO: 46), or a nucleic acid having a sequence homologous thereto. Polynucleotide. According to another embodiment, the isolated polynucleotide has at least 95, 96, 97, 98 or 99% homology to any one of H19011_1_T8 (SEQ ID NO: 45), H19011_1_T9 (SEQ ID NO: 46) .

  According to yet another embodiment, the novel splice LOC387597 mutant is an isolated having the amino acid sequence set forth in any one of H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), or a sequence homologous thereto. Protein or polypeptide. According to another embodiment, the isolated polypeptide has at least 95, 96, 97, 98 or 99% homology to any one of H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50). is there.

  Another object of the present invention is to provide molecules and isolated polypeptides comprising the soluble extracellular region (ECD) of C1ORF32 protein and fragments thereof, as well as nucleic acid sequences encoding said soluble extracellular region, and fragments and inclusions thereof. As well as its use as a therapeutic agent, including its use in immunotherapy (which promotes or inhibits immune co-stimulation).

  According to a further embodiment of the present invention, residues 21-186 of sequence H19011_1_P8 (SEQ ID NO: 48) corresponding to the amino acid sequence shown in SEQ ID NO: 147, or sequence H19011_1_P9 corresponding to the amino acid sequence shown in SEQ ID NO: 148 A different portion of the extracellular region corresponding to residue 1-184 of sequence H19011_1_P8 (SEQ ID NO: 48), corresponding to residues 21-169 of (SEQ ID NO: 50), or the amino acid sequence shown in SEQ ID NO: 299, or Another C1ORF32 protein comprising a variant thereof having at least 80% sequence identity, more preferably at least 90% sequence identity, more preferably 95, 96, 97, 98 or 99% sequence identity thereto There is a part.

  Another object of the present invention is an isolated or having or encoding an extracellular region of a C1ORF32 protein that may be added directly or indirectly to a non-C1ORF32 protein or nucleic acid sequence, such as a soluble immunoglobulin region or fragment To provide a purified soluble protein or nucleic acid sequence.

  According to certain embodiments, the invention provides a diagnostic marker that agonizes or antagonizes binding of other sites to FXYD3 protein or modulates (agonizes or antagonizes) at least one of FXYD3-related biological activities. FXYD3, a FXYD domain-containing ion transport regulator 3 precursor (SEQ ID NO: 70) (SwissProt accession id (accession ID)) that is a known protein that can be used as a therapeutic agent ) FXYD3jHUMAN, synonyms chlorine conductance inducer (Chloride conduit) ance inducer) for supplying protein Mat-8, Mammary tumor 8kDa protein, Hosuhoreman like (Phospholemman-like) also known as is) of the novel splice variant or the encoding polynucleotide.

  According to one embodiment, the novel FXYD3 splice variants are R31375_T19 (SEQ ID NO: 65), R31375_T25 (SEQ ID NO: 66), R31375_T26 (SEQ ID NO: 67), R31375_T29 (SEQ ID NO: 68), R31375_T39 (SEQ ID NO: 69). An isolated polynucleotide having the nucleic acid sequence of any one or a sequence homologous thereto. According to another embodiment, the isolated polynucleotide is of R31375_T19 (SEQ ID NO: 65), R31375_T25 (SEQ ID NO: 66), R31375_T26 (SEQ ID NO: 67), R31375_T29 (SEQ ID NO: 68), R31375_T39 (SEQ ID NO: 69). Any one has at least 95, 96, 97, 98 or 99% homology.

  According to yet another embodiment, the novel FXYD3 splice variant has the amino acid sequence set forth in any one of R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73), R31375_P33 (SEQ ID NO: 74) or a homologous thereto. An isolated protein or polypeptide having a sequence. According to another embodiment, the isolated polypeptide is at least 95, 96, 97, 98 in any one of R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73), R31375_P33 (SEQ ID NO: 74). Or 99% homology.

  Another object of the present invention is to provide molecules and isolated polypeptides comprising the soluble extracellular region (ECD) of the FXYD3 protein and fragments thereof, as well as nucleic acid sequences encoding said soluble extracellular region, and fragments and inclusions thereof. Combined as well as providing its use as a therapeutic agent, including its use in cancer immunotherapy.

  According to a further embodiment of the invention, residues 21-36 of sequence R31375_P0 (SEQ ID NO: 70) or R31375_P31 (SEQ ID NO: 73), corresponding to the amino acid sequence set forth in SEQ ID NO: 149, or amino acids set forth in SEQ ID NO: 150 Residues 21-65 of sequence R31375_P14 (SEQ ID NO: 72) corresponding to the sequence, or residues 21-25 of sequence R31375_P33 (SEQ ID NO: 74) corresponding to the amino acid sequence shown in SEQ ID NO: 151, or SEQ ID NO: 297 A different portion of the extracellular region corresponding to residues 1-63 of the sequence R31375_P14 (SEQ ID NO: 72), corresponding to the amino acid sequence shown in FIG. 2, or at least 80% sequence identity thereto, more preferably at least 90% Sequence identity, more preferably 95 There is another part of FXYD3 protein comprising 96, 97, 98 or a variant thereof having 99% sequence identity.

  Another object of the invention may be added directly or indirectly to a non-VSIG1, non-ILDR1, non-LOC253012, non-AI216611, non-C1ORF32 or non-FXYD3 protein or nucleic acid sequence, such as a soluble immunoglobulin region or fragment To provide an isolated or purified soluble protein or nucleic acid sequence with or encoding the extracellular region of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 protein.

  Another object of the present invention is the edge, tail or head of any one of the novel variants of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 of the present invention or a homologue or fragment thereof. molecules and isolated polypeptides comprising a head portion and nucleic acid sequences encoding said edge, tail or head portion and fragments and conjugates thereof and as therapeutic agents and / or It is to supply its use for diagnosis.

  A further object of the present invention is to provide a molecule comprising the bridge, edge, tail or head shown in any one of SEQ ID NOs: 284-295 and an isolated poly (bridge, edge, head or head portion). Peptides or homologues or fragments thereof, and nucleic acid sequences encoding edges, tails or heads and fragments and conjugates thereof, and their use as therapeutic agents and / or for diagnosis Is to supply

  Another object of the present invention is to provide any one of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3, secreted or soluble forms thereof and / or ECD of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 protein and To provide vectors, such as plasmids and recombinant viral vectors, which express a polypeptide conjugate comprising the mutant or any of the foregoing, and host cells containing the vectors.

  Another object of the present invention is to produce VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 protein, fragments or variants thereof and / or conjugates comprising any one of the foregoing. , LOC253012, AI216611, C1ORF32, FXYD3, secreted or soluble forms thereof and / or VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 ECD, or FXYD3 protein and variants thereof or any of the foregoing The use of these vectors and host cells containing them, such as plasmids and recombinant viral vectors that express polypeptide conjugates.

  Another object of the present invention is to provide a pharmaceutical or diagnostic composition comprising the foregoing.

  Another object of the invention is to treat or prevent cancer, autoimmune deficiency, transplant rejection, graft-versus-host disease, and / or immune co-mediated by VSIG1, ILDR1, LOC253012, AI216611, FXYD3 or C1ORF32 polypeptides. Supplying and utilizing a compound comprising a VSIG1 extracellular region or fragment thereof or variant thereof suitable for blocking or promoting stimulation.

  Specific objects of the present invention include AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16). , AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (sequence) No. 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19O11_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 72) 73), specifically binding to a full length VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen, its secreted form and / or its ECD or its conjugate or fragment thereof selected from the group consisting of R31375_P33 (SEQ ID NO: 74) To develop new monoclonal or polyclonal antibodies and antibody fragments and containing conjugates. These antibodies are potentially useful as therapeutic and / or diagnostic agents (also in vitro and in vivo diagnostic methods), in particular include ADCC (antibody dependent cytotoxic activity) or CDC (complement dependent cytotoxic activity) ) Antibodies and fragments that are immunostimulatory or immunosuppressive, such as antibodies or fragments that target cells through activity.

  Another object of the present invention is, for example, immunohistochemical assay, radioimaging assay, in vivo imaging, radioimmunoassay (RIA), ELISA, slot blot, competitive binding assay, fluorometric imaging assay, western Provide diagnostic methods including the use of any of the foregoing, including blots, FACS, etc. In particular, AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 16). 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P7 (SEQ ID NO: 37), P68654 ), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40), I216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73), R375 Specifically binds to a complete VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 protein, its soluble form, its ECD, and / or its conjugate, fragment or variant selected from the group consisting of (SEQ ID NO: 74) Assays using chimeric or non-human antibodies or fragments.

  Another object of the present invention includes, but is not limited to, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, breast, prostate, Various cancers such as spleen, kidney, bladder, head and neck, uterus, testis, stomach, cervix, liver, bone, skin, pancreas, brain cancer, and other non-solid and solid tumors, sarcomas, hematologic malignancies, etc. And VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen or secreted or soluble forms thereof or ECD and / or parts thereof, such as cancers that are non-metastatic, invasive, or metastatic AI581519_P3 (SEQ ID NO: 11), AI581 to treat a condition in which the variant is differentially expressed 19_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), A42 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 54) Sequence number 40), AI216611_P0 (sequence number 43), AI216611_P1 SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375 ^ P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73), R31375_P33 (SEQ ID NO: 74) Utilizing novel therapeutically effective polyclonal or monoclonal antibodies and fragments, conjugates and variants against any of the VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigens selected from the group.

  Another object of the invention is AI581519_P3 (SEQ ID NO: 11) for treating non-malignant deficiency diseases such as immunodeficiency, including but not limited to autoimmune diseases, transplant rejection and graft-versus-host disease. AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (A424839_P7 (A424839_P7) (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P7 (SEQ ID NO: 37), 68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 48) (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73), R31375_P33 (SEQ ID NO: 74), VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen, and fragments thereof Novel therapeutically effective polyclonal or monoclonal for any one of conjugates and variants It is to use the body.

  A specific object of the present invention is that antibodies and antibody fragments to VSIG1 antigen, secreted or soluble forms thereof or ECD and / or variants, conjugates or fragments thereof, and fragments and variants thereof are differentially expressed by the antigen. For use in treating or diagnosing lung cancer and / or ovarian cancer expressed in the body.

  Specific embodiments of the invention include antibodies and antibody fragments to ILDR1 antigen, secreted or soluble forms thereof or ECD and / or variants, conjugates or fragments thereof, and fragments and variants thereof, wherein Use for treating or diagnosing colon cancer and / or ovarian cancer that are expressed in an isolated manner.

  A specific object of the present invention is that the LOC253012 or C1ORF32 antigen, secreted or soluble forms thereof or antibodies and antibody fragments to ECD and / or variants, conjugates or fragments thereof and fragments and variants thereof It is to be used for treating or diagnosing subsequently expressed lung cancer, particularly small cell lung cancer.

  A specific object of the present invention is that antibodies and antibody fragments to AI216611 antigen, secreted or soluble forms thereof, or ECD and / or variants, conjugates or fragments thereof, and fragments and variants thereof are differentially expressed by the antigen. It is used for treating or diagnosing colon cancer that is expressed in cancer.

  A specific object of the present invention is to provide antibodies and antibody fragments against FXYD3 wild type antigen (R31375_P0 (SEQ ID NO: 70)), or secreted or soluble forms thereof, or antibodies and antibody fragments against ECD and containing conjugates. It is used to treat or diagnose ovarian cancer that is differentially expressed.

  Another object of the present invention is AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P_P (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 0), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73) ), VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 antigens and conjugates selected from the group consisting of R31375_P33 (SEQ ID NO: 74) to modulate (stimulate or inhibit) immunity. The antibody activates or suppresses immune co-stimulation, particularly B7-related immune co-stimulation, positive stimulation of T cell activity against cancer cells and autoimmunity and Through negative stimulation of T cell activity for the treatment of other immune deficiency, including antibodies which can handle associated therapeutic applications.

  A specific object of the present invention is to provide residues 23-234 of the VSIG1 protein sequence contained in the sequence of AI581519_P3 (SEQ ID NO: 11) corresponding to the amino acid sequence shown in SEQ ID NO: 138, or the amino acid shown in SEQ ID NO: 139 Residues 23-270 of the VSIG1 protein sequence included in the sequence of AI581519_P4 (SEQ ID NO: 12) corresponding to the sequence, or included in the sequence of AI581519_P5 (SEQ ID NO: 13) corresponding to the amino acid sequence shown in SEQ ID NO: 140 Residues 23-296 of the VSIG1 protein sequence, or residues 23-193 of the VSIG1 protein sequence contained in the sequence of AI581519_P7 (SEQ ID NO: 14) corresponding to the amino acid sequence shown in SEQ ID NO: 141, or shown in SEQ ID NO: 142 Amino acid composition VSIG1 included in the sequence of AI581519_P10 (SEQ ID NO: 16) corresponding to residues 23-203 of the VSIG1 protein sequence included in the sequence of AI581519_P9 (SEQ ID NO: 15) or the amino acid sequence shown in SEQ ID NO: 143 The extracellular region corresponding to residues 26-293 of the VSIG1 protein sequence included in the sequence of AI581519_P5 (SEQ ID NO: 13) corresponding to the amino acid sequence shown in residues 23-231 of the protein sequence or SEQ ID NO: 302 The production of antibodies and antibody fragments against another part of the VSIG1 protein containing the part.

Another specific embodiment of the invention resides in residues 24-162 of sequences AA424839_P3 (SEQ ID NO: 22) and AA424839_P5 (SEQ ID NO: 11), or SEQ ID NO: 76, corresponding to the amino acid sequence shown in SEQ ID NO: 75. Residues 24-105 of AA424839_P7 (SEQ ID NO: 23) corresponding to the amino acid sequence shown, or residues 24-105 of AA424839_1_P11 (SEQ ID NO: 24) corresponding to the amino acid sequence shown in SEQ ID NO: 296, or SEQ ID NO: Generating antibodies and antibody fragments against another part of the ILDR1 protein comprising a different part of the extracellular region corresponding to residues 50-160 of AA424839_1_P3 (SEQ ID NO: 22), corresponding to the amino acid sequence shown in 301 .

  Another specific object of the present invention is residues 38-349 of sequence H68654_1_P2 (SEQ ID NO: 35), or sequences H68654_1_P5 (SEQ ID NO: 36), H68665_1_P7 (SEQ ID NO: 36) corresponding to the amino acid sequence shown in SEQ ID NO: 144 37), residues 19-337 of H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40), or the amino acid sequence set forth in SEQ ID NO: 300 of the LOC25303012 protein sequence disclosed herein To produce an antibody or antibody fragment against another portion of the LOC2530112 protein, including a different portion of the extracellular region corresponding to residues 1-335 of H68654_1_P5 (SEQ ID NO: 36).

  Another specific object of the invention is residues 29-147 of sequence AI216611_P0 (SEQ ID NO: 43) or AI216611_P1 (SEQ ID NO: 44), corresponding to the amino acid sequence shown in SEQ ID NO: 146, or disclosed herein An antibody against another part of AI216611 protein comprising a different part of the extracellular region corresponding to residues 1-145 of sequence AI216611_P0 (SEQ ID NO: 43), corresponding to the amino acid sequence shown in SEQ ID NO: 298 It is to produce antibody fragments.

  Another specific object of the present invention is shown in residues 21-186 of the C1ORF32 protein sequence contained in the sequence of H19011_1_P8 (SEQ ID NO: 48), corresponding to the amino acid sequence shown in SEQ ID NO: 147, or SEQ ID NO: 148 Residues 21-169 of the C1ORF32 protein sequence included in the sequence of H19011_1_P9 (SEQ ID NO: 50) corresponding to the amino acid sequence described above, or the rest of the sequence H19011_1_P8 (SEQ ID NO: 48) corresponding to the amino acid sequence shown in SEQ ID NO: 299 To produce an antibody or antibody fragment against another part of the C1ORF32 protein, including a different part of the extracellular region corresponding to group 1-184.

  Another specific object of the invention is residues 21-36 of the FXYD3 protein sequence contained in the sequence of R31375_P0 (SEQ ID NO: 70) or R31375_P31 (SEQ ID NO: 73), corresponding to the amino acid sequence shown in SEQ ID NO: 149. Or R31375_P33 (corresponding to the amino acid sequence shown in SEQ ID NO: 151, or residues 21-65 of the FXYD3 protein sequence contained in the sequence of R31375_P14 (SEQ ID NO: 72) corresponding to the amino acid sequence shown in SEQ ID NO: 150 Residues 21-25 of the FXYD3 protein sequence included in the sequence of SEQ ID NO: 74) or residues 1-25 of the FXYD3 protein sequence included in the sequence R31375_P14 (SEQ ID NO: 72) corresponding to the amino acid sequence shown in SEQ ID NO: 297 63, extracellular equivalent Including different portions of band, it is to produce for another part of the FXYD3 protein, an antibody or antibody fragment.

  A specific object of the present invention is a polyclonal and antigen-binding site that specifically binds to VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 protein, soluble forms thereof, ECD and / or variants and fragments thereof Supplying monoclonal antibodies as well as fragments or antigen-binding fragments thereof.

  A specific object of the present invention is to utilize such antibodies and fragments thereof to modulate (activate or inhibit) the activity of a target in the treatment or prevention of cancer and / or immune co-stimulatory systems. .

  A related object of the present invention is to treat or prevent autoimmune deficiency, transplant rejection, GVHD, and / or block or promote immune co-stimulation mediated by VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 polypeptides. Suitable is to select monoclonal and polyclonal antibodies against VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 and fragments thereof.

  Specific objects of the present invention include, but are not limited to, lung cancer, ovarian cancer, colon cancer, and acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Other non-solid and solid tumors, such as Hodgkin lymphoma, non-Hodgkin lymphoma, breast, prostate, spleen, kidney, bladder, head and neck, uterus, testes, stomach, neck, liver, bone, skin, pancreas, brain cancer VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen, soluble form for the treatment and diagnosis of cancers such as sarcomas, hematologic malignancies, etc., which are non-metastatic, invasive or metastatic , Utilizing antibodies against either ECD or fragments or variants thereof.

  With respect to lung cancer, the disease is selected from the group consisting of squamous cell lung cancer, lung adenocarcinoma, carcinoid, small cell lung cancer or non-small cell lung cancer.

  Another object of the present invention is immunomodulation, such as treatment of autoimmunity, and preferably autoimmune diseases: multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosis, ulcerative colitis, Crohn's disease, Immunodeficiency related to transplant rejection, benign lymphocytic vasculitis, lupus erythematosus, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin-dependent diabetes mellitus, Good Pasteur syndrome, myasthenia gravis, pemphigus, sympathetic ophthalmitis, autoimmune uveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic hepatitis, ulcerative colitis colitis), Sjogren's syndrome, rheumatic disease, polymyositis, scleroderma, mixed connective tissue disease, inflammatory rheumatism, decadent rheumatism, non-articular rheumatism, Primary disease, chronic polyarthritis, psoriatic arthritis, ankylosing spondylitis, juvenile rheumatoid arthritis, shoulder periarthritis, polyarteritis nodosa, progressive systemic sclerosis, urate arthritis, dermatomyositis, rheumatoid muscle Any of the VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigens, soluble forms thereof, or ECD and mutations, useful in treating autoimmune diseases selected from myositis, myosclerosis and cartilage calcification To provide and utilize antibodies and antibody fragments against the body or fragments thereof, as well as soluble polypeptides or portions thereof comprising the extracellular region of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen.

  Another object of the invention is useful for the treatment or prevention of cancer, autoimmune deficiency, transplant rejection, GVHD, and / or immune co-stimulation mediated by VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 polypeptide VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3, agents such as small molecules, peptides, antibodies and fragments, and VSIG1, ILDR1, LOC253012, AI216611 Supplying and utilizing compounds such as ribozymes or antisense or siRNA targeting the C1ORF32 or FXYD3 nucleic acid sequences, or fragments or variants thereof.

  Another object of the invention is to block cancer, autoimmune deficiency disease, transplant rejection, treatment or prevention of GVHD, and / or immune co-stimulation mediated by VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 polypeptides Or small molecules, peptides, antibodies and fragments that bind to any of the VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigens, and VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 nucleic acid sequences or It is to provide and utilize compounds comprising drugs such as ribozymes or antisense or siRNA targeting the fragments or variants thereof.

  A preferred object corresponds to residues 23-234 of AI581519_P3 (SEQ ID NO: 11), corresponding to the amino acid sequence shown in SEQ ID NO: 138 of the VSIG1 protein sequence disclosed herein, or to the amino acid sequence shown in SEQ ID NO: 139 AI581519_P4 (SEQ ID NO: 12) residues 23-270, or AI581519_P5 (SEQ ID NO: 13) residues 23-296, or the amino acid sequence shown in SEQ ID NO: 141, corresponding to the amino acid sequence shown in SEQ ID NO: 140 AI581519_P7 (SEQ ID NO: 14) corresponding to residues 23-193, or AI581519_P9 (SEQ ID NO: 15) corresponding to residues 23-203, or SEQ ID NO: 143 corresponding to the amino acid sequence shown in SEQ ID NO: 142 Corresponds to amino acid sequence Any of the foregoing that specifically binds to residues 23-231 of AI581519_P10 (SEQ ID NO: 16), or residues 26-293 of AI581519_P5 (SEQ ID NO: 13) corresponding to the amino acid sequence set forth in SEQ ID NO: 302 It is intended to provide therapeutic and diagnostic antibodies and fragments and containing conjugates useful in treating or diagnosing.

  A preferred embodiment is shown in residues 24-162 of the ILDR1 protein sequence contained in the sequences of AA424839_P3 (SEQ ID NO: 22) and AA424839_P5 (SEQIDNOSl), corresponding to the amino acid sequence shown in SEQ ID NO: 75, or SEQ ID NO: 76 Included in the sequence of AA424839_1_P11 (SEQ ID NO: 24) corresponding to the amino acid sequence shown in SEQ ID NO: 296, or residues 24-457 of the ILDR1 protein sequence included in the sequence of AA424839_P7 (SEQ ID NO: 23) corresponding to the amino acid sequence A different part of the extracellular region corresponding to residues 50-160 of AA424839_1_P3 (SEQ ID NO: 22) corresponding to residues 24-105 of the ILDR1 protein sequence or amino acid sequence set forth in SEQ ID NO: 301 That specifically binds to, it is to provide a useful therapeutic and diagnostic antibodies as well as fragments and containing conjugates in treating or diagnosing any foregoing.

  A preferred object corresponds to residues 38-349 of the LOC253012 protein sequence contained in the sequence of H68654_1_P2 (SEQ ID NO: 35), which corresponds to the amino acid sequence shown in SEQ ID NO: 144, or the amino acid sequence shown in SEQ ID NO: 145, Residues 19-337 of the LOC253012 protein sequence included in the sequence of H68654_1_P5 (SEQ ID NO: 36), H68654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40), or Treat or diagnose any of the foregoing that specifically bind to residues 1-335 of the sequence H68654_1_P5 (SEQ ID NO: 36), corresponding to the amino acid sequence set forth in SEQ ID NO: 300 Hit and to provide a useful therapeutic and diagnostic antibodies as well as fragments and containing conjugate.

  A preferred purpose is shown in residues 29-147 of the AI216611 protein sequence contained in the sequence of AI216611_P0 (SEQ ID NO: 43) or AI216611_P1 (SEQ ID NO: 44), corresponding to the amino acid sequence shown in SEQ ID NO: 146, or SEQ ID NO: 298 Therapeutic and diagnostic antibodies and fragments useful for treating or diagnosing any of the foregoing that specifically bind to residues 1-145 of sequence AI216611_P0 (SEQ ID NO: 43), corresponding to the amino acid sequence It is to provide a containing conjugate.

  A preferred object corresponds to residues 21-186 of the C1ORF32 protein sequence contained in the sequence of H19011_1_P8 (SEQ ID NO: 48), corresponding to the amino acid sequence shown in SEQ ID NO: 147, or the amino acid sequence shown in SEQ ID NO: 149, A cell corresponding to residues 1 to 184 of the sequence H19011_1_P8 (SEQ ID NO: 48), corresponding to residues 21-169 of the C1ORF32 protein sequence contained in the sequence of H19011_1_P9 (SEQ ID NO: 50), corresponding to the amino acid sequence shown in SEQ ID NO: 299 It is to provide therapeutic and diagnostic antibodies and fragments and containing conjugates useful in treating or diagnosing any of the foregoing that specifically bind to different portions of the outer region.

  A preferred object is shown in residues 21-36 of the FXYD3 protein sequence contained in the sequence of R31375_P0 (SEQ ID NO: 70) or R31375_P31 (SEQ ID NO: 73), corresponding to the amino acid sequence shown in SEQ ID NO: 149, or SEQ ID NO: 150 R31375_P33 (SEQ ID NO: 74) corresponding to residues 21-65 of the FXYD3 protein sequence included in the sequence of R31375_P14 (SEQ ID NO: 72) or the amino acid sequence shown in SEQ ID NO: 151 ) That specifically binds to residues 21-25 of the FXYD3 protein sequence contained in the sequence of), or residues 1-63 of the sequence R31375_P14 (SEQ ID NO: 72) corresponding to the amino acid sequence shown in SEQ ID NO: 297, Treat or examine any of those And to provide useful therapeutic and diagnostic antibodies and fragments and containing conjugate order to.

  Also preferred is that the antibody is a chimeric, humanized, fully human antibody and / or an antibody or antibody fragment having CDC or ADCC activity in the target cell, VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 and above It is to provide antibodies and fragments thereof that bind to the specific residues identified and fragments thereof.

  A preferred object is also to provide chimeric and human antibodies and fragments and containing conjugates that bind to VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 and the specific residues identified above and fragments thereof. .

  Another specific object of the present invention is to include antibody fragments and containing conjugates useful in the above therapies and related diagnostic methods, including but not limited to Fab, F (ab ′) 2, Fv or scFv fragments. Is to provide.

  It is also an object of the present invention to direct or indirectly target antibodies and fragments directly to markers and other effector sites such as detectable markers, or effector sites such as enzymes, toxins, therapeutic agents, or chemotherapeutic agents. Is to add.

  In preferred embodiments, inventive antibodies or fragments of the invention may be added directly or indirectly to radioisotopes, metal chelators, enzymes, fluorescent compounds, bioluminescent compounds or chemiluminescent compounds.

  It is also an object of the present invention to provide pharmaceuticals and diagnostic compositions comprising forms effective for the treatment or diagnosis of the antibodies or antibody fragments of the present invention.

  Another specific object of the present invention is to inhibit the growth of VSIG1-expressing cells in a patient, which is said to be AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 ( Administering an antibody that specifically binds to an antigen referred to herein as SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16) or VSIG1 .

  Another specific object of the present invention is to provide AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10. (SEQ ID NO: 16) or to provide a method of treating or preventing cancer comprising administering an effective amount of a monoclonal antibody that specifically binds to VSIG1.

  A more preferred object of the present invention is to utilize these antibodies to treat a cancer selected from the group consisting of lung cancer and ovarian cancer, wherein the lung cancer or ovarian cancer is non-metastatic, invasive or metastatic. Yes, preferably the antibodies are of AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16) It has a specific antigen-binding region in the extracellular region.

  Another object of the present invention is to provide AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 11). 16) to provide a method of treating or preventing autoimmune diseases comprising administering an effective amount of a polyclonal or monoclonal antibody or fragment or containing conjugate that specifically binds to 16).

  Another specific embodiment of the present invention is to inhibit the growth of ILDR1-expressing cells in a patient, which includes AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7. (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24) or ILDR1, comprising administering an antibody that specifically binds to an antigen referred to herein.

  Another specific embodiment of the invention specifically binds to the patient to AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24) or ILDR1. It is to provide a method of treating or preventing cancer comprising administering an effective amount of a monoclonal antibody.

  A more preferred embodiment of the present invention is selected from the group consisting of colon cancer and ovarian cancer, and uses these antibodies to treat cancer in which colon cancer or ovarian cancer is non-metastatic, invasive or metastatic Preferably, the antibody has a specific antigen binding region in the extracellular region of AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23) or AA424839_1_P11 (SEQ ID NO: 24). Have.

  The anti-cancer vaccine of the present invention provides a patient with a polyclonal or monoclonal antibody or fragment that specifically binds to AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23) or AA424839_1_P11 (SEQ ID NO: 24) Is to provide a method of treating or preventing autoimmune disease comprising administering an effective amount of.

  Another specific object of the present invention is to inhibit the growth of cells expressing LOC253012 in a patient, which said patient has H68654_1_P2 (SEQ ID NO: 35), H68665_1_P5 (SEQ ID NO: 36), H68654_1_P7 ( Administering an antibody that specifically binds to an antigen referred to herein as SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40) or LOC253012. .

  Another specific object of the present invention is to provide patients with H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68665_1_P13 (SEQ ID NO: 39), H68654_1_P14. (SEQ ID NO: 40) or to provide a method of treating or preventing cancer comprising administering an effective amount of a monoclonal antibody that specifically binds to LOC253012.

  A more preferred object of the present invention is to utilize these antibodies to treat lung cancer, particularly small cell lung cancer, which is selected from the group consisting of non-metastatic, invasive or metastatic lung cancer. Yes, preferably the antibodies are H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40) It has a specific antigen-binding region in the extracellular region.

  Another object of the present invention is to provide patients with H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 39). 40) to provide a method for treating or preventing an autoimmune disease comprising administering an effective amount of a polyclonal or monoclonal antibody or fragment or containing conjugate that specifically binds to 40).

  Another specific object of the present invention is to treat or prevent cancer comprising administering to a patient an effective amount of a monoclonal antibody that specifically binds AI216611_P0 (SEQ ID NO: 43) or AI216611_P1 (SEQ ID NO: 44). Is to provide a way to do.

  Another object of the present invention includes administering to a patient an effective amount of a polyclonal or monoclonal antibody or fragment or containing conjugate that specifically binds AI216611_P0 (SEQ ID NO: 43) or AI216611_P1 (SEQ ID NO: 44), It is to provide a method for treating or preventing autoimmune diseases.

  Another specific object of the present invention is to inhibit the growth of cells expressing C1ORF32 in a patient, which may include H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), or C1ORF32 As administering an antibody that specifically binds to an antigen referred to herein.

  Another specific object of the invention is treating cancer comprising administering to a patient an effective amount of a monoclonal antibody that specifically binds to AH19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50) or C1ORF32. Or to provide a way to prevent.

  A more preferred object of the present invention is to utilize these antibodies for treating cancers selected from the group consisting of lung cancer, in particular small cell lung cancer, wherein the lung cancer is non-metastatic, invasive or metastatic. Preferably, the antibody has an antigen-binding region specific to the extracellular region of H19011_1_P8 (SEQ ID NO: 48) and H19011_1_P9 (SEQ ID NO: 50).

  Another object of the present invention includes administering to a patient an effective amount of a polyclonal or monoclonal antibody or fragment or containing conjugate that specifically binds to H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), It is to provide a method for treating or preventing autoimmune diseases.

  Another specific object of the present invention is specific to the antigen referred to herein as R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73), R31375_P33 (SEQ ID NO: 74). Administering an antibody that binds selectively.

  Another specific object of the present invention is the cells of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 for administration as anti-cancer vaccines for immunotherapy of cancer, including but not limited to ovarian cancer. The use of part or all of the outer region or its variants and containing conjugates.

  Another specific object of the present invention is to provide a patient with a monoclonal antibody that specifically binds to R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73), R31375_P33 (SEQ ID NO: 74). It is to provide a method of treating or preventing cancer comprising administering an effective amount.

  A more preferred object of the present invention is to utilize these antibodies to treat lung cancer, where the lung cancer is non-metastatic, invasive or metastatic, preferably the antibody is R31375_P0 (SEQ ID NO: 70). , R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73), and R31375_P33 (SEQ ID NO: 74) have a specific antigen-binding region.

  In another embodiment of the invention, the cancer is not limited to acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, Non-solid and solid tumors, sarcoma, blood, including lung, ovary, breast, prostate, colon, spleen, kidney, bladder, head and neck, uterus, testis, stomach, cervix, liver, bone, skin, pancreas, brain cancer Selected from the group consisting of malignant tumors, the cancer can be non-metastatic, invasive, or metastatic.

  In a preferred embodiment, the autoimmune disease is multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, immunodeficiency associated with transplant rejection, benign lymphocyte vasculitis, erythematous Lupus, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin-dependent diabetes, Goodpasture syndrome, myasthenia gravis, pemphigus, sympathetic ophthalmitis, self Immunouveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic hepatitis, ulceritis colitis, Sjogren's syndrome, rheumatic disease, polymyositis, scleroderma Mixed connective tissue disease, inflammatory rheumatism, degenerative rheumatism, non-articular rheumatism, collagen disease, chronic polyarthritis, arthritic psoriasis, ankylosing spondylitis, Year rheumatoid arthritis, shoulder periarthritis, nodosa, including progressive systemic scleroderma, urate arthritis, dermatomyositis, muscular rheumatism, myositis, a muscle hardness disease and cartilage calcification.

  A specific object of the present invention is to provide AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 11). No. 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68764 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P1 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 ( SEQ ID NO: 73), a method of treating or preventing any organ transplant rejection and / or graft host disease comprising administering an effective amount of an antibody that specifically binds to R31375_P33 (SEQ ID NO: 74) Is to provide. In addition, in the above method, the antibody is AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16). ), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), or AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1 , H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P1 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 ( It is preferable to have a specific antigen-binding region in the extracellular region of SEQ ID NO: 73) and R31375_P33 (SEQ ID NO: 74).

  According to the present invention, each of the following may be used in conjunction with simultaneous blockade of several costimulatory pathways or in combination therapy using conventional drugs such as immunosuppressants or cytotoxic drugs for cancer and cytotoxic drugs. VSIG1 extracellular region, ILDR1 extracellular region, LOC25303012 extracellular region, AI216611 extracellular region, C1ORF32 extracellular region or FXYD3 extracellular region, FXYD3 antigen VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 of the present invention Antibody and fragment, cancer, autoimmune deficiency disease, transplant rejection, treatment or prevention of GVHD and / or immunity mediated by VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 polypeptide Useful in blocking or enhancing costimulation, such as small molecules, peptides, and ribozymes or antisense or siRNA targeting VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 nucleic acid sequences or fragments or variants thereof A compound containing a drug.

  Another object of the present invention is AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23) or AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P (SEQ ID NO: 38), H68654J_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73) ) Or R31375_P33 (SEQ ID NO: 74) to provide an assay for detecting the presence or absence of a protein in a biological sample or individual in vitro or in vivo, which includes AI581519_P3 (SEQ ID NO: 11), AI581519. P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839 (SEQ ID NO: 23) or AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 54) SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (arrangement No. 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73) or R31375_P33 (SEQ ID NO: 74) polypeptide or a combination thereof Contacting with an antibody having specificity for, and AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839 P7 (SEQ ID NO: 23) or AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (68) (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 ( SEQ ID NO: 73) or R31375_P33 (SEQ ID NO: 74) protein binding is detected in the sample Including that.

  Another object of the present invention is AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23) or AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (sequence) No. 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73) or a method of detecting a disease, diagnosing the disease, observing the progression of the disease or the effect of treatment or the recurrence of the disease, or selecting a therapy for the disease, including detecting the expression of R31375_P33 (SEQ ID NO: 74) Is to provide.

  For related purposes, the diseases detected include cancers that are non-metastatic, invasive, or metastatic, such as lung cancer, ovarian cancer, colon cancer, and but are not limited to acute lymphocytic leukemia, chronic lymphoma Spherical leukemia, acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, breast, prostate, spleen, kidney, bladder, head and neck, uterus, testicles, stomach, neck, liver, bone Other non-solid and solid tumors, including skin, pancreas, brain cancer, sarcomas, hematologic malignancies.

  With regard to lung cancer, the disease is non-metastatic, invasive, metastatic lung cancer, squamous cell lung cancer, lung adenocarcinoma, carcinoid, small cell lung cancer or non-small cell lung cancer, overexpression in lung metastasis (to primary tumor) Detection, for example, detection of overexpression in lung cancer such as non-small cell lung cancer (eg, adenocarcinoma, squamous cell carcinoma, or carcinoid, or large cell carcinoma), identification of unexplained metastases from primary lung cancer, Not present in non-lung lungs, including osteosarcoma and soft tissue sarcomas, colorectal, uterus, cervical and body tumors, head and neck, breast, testicular and salivary gland cancer, melanoma, and bladder and kidney tumors Assessment of malignant tissue, potentially identifying different types of lung cancer (eg, small cell versus non-small cell tumor) (thus affecting treatment choice), unexplained dyspnea, chronic cough and / or Analysis of hemoptysis, differential diagnosis of pleural effusion origin, but not limited to, lung lesions and infiltrates, wheezing, wheezing, tracheal obstruction, esophageal compression, dysphagia, recurrent nerve palsy, hoarseness, diaphragm with unilateral diaphragm elevation Have similar symptoms, signs and complications as lung cancer, including but not limited to non-malignant causes of lung symptoms and signs, including neurological paralysis and Horner syndrome, and differential diagnosis between them and lung cancer is clinical Diagnosis of disease conditions that are important to, or not limited, anorexia, cachexia, weight loss, fever, hypercalcemia, hypophosphatemia, hyponatremia, antidiuretic hormone secretion syndrome, high Selected from the group consisting of ANP, high ACTH, hypokalemia, finger repellent formation, detection of the cause of any medical condition suggestive of malignant tumors, including neuromuscular disorder syndrome and thrombophlebitis.

  With regard to ovarian cancer, the compounds of the present invention are useful for the diagnosis, treatment or prognostic assessment of non-metastatic, invasive, or metastatic ovarian cancer, associating stage with malignancy, unknown cause from primary ovarian cancer Identification of metastases, differential diagnosis between benign and malignant ovarian cysts, analysis of the cause of infertility causes (eg, differential diagnosis of its various causes), serum levels of markers associated with ovary can be raised, limitations are One or more including non-malignant conditions such as endometrium, cervix, faropian tube, pancreas, breast, lung and colon cancer, pregnancy, endometriosis, pelvic inflammatory disease and uterine fibroids Detection of non-ovarian cancer pathology, including but not limited to benign ovarian cysts, uterine fibroids, endometriosis, benign ovarian neoplasms and inflammatory bowel disorders With ovarian cancer, including non-malignant causes Diagnosis of, but not limited to, anorexia, cachexia, weight loss, fever, high, with similar symptoms, signs and complications and differential diagnosis between them and lung cancer is clinically important Malignant, including calciumemia, hypophosphatemia, hyponatremia, antidiuretic hormone secretion syndrome, high ANP, high ACTH, hypokalemia, finger repellent formation, neuromuscular disorder syndrome and thrombophlebitis It can be used in determining the cause of any disease state that suggests a tumor.

  For another related purpose, the disease detected is multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, immunodeficiency associated with transplant rejection, benign lymphocyte vasculitis Erythematous lupus, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin-dependent diabetes, Goodpasture syndrome, myasthenia gravis, pemphigus, sympathetic eye Inflammation, autoimmune uveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic hepatitis, ulceritis colitis, Sjogren's syndrome, rheumatic disease, polymyositis, Scleroderma, mixed connective tissue disease, inflammatory rheumatism, degenerative rheumatism, non-articular rheumatism, collagen disease, chronic polyarthritis, arthritic psoriasis, ankylosing spondylitis Selected from the group consisting of juvenile rheumatoid arthritis, shoulder periarthritis, polyarteritis nodosa, progressive systemic scleroderma, urate arthritis, dermatomyositis, rheumatoid muscle, myositis, myosclerosis and cartilage calcification Will include autoimmunity and neoplastic disorders.

  For another related purpose, the disease detected will include any organ transplant rejection and / or graft host disease.

  In a related aspect, the assays described above include AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO. 16). ), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23) or AA424839J_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P , H68654_1_P12 (SEQ ID NO: 38), H68654J_P13 (SEQ ID NO: 39), H6865 _1_P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19O11_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R1375 An antibody that specifically binds to the (SEQ ID NO: 73) or R31375_P33 (SEQ ID NO: 74) protein would be used to detect cells affected by the disease and the assay could be accomplished in vitro or in vivo; Includes RIA, ELISA, fluorometric assay, FACS, slot blot, Western blot, immunohistochemical assay, radioimaging assay and the like. In some embodiments, the present invention provides a method of diagnosing a disease in a patient, wherein in a sample obtained from that patient or said patient, a polypeptide or polynucleotide selected from the group consisting of: Detecting at least one.

  A polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 11-16, 21-34, 35-40, 43-44, 48-50, 70-76, 138-151, 296, 298-302.

  A polypeptide comprising any one of SEQ ID NOs: 284 to 295, a bridge, an edge, a tail or a head (bridge, edge portion, tail or head portion) or a homologue thereof or a fragment thereof.

  A polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-10, 17-20, 25-34, 41-42, 45-46, 51-69.

  A polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising a bridge, edge, tail, or head or head or head portion of any one of SEQ ID NOs: 284-295.

  SEQ ID NOs: 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253 An oligonucleotide having a nucleic acid sequence.

  According to a further embodiment, detecting a polypeptide of the invention comprises a bridge, edge, tail or head of any one of SEQ ID NOs: 284-295 (bridge, edge portion, tail or head portion). Using an antibody capable of specifically binding to at least one epitope of the polypeptide comprising the amino acid sequence of the polypeptide. According to one embodiment, detecting the presence of the polypeptide or polynucleotide indicates the presence and / or severity and / or progression of the disease. According to another embodiment, a change in the expression and / or level of a polynucleotide or polypeptide relative to its expression and / or level in a healthy subject or a sample obtained therefrom is the presence of a disease and / or Indicates its severity and / or progression. According to further embodiments, an initial change in the expression and / or level of a polynucleotide or polypeptide relative to its expression and / or level in a healthy subject or a sample derived therefrom is indicative of disease progression. . According to further embodiments, the presence of the polynucleotide or polypeptide and / or the relative change in its expression and / or level is useful for treatment selection and / or observation of disease samples.

  According to one embodiment, detecting the polypeptide of the present invention comprises SEQ ID NOs: 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, An isolated oligonucleotide capable of specifically hybridizing to at least a part of a polynucleotide having the nucleic acid sequence of 232, 235, 238, 241, 244, 247, 250, 253 or a homologous polynucleotide thereto Using a primer pair comprising a pair of

  According to another embodiment, detecting the polypeptide of the present invention comprises SEQ ID NOs: 185-186, 188-189, 191-192, 194-195, 197-198, 200-201, 203-204, 206- 207, 209-210, 212-213, 215-216, 218-219, 221-222, 224-225, 227-228, 230-231, 233-234, 236-237, 239-240, 242-243, Using a primer pair comprising the pair of isolated oligonucleotides described in 245-246, 248-249, 251-252.

  The present invention also includes the following specific embodiments.

  In one embodiment, the present invention relates to AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 38) ), H68654_1_P14 (SEQ ID NO: 40), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73), R31375_P33 (SEQ ID NO: 74) Or isolated from a fragment or variant having at least 95, 96, 97, 98 or 99% sequence identity thereto. Comprising a polypeptide.

  In another embodiment, the invention includes a fragment or conjugate comprising any one of the aforementioned polypeptides.

  In another embodiment, the invention includes any one of the aforementioned polypeptides fused to an immunoglobulin region.

  In another embodiment, the invention includes any one of the aforementioned polypeptides attached to a detectable or therapeutic site.

  In another embodiment, the present invention comprises a nucleic acid sequence encoding any one of the aforementioned polypeptides.

  In another embodiment, the invention provides AI581519_T10 (SEQ ID NO: 9), AI581519_T11 (SEQ ID NO: 10), AA424839_1_T7 (SEQ ID NO: 20), H68654_1_T8 (SEQ ID NO: 28), H68654_1_T15 (SEQ ID NO: 29), H68654_1_T16 (SEQ ID NO: 30). ), H68654_1_T17 (SEQ ID NO: 31), H68654_1_T18 (SEQ ID NO: 32), H68654_1_T19 (SEQ ID NO: 33), H68654_1_T20 (SEQ ID NO: 34), AI216611_T1 (SEQ ID NO: 42), H19011_1_T8 (SEQ ID NO: 45), H19011_1_9 , R31375_T19 (SEQ ID NO: 65); R31375_T25 (SEQ ID NO: 66), R31375_T26 (arrangement) No. 67), nucleic acid sequences or fragments selected from R31375_T29 (SEQ ID NO: 68), R31375_T39 (SEQ ID NO: 69) or variants and conjugates thereof comprising at least 95, 96, 97, 98 or 99% sequence identity Including either

  In another embodiment, the present invention comprises an isolated VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 extracellular region polypeptide, or fragment or conjugate thereof.

  In another embodiment, the invention is shown in residues 23-234 of the VSIG1 protein sequence contained in the sequence of AI581519_P3 (SEQ ID NO: 11), corresponding to the amino acid sequence shown in SEQ ID NO: 138, or SEQ ID NO: 139 Residues 23-270 of the VSIG1 protein sequence included in the sequence of AI581519_P4 (SEQ ID NO: 12) corresponding to the amino acid sequence, or included in the sequence of AI581519_P5 (SEQ ID NO: 13) corresponding to the amino acid sequence shown in SEQ ID NO: 140 Residues 23-296 of the VSIG1 protein sequence, or residues 23-193 of the VSIG1 protein sequence contained in the sequence of AI581519_P7 (SEQ ID NO: 14) corresponding to the amino acid sequence shown in SEQ ID NO: 141, or SEQ ID NO: 142 Amino indicated Residues 23-203 of the VSIG1 protein sequence included in the sequence of AI581519_P9 (SEQ ID NO: 15) corresponding to the sequence, or included in the sequence of AI581519_P10 (SEQ ID NO: 16) corresponding to the amino acid sequence shown in SEQ ID NO: 143 Residues 23-231 of the VSIG1 protein sequence, or residues 26-293 of the VSIG1 protein sequence contained in the sequence of AI581519_P5 (SEQ ID NO: 13) corresponding to the amino acid sequence shown in SEQ ID NO: 302, or shown in SEQ ID NO: 75 AA424839_P3 (SEQ ID NO: 22) or AA424839_P5 (SEQIDNOSl), or AA424839_P corresponding to the amino acid sequence shown in SEQ ID NO: 76 Amino acid residues 24-105 of AA424839_1_P11 (SEQ ID NO: 24) corresponding to amino acid residues 24-456 of (SEQ ID NO: 23) or the amino acid sequence shown in SEQ ID NO: 296, or the amino acid sequence shown in SEQ ID NO: 301 Corresponding residues 50-160 of AA424839_1_P3 (SEQ ID NO: 22) or amino acid residues 29-147 of sequence AI216611_P0 (SEQ ID NO: 43) or AI216611_P1 (SEQ ID NO: 44) corresponding to the amino acid sequence shown in SEQ ID NO: 146 Or residue 1-145 of sequence AI216611_P0 (SEQ ID NO: 43) corresponding to the amino acid sequence shown in SEQ ID NO: 298, or sequence H19011_1_P8 (SEQ ID NO: 48) corresponding to the amino acid sequence shown in SEQ ID NO: 147 Amino acid residues 21-186 or residues 21-169 of the sequence H19011_1_P9 (SEQ ID NO: 50) corresponding to the amino acid sequence shown in SEQ ID NO: 148, or a sequence H19011_1_P8 (corresponding to the amino acid sequence shown in SEQ ID NO: 299) SEQ ID NO: 48) at residues 1-184, or at residues 21-36 of sequence R31375_P0 (SEQ ID NO: 70) or R31375_P31 (SEQ ID NO: 73), corresponding to the amino acid sequence set forth in SEQ ID NO: 149, or SEQ ID NO: 150 Residues 21-25 of sequence R31375_P14 (SEQ ID NO: 72) corresponding to the amino acid sequence shown, or residues 21-25 of sequence R31375_P33 (SEQ ID NO: 74) corresponding to the amino acid sequence shown in SEQ ID NO: 151, or Amy shown in SEQ ID NO: 297 The above polypeptide comprising a sequence of amino acid residues having at least 95, 96, 97, 98 or 99% sequence identity with residues 1-63 of sequence R31375_P14 (SEQ ID NO: 72), which corresponds to an acid sequence One of these.

  In another embodiment, the invention provides a polypeptide, AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 ( SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), P6876 No. 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654 1_P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375 (SEQ ID NO: 73) or any of the aforementioned polypeptides comprising the extracellular region of R31375_P33 (SEQ ID NO: 74).

  In another embodiment, the invention includes any of the aforementioned polypeptides that are added to a detectable or therapeutic site.

  In another embodiment, the invention includes any of the aforementioned nucleic acid sequences encoding any one of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 extracellular region polypeptide and containing conjugates.

  In another embodiment, the present invention includes an expression vector comprising the nucleic acid sequence.

  In another embodiment, the invention is a host cell comprising said expression vector or a virus comprising a nucleic acid sequence encoding VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 extracellular region polypeptide or fragment or conjugate thereof. Thus, the cell includes a host cell that expresses the polypeptide encoded by the DNA section.

  In another embodiment, the invention includes a method of generating either VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 extracellular region polypeptide or fragment or conjugate thereof, which is encoded by a DNA section or nucleic acid. Culturing the host cell expressing the polypeptide being recovered, and recovering the polypeptide.

  In another embodiment, the present invention comprises any of the isolated soluble VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 extracellular region, and the polypeptide comprises AI581519_P3 (SEQ ID NO: 11), AI581519LP4 ( Sequence number 12), AI581519_P5 (sequence number 13), AI581519_P7 (sequence number 14), AI581519_P9 (sequence number 15), AI581519_P10 (sequence number 16), AA424839_P3 (sequence number 22), AA424839JP5 (sequence number 21), AA424839P7 No. 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 3) ), H68654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1P , H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73), R31375JP33 (SEQ ID NO: 74) or a fragment or variant thereof Block or inhibit the interaction.

  In another embodiment, the invention comprises the isolated soluble VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 extracellular region, wherein the polypeptide is AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12 ), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 21) , AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H6 654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 44) (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73) or R31375_P33 (SEQ ID NO: 74) or a fragment or variant thereof with the corresponding functional counterpart Replaces or enhances action.

  In another embodiment, the invention relates to said isolated soluble VSIG1, ILDR1, LOC253012, AI216611, non-VSIG1, non-ILDR1, non-LOC253012, non-AI216611, non-C1ORF32, non-FXYD3 protein sequences, respectively A fusion protein containing either C1ORF32 or FXYD3 extracellular region is included.

  In another embodiment, the invention comprises any of the aforementioned fusion proteins, wherein the non-VSIG1, non-ILDR1, non-LOC253012, non-AI216611, non-C1ORF32, non-FXYD3 protein is at least part of an immunoglobulin molecule.

  In another embodiment, the invention includes any of the aforementioned fusion proteins, wherein a polyalkyloxide moiety, such as polyethylene glycol, is appended to the polypeptide.

  In another embodiment, the invention includes any of the aforementioned fusion proteins, wherein the immunoglobulin heavy chain constant region is an Fc fragment.

  In another embodiment, the invention provides a protein sequence of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 and FXYD3ECD fused to mouse Fc according to any one of the amino acid sequences shown in SEQ ID NOs: 103-108. Nucleic acid sequences encoding any one or VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 and FXYD3ECD fused to mouse Fc. The invention further comprises a nucleic acid sequence encoding VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 and FXYD3ECD fused to a mouse Fc according to any one of the sequences shown in SEQ ID NOs: 97-102.

  In another embodiment, the invention comprises any of the aforementioned fusion proteins, wherein the immunoglobulin heavy chain constant region is an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA, and IgD. .

  In another embodiment, the invention includes any of the aforementioned fusion proteins, wherein the polypeptide is fused to the VASP region.

  In another embodiment, the invention includes any of the aforementioned fusion proteins, wherein the fusion protein modulates lymphocyte activation.

  In another embodiment, the invention includes a pharmaceutical composition comprising any of the aforementioned polynucleotide sequences and further comprising a pharmacologically acceptable diluent or carrier.

  In another embodiment, the present invention comprises a pharmaceutical composition comprising said vector and further comprising a pharmacologically acceptable diluent or carrier.

  In another embodiment, the present invention includes a pharmaceutical composition comprising said host cell and further comprising a pharmacologically acceptable diluent or carrier.

  In another embodiment, the present invention includes a pharmaceutical composition comprising any of the aforementioned VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 extracellular region, and further comprising a pharmaceutically acceptable diluent or carrier. .

  In another embodiment, the invention includes a pharmaceutical composition comprising any of the aforementioned polypeptides and further comprising a pharmacologically acceptable diluent or carrier.

  In another embodiment, the present invention includes a pharmaceutical composition comprising said fusion protein and further comprising a pharmacologically acceptable diluent or carrier.

  In another embodiment, the invention includes a method of treating or preventing cancer, which is included in the sequence of AI581519_P3 (SEQ ID NO: 11) corresponding to the amino acid sequence set forth in SEQ ID NO: 138 to a patient in need thereof. Residues 23-234 of the VSIG1 protein sequence, or residues 23-270 of the VSIG1 protein sequence contained in the sequence of AI581519_P4 (SEQ ID NO: 12) corresponding to the amino acid sequence shown in SEQ ID NO: 139, or SEQ ID NO: 140 Residues 23-296 of the VSIG1 protein sequence included in the sequence of AI581519_P5 (SEQ ID NO: 13) corresponding to the amino acid sequence shown, or the sequence of AI581519_P7 (SEQ ID NO: 14) corresponding to the amino acid sequence shown in SEQ ID NO: 141 Of VSIG1 protein contained in Residues 23-193 of VSIG1 protein sequence contained in the sequence of AI581519_P9 (SEQ ID NO: 15), corresponding to the amino acid sequence shown in SEQ ID NO: 142, or the amino acid sequence shown in SEQ ID NO: 143 VSIG1 included in the sequence of AI581519_P5 (SEQ ID NO: 13) corresponding to the amino acid sequence shown in SEQ ID NO: 302 or residues 23-231 of the VSIG1 protein sequence included in the sequence of AI581519_P10 (SEQ ID NO: 16) Residues 26-293 of the protein sequence, or amino acid residues 24-162 of AA424839_P3 (SEQ ID NO: 22) or AA424839_P5 (SEQIDNOSl), corresponding to the amino acid sequence shown in SEQ ID NO: 75, or SEQ ID NO: Amino acid residues 24-105 of AA424839_P7 (SEQ ID NO: 23) corresponding to the amino acid sequence shown in 6, or AA424839_1_P11 (SEQ ID NO: 24) corresponding to the amino acid sequence shown in SEQ ID NO: 296 , Or residues 50-160 of AA424839_1_P3 (SEQ ID NO: 22) corresponding to the amino acid sequence shown in SEQ ID NO: 301, or sequence AI216611_P0 (SEQ ID NO: 43) or AI216611_P1 (corresponding to the amino acid sequence shown in SEQ ID NO: 146) SEQ ID NO: 44), amino acid residues 29-147, or residues 1-145 of sequence AI216611_P0 (SEQ ID NO: 43) corresponding to the amino acid sequence shown in SEQ ID NO: 298, or the amino acid sequence shown in SEQ ID NO: 147 Amino acid residues 21-186 of sequence H19011_1_P8 (SEQ ID NO: 48) corresponding to the columns, or residues 21-169 of sequence H19011_1_P9 (SEQ ID NO: 50) corresponding to the amino acid sequence shown in SEQ ID NO: 148, or SEQ ID NO: Sequence R31375_P0 (SEQ ID NO: 70) or R31375_P31 (SEQ ID NO: 73) corresponding to the amino acid sequence shown in SEQ ID NO: 149, or residue 1-184 of sequence H19011_1_P8 (SEQ ID NO: 48), corresponding to the amino acid sequence shown in 299 ) Residues 21-36, or residues 21-65 of sequence R31375_P14 (SEQ ID NO: 72) corresponding to the amino acid sequence shown in SEQ ID NO: 150, or sequences R31375_P33 corresponding to the amino acid sequence shown in SEQ ID NO: 151 (Sequence number 4) residues 21-25, or residues 1-63 of sequence R31375_P14 (SEQ ID NO: 72), corresponding to the amino acid sequence shown in SEQ ID NO: 297, at least 95, 96, 97, 98 or 99% sequence VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 polypeptide extracellular region or fragment or conjugate thereof, including a sequence of amino acid residues having identity, or a polypeptide, or a nucleic acid sequence encoding the same Administering a pharmaceutical composition comprising a soluble molecule.

  In another embodiment, the invention provides that the cancer is a hematological malignancy such as acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma And selected from the group consisting of soft or solid tumors such as breast, prostate, lung, ovary, colon, spleen, kidney, bladder, head and neck, uterus, testes, stomach, neck, liver, bone, skin, pancreas, brain cancer, etc. Wherein the cancer is non-metastatic, invasive, or metastatic.

  In another embodiment, the invention provides the method, wherein the cancer is selected from the group consisting of lung cancer, ovarian cancer or colon cancer, and the lung cancer, ovarian cancer or colon cancer is non-metastatic, invasive or metastatic. including.

  In another embodiment, the invention includes a method for treating or preventing an immune-related condition such as an autoimmune disease or transplant rejection, corresponding to the amino acid sequence set forth in SEQ ID NO: 138 for a patient in need thereof. , Residues 23-234 of the VSIG1 protein sequence contained in the sequence of AI581519_P3 (SEQ ID NO: 11), or the VSIG1 protein sequence contained in the sequence of AI581519_P4 (SEQ ID NO: 12) corresponding to the amino acid sequence shown in SEQ ID NO: 139 Residues 23-270, or residues 23-296 of the VSIG1 protein sequence contained in the sequence of AI581519_P5 (SEQ ID NO: 13), corresponding to the amino acid sequence shown in SEQ ID NO: 140, or the amino acid sequence shown in SEQ ID NO: 141 Corresponding AI581519_P7 (sequence number 14) residues 23-193 of the VSIG1 protein sequence contained in the sequence of 14), or residues 23-203 of the VSIG1 protein sequence contained in the sequence of AI581519_P9 (SEQ ID NO: 15) corresponding to the amino acid sequence shown in SEQ ID NO: 142 Or AI581519_P5 (corresponding to the amino acid sequence shown in SEQ ID NO: 302, or residues 23-231 of the VSIG1 protein sequence contained in the sequence of AI581519_P10 (SEQ ID NO: 16) corresponding to the amino acid sequence shown in SEQ ID NO: 143 Of the sequence AA424839_P3 (SEQ ID NO: 22) or AA424839_P5 (SEQ ID NO: 21) corresponding to residues 26-293 of the VSIG1 protein sequence included in the sequence of SEQ ID NO: 13) or the amino acid sequence shown in SEQ ID NO: 75 Amino acid residues 24-162, or AA424839_P7 (SEQ ID NO: 23) corresponding to the amino acid sequence shown in SEQ ID NO: 76, or AA424839_1_P11 (corresponding to the amino acid sequence shown in SEQ ID NO: 296) SEQ ID NO: 24) corresponding to amino acid residues 24-105 of AA424839_1_P3 (SEQ ID NO: 22) corresponding to the amino acid sequence shown in SEQ ID NO: 301, or corresponding to the amino acid sequence shown in SEQ ID NO: 146 Amino acid residues 29-147 of sequence AI216611_P0 (SEQ ID NO: 43) or AI216611_P1 (SEQ ID NO: 44), or residues 1-145 of sequence AI216611_P0 (SEQ ID NO: 43) corresponding to the amino acid sequence shown in SEQ ID NO: 298, Alternatively, amino acid residues 21-186 of the sequence H19011_1_P8 (SEQ ID NO: 48) corresponding to the amino acid sequence shown in SEQ ID NO: 147, or the remainder of the sequence H19011_1_P9 (SEQ ID NO: 50) corresponding to the amino acid sequence shown in SEQ ID NO: 148 Group 21-169, or residue 1-184 of sequence H19011_1_P8 (SEQ ID NO: 48), corresponding to the amino acid sequence shown in SEQ ID NO: 299, or sequence R31375_P0 (SEQ ID NO: 149 corresponding to the amino acid sequence shown in SEQ ID NO: 149 70) or residues 21-36 of R31375_P31 (SEQ ID NO: 73), or residues 21-65 of sequence R31375_P14 (SEQ ID NO: 72), corresponding to the amino acid sequence shown in SEQ ID NO: 150, or SEQ ID NO: 151 Equivalent to amino acid sequence At least 95, 96, 97 to residues 21-25 of sequence R31375_P33 (SEQ ID NO: 74), or residues 1-63 of sequence R31375_P14 (SEQ ID NO: 72) corresponding to the amino acid sequence set forth in SEQ ID NO: 297 An extracellular region or fragment of a VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 polypeptide, or a conjugate thereof, or a polypeptide or a coding sequence thereof, comprising a sequence of amino acid residues having 98 or 99% sequence identity Administering a pharmaceutical composition comprising a soluble molecule having the nucleic acid sequence to be treated.

  In another embodiment, the invention provides that the autoimmune disease is multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, transplant rejection, benign lymphocytes; Vasculitis, lupus erythematosus, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin-dependent diabetes, Goodpasture syndrome, myasthenia gravis, pemphigus, cross Sensitive ophthalmitis, autoimmune uveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic hepatitis, ulcerative colitis, Sjogren's syndrome, rheumatic disease, multiple Myositis, scleroderma, mixed connective tissue disease, inflammatory rheumatism, degenerative rheumatism, non-articular rheumatism, collagen disease, chronic polyarthritis, arthritic psoriasis, ankylosing spine Selected from the group consisting of inflammation, juvenile rheumatoid arthritis, peri-shoulderitis, polyarteritis nodosa, progressive systemic sclerosis, urate arthritis, dermatomyositis, rheumatoid myositis, myositis, myopathy and cartilage calcification Said method.

  In another embodiment, the invention includes the aforementioned method, wherein the immune-related disorder is selected from transplant rejection or graft host disease.

  In another embodiment, the invention provides AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16). ), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1 , H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 ( Column number 40), AI216611_P0 (sequence number 43), AI216611_P1 (sequence number 44), H19011_1_P8 (sequence number 48), H19011_1_P9 (SEQIDNOSO), R31375_P0 (sequence number 70), R31375_P14 (sequence number 72), R31375_P31 (sequence number 72) ), A siRNA that binds to and inhibits the expression of a transcription product encoding any one of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 polypeptide or a variant thereof selected from R31375_P33 (SEQ ID NO: 74) , Antisense RNA or ribozyme.

  In another embodiment, the invention provides AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16). ), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839JJ7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1 , H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 ( (Column number 40), AI216611_P0 (sequence number 43), AI216611_P1 (sequence number 44), H19011_1_P8 (sequence number 48), H19011_1_P9 (sequence number 50), R31375_P0 (sequence number 70), R31375_P14 (sequence number 72), R31375_P31 (sequence) No. 73), specifically binds to any one or fragment of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 polypeptide selected from R31375_P33 (SEQ ID NO: 74), or variants and containing conjugates thereof, and / or Or polyclonal or monoclonal antibodies that modulate the activity induced thereby.

  In another embodiment, the invention provides AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16). ), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1 , H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 ( Column number 40), AI216611_P0 (sequence number 43), AI216611_P1 (sequence number 44), H19011_1_P8 (sequence number 48), H19011_1_P9 (sequence number 50), R31375_P0 (sequence number 70), R31375_P14 (sequence number 72), R31375_P31 (sequence) No. 73), specifically binds to any one or fragment of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 polypeptide contained in R31375_P33 (SEQ ID NO: 74) or a variant thereof that is at least 80% identical thereto A monoclonal or polyclonal antibody or antigen-binding fragment thereof containing an antigen-binding site is included.

  In another embodiment, the invention provides that the antibody comprises AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), P6876 SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_ _P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_31 (SEQ ID NO: 73), one of R31375_P33 (SEQ ID NO: 74), or any of the aforementioned antibodies or fragments thereof that block or inhibit interaction with the opposite activity or function of the fragment or variant thereof.

  In another embodiment, the invention provides that the antibody comprises AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), P6876 SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_ _P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73), including any of the aforementioned antibodies or fragments thereof that replace or enhance interaction with one of the R31375_P33 (SEQ ID NO: 74) or fragment or a variant activity or function thereof.

  In another embodiment, the invention includes a method of modulating lymphocyte activity, comprising AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 24) (SEQ ID NO: 36), H68654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 ( Column number 39), H68654_1_P14 (sequence number 40), AI216611_P0 (sequence number 43), AI216611_P1 (sequence number 44), H19011_1_P8 (sequence number 48), H19011_1_P9 (sequence number 50), R31375_P0 (sequence number 70), R31375_P14 (sequence number) 72), R31375_P31 (SEQ ID NO: 73), R31375_P33 (SEQ ID NO: 74) positive lymphocytes with bioactive agents capable of modulating VSIG1-mediated, ILDR1-mediated, LOC253012-mediated, AI216611-mediated, C1ORF32-mediated or FXYD3-mediated signaling Contacting with an amount effective to modulate at least one lymphocyte activity.

  In another embodiment, the invention provides that the agent comprises VSIG1-mediated, ILDR1-mediated, LOC253012-mediated, AI216611-mediated, C1ORF32-mediated signaling antagonists or FXYD3-mediated signaling, wherein the contact is mediated by such signaling The method of inhibiting the attenuation of lymphocyte activity.

  In another embodiment, the invention includes the method, wherein the contacting increases lymphocyte activity.

  In another embodiment, the invention provides the method, wherein the antagonist comprises a blocking agent capable of preventing a functional interaction of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 antigens with each other. Including.

  In another embodiment, the present invention is suitable for the treatment or prevention of cancer by modulating the activity of any one of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 protein in the B7-like costimulatory system. Includes antibodies or fragments.

  In another embodiment, the present invention includes the aforementioned methods wherein the administered antibody or fragment inhibits negative stimulation of T cell activity against cancer cells.

  In another embodiment, the invention provides that the cancer is a hematological malignancy such as acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma And from the group consisting of soft tissue or solid tumors such as breast, prostate, lung, ovary, colon, spleen, kidney, bladder, head and neck, uterus, testis, stomach, neck, liver, bone, skin, pancreas, brain cancer, etc. A selected cancer, as well as any of the aforementioned antibodies or fragments, wherein the cancer is non-metastatic, invasive, or metastatic.

  In another embodiment, the invention relates to an immune-related disorder, such as an autoimmune disease or transplant rejection, by modulating the activity of any of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 protein in a B7-like costimulatory system. Any of the aforementioned antibodies or fragments suitable for the treatment or prevention of

  In another embodiment, the invention relates to multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, immunodeficiency associated with transplant rejection, benign lymphocyte vasculitis, erythema Lupus, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin-dependent diabetes, Goodpascher's syndrome, myasthenia gravis, pemphigus, sympathetic ophthalmitis, Autoimmune uveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic hepatitis, Sjogren's syndrome, rheumatic disease, polymyositis, scleroderma, mixed connective tissue disease, inflammatory Rheumatism, degenerative rheumatism, non-articular rheumatism, collagen disease, chronic polyarthritis, arthritic psoriasis, ankylosing spondylitis, juvenile rheumatoid arthritis, shoulder periarthritis, nodular polyarteritis, progressive Including only scleroderma, arthritis Uratica, dermatomyositis, muscular rheumatism, myositis, muscle hardening diseases and one of the antibodies or fragments are suitable for the treatment of autoimmune diseases selected from the cartilage calcification.

  In another embodiment, the invention includes any of the aforementioned antibodies or fragments that are suitable for the treatment of transplant rejection or graft host disease.

  In another embodiment, the invention provides amino acids: residues 23-234 of the VSIG1 protein sequence contained in the sequence of AI581519_P3 (SEQ ID NO: 11), corresponding to the amino acid sequence shown in SEQ ID NO: 138, or SEQ ID NO: 139 Residues 23-270 of the VSIG1 protein sequence included in the sequence of AI581519_P4 (SEQ ID NO: 12) corresponding to the amino acid sequence shown, or the sequence of AI581519_P5 (SEQ ID NO: 13) corresponding to the amino acid sequence shown in SEQ ID NO: 140 Residues 23-296 of the VSIG1 protein sequence contained in VSIG1, or residues 23-193 of the VSIG1 protein sequence contained in the sequence of AI581519_P7 (SEQ ID NO: 14) corresponding to the amino acid sequence shown in SEQ ID NO: 141, or SEQ ID NO: Shown in 142 In the sequence of AI581519_P10 (SEQ ID NO: 16) corresponding to the amino acid sequence shown in SEQ ID NO: 143, or residues 23-203 of the VSIG1 protein sequence included in the sequence of AI581519_P9 (SEQ ID NO: 15) Residues 23-231 of the included VSIG1 protein sequence, or residues 26-293 of the VSIG1 protein sequence included in the sequence of AI581519_P5 (SEQ ID NO: 13) corresponding to the amino acid sequence shown in SEQ ID NO: 302, or SEQ ID NO: 75 AA424 corresponding to the amino acid sequence shown in SEQ ID NO: 76, or AA424 corresponding to the amino acid sequence shown in SEQ ID NO: 76, or AA424248_P3 (SEQ ID NO: 22) or AA424839_P5 (SEQ ID NOSl) Amino acid residues 24-105 of AA424839_1_P11 (SEQ ID NO: 24) corresponding to amino acid residues 24-456 of 39_P7 (SEQ ID NO: 23) or the amino acid sequence shown in SEQ ID NO: 296, or the amino acid sequence shown in SEQ ID NO: 301 Corresponding to residues 50-160 of AA424839_1_P3 (SEQ ID NO: 22), or amino acid residues 29- 147, or a residue H-14511_1_P8 (SEQ ID NO: 298) corresponding to the residue 1-145 of the sequence AI216611_P0 (SEQ ID NO: 43), which corresponds to the amino acid sequence shown in SEQ ID NO: 298, or the amino acid sequence shown in SEQ ID NO: 147 No. 48) corresponding to amino acid residues 21-186 of SEQ ID NO: 148, or amino acid sequences shown in SEQ ID NO: 148, corresponding to residues 21-169 of sequence H19011_1_P9 (SEQ ID NO: 50), or SEQ ID NO: 299 , Residues 1-184 of sequence H19011_1_P8 (SEQ ID NO: 48), or residues 21-36 of sequence R31375_P0 (SEQ ID NO: 70) or R31375_P31 (SEQ ID NO: 73) corresponding to the amino acid sequence set forth in SEQ ID NO: 149, or Residues 21-65 of sequence R31375_P14 (SEQ ID NO: 72) corresponding to the amino acid sequence shown in SEQ ID NO: 150, or residue 21 of sequence R31375_P33 (SEQ ID NO: 74) corresponding to the amino acid sequence shown in SEQ ID NO: 151 -25, or as set forth in SEQ ID NO: 297 Corresponding to amino acid sequence, including any of the antibodies or fragments that specifically bind to residues 1-63, or variant or fragment or epitope thereof of SEQ R31375_P14 (SEQ ID NO: 72).

  In another embodiment, the invention provides an antibody or fragment of said antibody or fragment, wherein the antigen binding site comprises about 3-7 consecutive or non-contiguous amino acids, more typically at least 5 consecutive or non-contiguous amino acids. Including either These binding sites include conformational and nonconformational epitopes.

  In another embodiment, the invention includes any of the aforementioned antibodies or fragments, wherein the antibody is a fully human antibody.

  In another embodiment, the invention includes any of the aforementioned antibodies or fragments, wherein the antibody is a chimeric antibody.

  In another embodiment, the invention comprises any of the aforementioned antibodies or fragments, wherein the antibody is a humanized or primatized antibody.

  In another embodiment, the invention the fragment is selected from the group consisting of Fab, Fab ′, F (ab ′) 2, F (ab ′), F (ab), Fv or scFv fragments and minimal recognition units. Including either the antibody or fragment.

  In another embodiment, the invention includes any of said antibodies or fragments wherein the antibody or fragment is linked to a detectable marker or effector site.

  In another embodiment, the invention includes any of the aforementioned antibodies or fragments, wherein the effector site is an enzyme, toxin, therapeutic agent or chemotherapeutic agent.

  In another embodiment, the invention comprises any of the aforementioned antibodies or fragments, wherein the detectable marker is a radioisotope, metal chelator, enzyme, fluorescent compound, bioluminescent compound or chemiluminescent compound.

  In another embodiment, the invention includes a pharmaceutical composition comprising any of the antibodies or fragments thereof.

  In another embodiment, the present invention comprises a pharmaceutical composition comprising said antibody or fragment thereof.

  In another embodiment, the invention includes a method of eliciting or enhancing an immune response, the method comprising administering the antibody or fragment to a patient in need thereof and inducing or enhancing the immune response. Including detecting.

  In another embodiment, the invention includes a method of enhancing a secondary immune response to an antigen in a patient, the method comprising administering an effective amount of either the antibody or fragment.

  In another embodiment, the invention includes the above methods wherein the antigen is preferably a cancer antigen, Weil's antigen or bacterial antigen, preferably undergoing treatment with an anti-cancer vaccine or a viral vaccine.

  In another embodiment, the present invention provides a method for treating a patient with a VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 positive malignancy comprising administering to the patient an effective amount of any of the above antibodies or fragments. including.

  In another embodiment, the present invention includes the aforementioned methods further comprising co-administering a chemotherapeutic agent.

  In another embodiment, the invention provides that the malignancy is blood such as acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma Malignant tumors as well as soft or solid tumors such as breast, prostate, lung, ovary, colon, spleen, kidney, bladder, head and neck, uterus, testes, stomach, neck, liver, bone, skin, pancreas, brain cancer Including said method wherein the cancer is selected from the group and the cancer is non-metastatic, invasive or metastatic.

  In another embodiment, the invention provides that the malignant tumor is selected from the group consisting of lung cancer, ovarian cancer, colon cancer, and the lung cancer, ovarian cancer or colon cancer is non-metastatic, invasive or metastatic. Including the method.

  In another embodiment, the present invention relates to AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15) in a biological sample. AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654J_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 35) (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H686 4_1_P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R1375 (SEQ ID NO: 73), an assay that detects the presence of R31375_P33 (SEQ ID NO: 74) or a fragment or variant thereof, wherein the assay comprises contacting the sample with any one of the antibodies described above, and in the sample AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519 P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), 68 (SEQ ID NO: 36), H68654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011 SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 7) 0), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73), R31375_P33 (SEQ ID NO: 74) or a fragment or variant thereof.

  In another embodiment, the invention provides AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16). ), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1 , H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 ( Column number 40), AI216611_P0 (sequence number 43), AI216611_P1 (sequence number 44), H19011_1_P8 (sequence number 48), H19011_1_P9 (sequence number 50), R31375_P0 (sequence number 70), R31375_P14 (sequence number 72), R31375_P31 (sequence) No. 73), detecting a disease, detecting a disease, detecting the disease, including detecting the expression of R31375_P33 (SEQ ID NO: 74) or a fragment or variant thereof, or observing the progression of the disease or the effect of treatment or the recurrence of the disease, or A method of selecting a therapy for the disease.

  In another embodiment, the invention provides AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16). ), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1 , H68654_1_P12 (SEQ ID NO: 38), H68654J_P13 (SEQ ID NO: 39), H68654_1_P14 (arrangement) No. 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 72) 73), wherein the detection of expression of R31375_P33 (SEQ ID NO: 74) or fragment or variant thereof is performed in vivo or in vitro.

  In another embodiment, the invention provides the method, wherein the disease is selected from the group consisting of lung cancer, ovarian cancer, colon cancer and the lung cancer, ovarian cancer or colon cancer is non-metastatic, invasive or metastatic. including.

  In another embodiment, the invention provides that the disease is multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, immunodeficiency associated with transplant rejection, benign lymphocyte vasculitis Erythematous lupus, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin-dependent diabetes, Goodpasture syndrome, myasthenia gravis, pemphigus, sympathetic eye Inflammation, autoimmune uveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic hepatitis, ulceritis colitis, Sjogren's syndrome, rheumatic disease, polymyositis, Scleroderma, mixed connective tissue disease, inflammatory rheumatism, degenerative rheumatism, non-articular rheumatism, collagen disease, chronic polyarthritis, arthritic psoriasis, ankylosing spondylitis, young Sex rheumatoid arthritis, shoulder periarthritis, nodosa, progressive systemic scleroderma, urate arthritis, dermatomyositis, a muscular rheumatism, myositis, muscle hardness disease or cartilage calcification, including the method.

  In another embodiment, the invention provides AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 ( SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_I_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), P6876 No. 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654 1_P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19O11_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R3375 (SEQ ID NO: 73), R31375_P33 (SEQ ID NO: 74), a method of inhibiting the growth of cells expressing a polypeptide or fragment thereof, or a variant thereof, the method comprising: Administration.

  In another embodiment, the invention provides AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16). ), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1 , H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 ( Column number 40), AI216611_P0 (sequence number 43), AI216611_P1 (sequence number 44), H19011_1_P8 (sequence number 48), H19011_1_P9 (sequence number 50), R31375_P0 (sequence number 70), R31375_P14 (sequence number 72), R31375_P31 (sequence) No. 73), R31375_P33 (SEQ ID NO: 74) or fragment or a method of treating or preventing cancer comprising administration of a therapeutically effective amount of an antibody or binding fragment that specifically binds to a variant with 80% identity. Including.

  In another embodiment, the invention provides that the cancer is a hematological malignancy such as acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma And from the group consisting of soft or solid tumors such as breast, prostate, lung, ovary, colon, spleen, kidney, bladder, head and neck, uterus, testes, stomach, neck, liver, bone, skin, pancreas, brain cancer, etc. Selected, wherein the cancer is non-metastatic, invasive, or metastatic.

  In another embodiment, the invention provides the method, wherein the cancer is selected from the group consisting of lung cancer, ovarian cancer, colon cancer, and the lung cancer, ovarian cancer or colon cancer is non-metastatic, invasive or metastatic. including

  In another embodiment, the present invention includes the aforementioned methods wherein the antibody is a human, humanized or chimeric antibody or antigen-binding fragment.

  In another embodiment, the present invention includes the aforementioned methods, wherein the antibody or fragment is added directly or indirectly to the effector site.

  In another embodiment, the present invention includes the aforementioned methods wherein the effector is selected from a drug, toxin, radionuclide, fluorophore, and enzyme.

  In another embodiment, the invention includes a method of treating or preventing an immunodeficiency disease, such as an autoimmune or transplantation related disease, which comprises administering to the patient a therapeutically effective amount of an antibody that specifically binds next. Do: AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 16) AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H686 4_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 4811) (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73), R31375_P33 (SEQ ID NO: 74),

  Or a fragment or variant thereof having at least 80% sequence identity thereto.

  In another embodiment, the invention comprises the method of the above method, wherein the antibody has an antigen binding region specific for the extracellular region of any one of said VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 polypeptide. .

  In another embodiment, the invention comprises the method of the above method wherein the antibody or fragment modulates the B7 / costimulatory system in a manner that inhibits positive stimulation of T cell activity that produced an autoimmune effect.

  In another embodiment, the invention includes the method of the above methods, wherein the treatment is combined with a site that is useful in treating an autoimmune or transplant rejection condition.

  In another embodiment, the invention includes the method of the above methods, wherein the site is a cytokine antibody, cytokine receptor antibody, drug or other immunomodulator.

  In another embodiment, the invention provides that the autoimmune disease is multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, transplant rejection-related immune deficiency, benign lymphocytes Vasculitis, lupus erythematosus, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin-dependent diabetes, Goodpasture syndrome, myasthenia gravis, pemphigus, cross Sensitive ophthalmitis, autoimmune uveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic hepatitis, ulcerative colitis, Sjogren's syndrome, rheumatic disease, multiple Myositis, scleroderma, mixed connective tissue disease, inflammatory rheumatism, degenerative rheumatism, non-articular rheumatism, collagen disease, chronic polyarthritis, arthritic psoriasis, ankylosing spine Selected from the group consisting of inflammation, juvenile rheumatoid arthritis, peri-shoulderitis, polyarteritis nodosa, progressive systemic sclerosis, urate arthritis, dermatomyositis, rheumatoid myositis, myositis, myopathy and cartilage calcification Including the method of said method.

  In another embodiment, the invention includes the aforementioned methods, wherein the immunodeficiency disorder is transplant rejection or graft host disease.

  In another embodiment, the invention provides AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16). ), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1 , H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 ( (Column number 40), AI216611_P0 (sequence number 43), AI216611_P1 (sequence number 44), H19011_1_P8 (sequence number 48), H19011_1_P9 (sequence number 50), R31375_P0 (sequence number 70), R31375_P14 (sequence number 72), R31375_P31 (sequence) No. 73), AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12) for in vivo imaging of tumors or sites of inflammation characterized by differential expression of R31375_P33 (SEQ ID NO: 74) or fragments or variants thereof AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_37 ), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1P (H19011_1P) , R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375 This includes a method of using an antibody or antigen-binding fragment that specifically binds to _P31 (SEQ ID NO: 73), R31375_P33 (SEQ ID NO: 74), or a fragment or variant thereof.

  In another embodiment, the invention includes the method used to assess cancer prognosis or treatment protocol.

  In another embodiment, the invention includes a method of screening for disease in a patient, wherein the method comprises AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), in the patient or a sample obtained from said patient. AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (A1124823_P7 (A41123939)) (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P7 (SEQ ID NO: 37), H 8654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 48) (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73), R31375JP33 (SEQ ID NO: 74), a polypeptide having a sequence that is at least 85% homologous to the amino acid sequence, or AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_ SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 6611), A21 No. 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 5) 0), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73), R31375_P33 (SEQ ID NO: 74), and to detect a polypeptide having a sequence containing one of the extracellular regions Including.

  In another embodiment, the invention provides that screening for a disease comprises detecting the presence or severity of a disease, disorder, condition, or prognosis of a subject, or selecting a treatment for the subject, or observing treatment for the subject. Including methods.

  In another embodiment, the invention provides that the disease is a cancer selected from the group consisting of lung cancer, ovarian cancer, colon cancer, wherein lung cancer, ovarian cancer and colon cancer are non-metastatic, invasive or metastatic. Including the method.

  In another embodiment, the invention relates to an autoimmune disease wherein the disease is multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, transplant rejection Immunodeficiency, benign lymphocyte vasculitis, lupus erythematosus, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin-dependent diabetes mellitus, Goodpasture syndrome, myasthenia gravis Disease, pemphigus, sympathetic ophthalmitis, autoimmune uveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic acting hepatitis, ulcerative colitis, Sjogren's syndrome, Rheumatoid disease, polymyositis, scleroderma, mixed connective tissue disease, inflammatory rheumatism, degenerative rheumatism, non-articular rheumatism, collagen disease, chronic polyarthritis, joint Psoriasis, ankylosing spondylitis, juvenile rheumatoid arthritis, shoulder periarthritis, polyarteritis nodosa, progressive systemic sclerosis, urate arthritis, dermatomyositis, rheumatoid muscularitis, myositis, myosclerosis and cartilage lime Including the method selected from

  In another embodiment, the present invention includes the aforementioned methods, wherein the detection is performed by an immunoassay.

  In another embodiment, the invention provides that the immunoassay comprises AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10. (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), P6876 SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H6865 _1_P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R3375 (SEQ ID NO: 73), a polypeptide having a sequence that is at least 85% homologous to the amino acid sequence of any one of R31375_P33 (SEQ ID NO: 74), AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_37 ), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1P (H19011_1P) , R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375 The method includes using an antibody that specifically interacts with a polypeptide having a sequence including the extracellular region of any one of _P31 (SEQ ID NO: 73) and R31375_P33 (SEQ ID NO: 74).

  In another embodiment, the invention induces apoptosis or lysis of cancer cells expressing the protein, AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), H68654_1_P ), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H 8654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R1375_P0 (SEQ ID NO: 70) (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73), and R31375_P33 (SEQ ID NO: 74), or fragments or variants thereof.

  In another embodiment, the invention comprises said antibody or fragment wherein said apoptosis or lysis comprises the CDC or ADCC activity of the antibody.

  In another embodiment, the invention provides that the cancer cells are hematologic malignancies such as acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma. Groups of tumors and soft or solid tumors such as breast, prostate, lung, ovary, colon, spleen, kidney, bladder, head and neck, uterus, testis, stomach, neck, liver, bone, skin, pancreas, brain cancer, etc. Any one of said antibodies or fragments selected from.

  In another embodiment, the invention comprises any of the aforementioned antibodies or fragments, wherein the cancer cells are lung, ovarian or colon cancer cells.

  In another embodiment, the present invention provides the isolated soluble VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, wherein the polypeptide or fragment or variant thereof is used as an anti-cancer vaccine for cancer immunotherapy. , Any of FXYD3 extracellular region polypeptides.

  In another embodiment, the invention provides at least 80%, 85%, 90%, 95, 96, 97, 98 or 99%, 100% homology to the sequence set forth in any one of SEQ ID NOs: 284-295. Pertains to any isolated polypeptide or fragment thereof comprising a sex amino acid sequence.

  In another embodiment, the present invention provides 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, Relates to any isolated polynucleotide comprising an amplicon having a nucleic acid sequence selected from the group consisting of 247, 250, 253, or a polynucleotide homologous thereto.

  In another embodiment, the invention relates to any primer pair comprising a pair of isolated oligonucleotides capable of amplifying the amplicon.

  SEQ ID NOs: 185-186, 188-189, 191-192, 194-195, 197-198, 200-201, 203-204, 206-207, 209-210, 212-213, 215-216, 218-219, A sequence selected from the group consisting of 221-222, 224-225, 227-228, 230-231, 233-234, 236-237, 239-240, 242-243, 245-246, 248-249, and 251-252 A primer pair comprising a pair of isolated oligonucleotides having

  The present invention is a method of screening for a disease, disorder, or disease state in a patient, and the method is performed by using SEQ ID NOs: 1-10, 17-20, 25-34, 41 in the patient or a sample obtained from the patient. -42, 45-46, 51-69, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244 Detection of a polynucleotide having a sequence that is at least 85% homologous to the nucleic acid sequence of any one of 247, 250 and 253.

  In another embodiment, the invention relates to the method as described above, wherein disease screening comprises detection of the presence or severity of a disease, disorder, condition, or prognosis of a subject, or selection of treatment for the subject, or treatment observation.

  The aforementioned method, wherein the disease is a cancer selected from the group consisting of lung cancer, colon cancer and ovarian cancer, wherein lung cancer, colon cancer and ovarian cancer are non-metastatic, invasive or metastatic.

  Said method wherein the disease is an autoimmune disease.

  Detection is SEQ ID NO: 1-10, 17-20, 25-34, 41-42, 45-46, 51-69, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217 , 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, and 253, an oligonucleotide capable of hybridizing to at least a portion of the nucleic acid sequence that is at least 85% homologous to the nucleic acid sequence The above method carried out using pairs.

  Detection is SEQ ID NO: 185-186, 188-189, 191-192, 194-195, 197-198, 200-201, 203-204, 206-207, 209-210, 212-213, 215-216, 218 -219, 221-222, 224-225, 227-228, 230-231, 233-234, 236-237, 239-240, 242-243, 245-246, 248-249, and 251-252 The above method carried out using the oligonucleotide pair described in 1.

FIG. 1 shows a schematic summary of quantitative real-time PCR analysis. FIG. 2 is a scatter plot showing the expression of AI581519 transcript encoding VSIG1 protein on a virtual panel of all tissues and conditions using the MED discovery engine, in lung cancer compared to normal lung samples Shows overexpression of AI581519 transcript. FIG. 3A shows a comparative alignment of AI581519_P4 protein to the known VSIG1 protein NP_872413 (SEQ ID NO: 11) and Q86XK7_HUMAN. FIG. 3B shows a comparative alignment of the AI581519_P5 protein against the known VSIG1 protein NP_872413 (SEQ ID NO: 11) and Q86XK7_HUMAN. FIG. 3C shows a comparative alignment of the AI581519_P7 protein to the known VSIG1 protein NP_872413 (SEQ ID NO: 11) and Q86XK7_HUMAN. FIG. 3D shows a comparative alignment of AI581519_P9 protein to the known VSIG1 protein NP_872413 (SEQ ID NO: 11) and Q86XK7_HUMAN. FIG. 3E shows a comparative alignment of the AI581519_P10 protein against the known VSIG1 protein NP_872413 (SEQ ID NO: 11) and Q86XK7_HUMAN. FIG. 4 shows V-set and immunoglobulin domain containing 1 that can be detected in normal and cancerous ovarian tissues by the amplicon shown as SEQ ID AI581519_seg7 (SEQ ID NO: 190). (VSIG1) represents a histogram showing the expression of AI581519 transcript. FIG. 5 shows V-set and immunoglobulin domain containing 1 that can be detected by the amplicon shown in SEQ ID NO: AI581519_seg7 (SEQ ID NO: 190) in normal and cancerous lung tissue. (VSIG1) represents a histogram showing the expression of AI581519 transcript. FIG. 6A shows V-set and immunoglobulin domain containing 1 (VSIG1) that can be detected by the amplicon shown in SEQ ID NO: AI581519_seg7 (SEQ ID NO: 190) in various normal tissues. ) Represents a histogram showing the expression of AI581519 transcript. FIG. 6A shows the expression of each sample relative to the median value of the ovary sample. FIG. 6B shows V-set and immunoglobulin domain containing 1 (VSIG1) that can be detected by the amplicon shown in SEQ ID NO: AI581519_seg7 (SEQ ID NO: 190) in various normal tissues. ) Represents a histogram showing the expression of AI581519 transcript. FIG. 6B shows the expression of each sample relative to the median value of the lung sample. FIG. 7 shows that in normal and cancerous ovarian tissue, V-set and immunoglobulin domain containing 1 (V-set and immunoglobulin domain containing 1), detectable by the amplicon shown as SEQ ID AI581519_seg7-9 (SEQ ID NO: 187). 1) (VSIG1) represents a histogram showing the expression of AI581519 transcript. FIG. 8 shows V-set and immunoglobulin domain containing 1 detectable in normal and cancerous lung tissue by the amplicon shown as SEQ ID AI581519_seg7-9 (SEQ ID NO: 187). 1) (VSIG1) represents a histogram showing the expression of AI581519 transcript. FIG. 9 shows a V-set and immunoglobulin domain containing 1 that can be detected by the amplicon shown in SEQ ID NO: AI581519seg7-9 (SEQ ID NO: 196) in a blood-specific panel. (VSIG1) represents a histogram showing the expression of AI581519 transcript. FIG. 10 shows V-set and immunoglobulin domain containing 1 (V-set and immunoglobulin domain containing 1) that can be detected by the amplicon shown in SEQ ID NO: AI581519_junc7-11F2R2 (SEQ ID NO: 193) in normal and cancerous lung tissue. 1) (VSIG1) represents a histogram showing the expression of AI581519 transcript. FIG. 11 shows V-set and immunoglobulin domain containing 1 (V-set and immunoglobulin domain containing 1) that can be detected by the amplicon shown in SEQ ID NO: AI581519_junc7-11F2R2 (SEQ ID NO: 193) in normal and cancerous ovarian tissues. 1) (VSIG1) represents a histogram showing the expression of AI581519 transcript. FIG. 12A shows V-set and immunoglobulin domain containing 1 that can be detected by the amplicon shown in SEQ ID NO: AI581519_junc7-11F2R2 (SEQ ID NO: 193) in various normal tissues. (VSIG1) represents a histogram showing the expression of AI581519 transcript. FIG. 12A shows the expression of each sample relative to the median value of the lung sample. FIG. 12B shows V-set and immunoglobulin domain containing 1 that can be detected by the amplicon shown in SEQ ID NO: AI581519_junc7-11F2R2 (SEQ ID NO: 193) in various normal tissues. (VSIG1) represents a histogram showing the expression of AI581519 transcript. FIG. 12B shows the expression of each sample relative to the median value of the ovary sample. FIG. 13 shows a V-set and immunoglobulin domain containing 1 that can be detected by the amplicon shown in SEQ ID NO: AI581519_junc7-11F2R2 (SEQ ID NO: 193) in a blood-specific panel. (VSIG1) represents a histogram showing the expression of AI581519 transcript. FIG. 14 is a scatter plot showing expression of AA424839 transcript encoding ILDR1 protein on a virtual panel of all tissues and conditions using a MED discovery engine, compared to normal ovarian samples 2 shows overexpression of AA424839 transcript in FIG. FIG. 15 is a scatter plot showing the expression of AA424839 transcripts encoding ILDR1 protein on a virtual panel of all tissues and conditions using the MED discovery engine and colon cancer compared to normal colon samples 2 shows overexpression of AA424839 transcript in FIG. FIG. 16A shows a comparative alignment of the AA424839_P3 protein against the known ILDR1 proteins Q86SU0_HUMAN and NP_787120 (SEQ ID NO: 21). FIG. 16B shows a comparative alignment of the AA424839_P7 protein against the known ILDR1 proteins Q86SU0_HUMAN and NP_787120 (SEQ ID NO: 21). FIG. 16C shows a comparative alignment of the AA424839_1_P11 protein against the known ILDR1 proteins Q86SU0_HUMAN and NP_787120 (SEQ ID NO: 21). FIG. 17 shows an immunoglobulin-like domain-containing receptor 1 (ILDR1) that can be detected in normal and cancerous ovarian tissue by the amplicon represented by the sequence name AA424839_seg8wt (SEQ ID NO: 199). ) Represents a histogram showing expression of AA424839 transcript. FIG. 18 shows an immunoglobulin-like domain-containing receptor 1 (ILDR1) AA424839, which is detectable by the amplicon shown in SEQ ID NO: AA424839_seg8wt (SEQ ID NO: 199) in various normal tissues. 1 represents a histogram showing expression of transcripts. FIG. 19 shows an immunoglobulin-like domain containing receptor 1 that is detectable by the amplicon shown in SEQ ID NO: AA424839_seg4-16 (SEQ ID NO: 202) in normal and cancerous ovarian tissues (ILDR1) represents a histogram showing expression of AA424839 transcript. FIG. 20 shows an immunoglobulin-like domain-containing receptor 1 (ILDR1) that can be detected by the amplicon represented by SEQ ID NO: AA424839_seg4-16 (SEQ ID NO: 202) in various normal tissues. ) Represents a histogram showing the expression of AA424839 transcript. FIG. 21 shows an immunoglobulin-like domain containing receptor 1 (ILDR1) that is detectable by the amplicon shown in SEQ ID NO: AA424839segll-14F3R3 (SEQ ID NO: 205) in a blood-specific panel. ) Represents a histogram showing expression of AA424839 transcript. FIG. 22 represents a histogram showing the expression of AI216611 transcripts detectable in normal and cancerous colon tissue by the amplicon shown as SEQ ID AI216611_junc4-6F2R2 (SEQ ID NO: 208). FIG. 23 presents histograms showing the expression of AI216611 transcripts that can be detected by the amplicon shown in SEQ ID NO: AI216611_junc4-6F2R2 (SEQ ID NO: 208) in various normal tissues. FIG. 24 depicts a histogram showing the expression of AI216611 transcripts detectable in the normal and cancerous colon tissue by the amplicon shown as SEQ ID AI216611_seg2WT (SEQ ID NO: 211). FIG. 25 represents a histogram showing the expression of AI216611 transcripts detectable in various normal tissues by the amplicon represented by the sequence name AI216611_seg2WT (SEQ ID NO: 211). FIG. 26 presents a histogram showing the expression of AI216611 transcript that can be detected by the amplicon shown in SEQ ID NO: AI216611_junc4-6 (SEQ ID NO: 214) in the blood specific panel. FIG. 27 presents a histogram showing the expression of AI216611 transcripts detectable in the blood specific panel by the amplicon indicated by the sequence name AI216611_junc2-4seg5F2R2 (SEQ ID NO: 220). FIG. 28 presents a histogram showing the expression of AI216611 transcripts detectable in normal and cancerous colon tissue by the amplicon shown as SEQ ID AI216611_junc2-4seg5F2R2 (SEQ ID NO: 220). FIG. 29 is a scatter plot showing the expression of the H68654 transcript encoding the LOC253012 protein on a virtual panel of all tissues and conditions using the MED discovery engine, in lung cancer compared to normal lung samples Shows overexpression of the H68654 transcript. FIG. 30A shows a comparative alignment of the H68654_1_P7 protein to the known LOC253012 proteins NP_937794 and Q6UXIO_HUMAN (SEQ ID NO: 36). FIG. 30B shows a comparative alignment of the H68654_1_P7 protein to the known LOC253012 protein NP_937794 and Q6UXIO_HUMAN (SEQ ID NO: 36). FIG. 30C shows a comparative alignment of the H68654_1_P12 protein to the known LOC253012 protein NP_937794 and Q6UXIO_HUMAN (SEQ ID NO: 36). FIG. 30D shows a comparative alignment of the H68654_1_P12 protein to the known LOC253012 protein NP_937794 and Q6UXIO_HUMAN (SEQ ID NO: 36). FIG. 30E shows a comparative alignment of the H68654_1_P13 protein to the known LOC253012 proteins NP_937794 and Q6UXIO_HUMAN (SEQ ID NO: 36). FIG. 30F shows a comparative alignment of the H68654_1_P13 protein to the known LOC253012 proteins NP_937794 and Q6UXIO_HUMAN (SEQ ID NO: 36). FIG. 30G shows a comparative alignment of the H68654_1_P14 protein to the known LOC2530112 proteins NP_937794 and Q6UXIO_HUMAN (SEQ ID NO: 36). FIG. 30H shows a comparative alignment of the H68654_1_P14 protein to the known LOC253012 protein NP_937794 and Q6UXIO_HUMAN (SEQ ID NO: 36). FIG. 31 presents a histogram showing the expression of the hypothesis-based protein LOC253012 H68654 transcript that can be detected in normal and cancerous lung tissue by the amplicon set forth in SEQ ID NO: H68654_seg3WTF2R2 (SEQ ID NO: 226). FIG. 32 presents a histogram showing the expression of the hypothetical protein LOC253012 H68654 transcript that can be detected by the amplicon shown in SEQ ID NO: H68654_seg3WTF2R2 (SEQ ID NO: 226) in various normal tissues. FIG. 33 presents a histogram showing the expression of the hypothetical protein LOC253012 H68654 transcript that can be detected by the amplicon set forth in SEQ ID NO: H68654_seg7-12WT (SEQ ID NO: 223) in normal and cancerous lung tissue. FIG. 34 presents a histogram showing the expression of the hypothetical protein LOC253012 H68654 transcript, detectable by the amplicon shown in SEQ ID NO: H68654_seg7-12WT (SEQ ID NO: 223) in various normal tissues. FIG. 35A presents a histogram showing the expression of the hypothetical protein LOC253012 H68654 transcript that can be detected by the amplicon shown in SEQ ID NO: H68654seg3F2R2 (SEQ ID NO: 226) in a blood-specific panel. FIG. 35B presents a histogram showing the expression of the hypothetical protein LOC253012 H68654 transcript that can be detected by the amplicon shown in SEQ ID NO: H68654seg7-12F1R1 (SEQ ID NO: 223) in the blood specific panel. FIG. 36 presents a histogram showing the expression of the hypothesis-based protein LOC253012 H68654 transcript that is detectable by the amplicon set forth in SEQ ID NO: H68654_seg0-3 (SEQ ID NO: 229) in normal and cancerous lung tissue. FIG. 37 presents a histogram showing the expression of the hypothetical protein LOC253012 H68654 transcript that can be detected by the amplicon shown in SEQ ID NO: H68654_seg2-3 (SEQ ID NO: 232) in normal and cancerous lung tissue. FIG. 38A shows a comparative alignment of the H19011_1_P8 (FIG. 38A) protein to the known C1ORF32 proteins Q71H61_HUMAN and NP_955383 (SEQ ID NO: 47). FIG. 38B shows a comparative alignment of the H19011_1_P9 (FIG. 38B) protein to the known C1ORF32 proteins Q71H61_HUMAN and NP_955383 (SEQ ID NO: 47). FIG. 39 shows C1ORF32, chromosome 1 open reading frame 32, H19011 transcription, detectable by the amplicon shown in SEQ ID NO: H19011_segl3F2R2 (SEQ ID NO: 235) in normal and cancerous colon tissue. 1 represents a histogram showing product expression. FIG. 40 shows C1ORF32, chromosome 1 open reading frame 32, H19011 transcription, detectable by the amplicon shown in SEQ ID NO: H19011_segl3F2R2 (SEQ ID NO: 235) in normal and cancerous lung tissue. 1 represents a histogram showing product expression. FIG. 41A shows C1ORF32, chromosome 1 open reading frame 32, H19011 transcript, detectable by the amplicon shown in SEQ ID NO: H19011_segl3F2R2 (SEQ ID NO: 235) in various normal tissues. 1 represents a histogram showing expression. FIG. 41A shows the expression of each sample relative to the median value of the colon sample. FIG. 41B shows C1ORF32, Chromosome 1 open reading frame 32, H19011 transcript, detectable by the amplicon represented by SEQ ID NO: H19011_segl3F2R2 (SEQ ID NO: 235) in various normal tissues. 1 represents a histogram showing expression. FIG. 41B shows the expression of each sample relative to the median value of the lung sample. FIG. 42 shows C1ORF32, Chromosome 1 open reading frame 32 (chromosome 1 open reading frame 32), which is detectable in normal and cancerous lung tissue by the amplicon represented by SEQ ID NO: H19011_seg8-13FlRl (SEQ ID NO: 238), 2 represents a histogram showing expression of H19011 transcript. FIG. 43 shows C1ORF32, chromosome 1 open reading frame 32 (chromosome 1 open reading frame 32), which can be detected by the amplicon shown in SEQ ID NO: H19011_junc8-10segl3 (SEQ ID NO: 241) in normal and cancerous lung tissue. 2 represents a histogram showing expression of H19011 transcript. FIG. 44 shows C1ORF32, chromosome 1 open reading frame 32 (chromosome 1 open reading frame 32), which can be detected by the amplicon shown in SEQ ID NO: H19011_junc8-10segl3 (SEQ ID NO: 241) in normal and cancerous colon tissue. 2 represents a histogram showing expression of H19011 transcript. FIG. 45A shows C1ORF32, Chromosome 1 open reading frame 32, H19011 transcription, detectable by the amplicon shown in SEQ ID NO: H19011_junc8-10segl3 (SEQ ID NO: 241) in various normal tissues. 1 represents a histogram showing product expression. FIG. 45A shows the expression of each sample relative to the median value of the colon sample. FIG. 45B shows C1ORF32, chromosome 1 open reading frame 32, H19011 transcription, detectable by the amplicon shown in SEQ ID NO: H19011_junc8-10segl3 (SEQ ID NO: 241) in various normal tissues. 1 represents a histogram showing product expression. FIG. 45B shows the expression of each sample relative to the median value of the lung sample. FIG. 46 shows C1ORF32, Chromosome 1 open reading frame 32, H19011 transcription, which can be detected by the amplicon shown in SEQ ID NO: H19011_junc8-10segl3 (SEQ ID NO: 241) in the blood-specific panel. 1 represents a histogram showing product expression. FIG. 47 shows C1ORF32, chromosome 1 open reading frame 32 (chromosome 1 open reading frame 32), which can be detected by the amplicon shown in SEQ ID NO: H19011_junc6-10FlRl (SEQ ID NO: 244) in normal and cancerous lung tissue. 2 represents a histogram showing expression of H19011 transcript. FIG. 48 shows C1ORF32, chromosome 1 open reading frame 32 (chromosome 1 open reading frame 32), which can be detected in normal and cancerous colon tissue by the amplicon represented by SEQ ID NO: H19011_junc6-10FlRl (SEQ ID NO: 244), 1 represents a histogram showing expression of H19011 transcripts. FIG. 49A shows a comparative alignment of the R31375_P14 protein to the known FXYD3 protein NP_068710 (SEQ ID NO: 71), FXYD3_HUMAN (SEQ ID NO: 70), NP_005962 and Q6IB59_HUMAN (SEQ ID NO: 70). FIG. 49B shows a comparative alignment of the R31375_P31 protein against the known FXYD3 protein NP_068710 (SEQ ID NO: 71), FXYD3_HUMAN (SEQ ID NO: 70), NP_005962 and Q6IB59_HUMAN (SEQ ID NO: 70). FIG. 49C shows a comparative alignment of the R31375_P31 protein against the known FXYD3 proteins NP_068710 (SEQ ID NO: 71), FXYD3_HUMAN (SEQ ID NO: 70), NP_005962 and Q6IB59_HUMAN (SEQ ID NO: 70). FIG. 49D shows a comparative alignment of the R31375_P33 protein against the known FXYD3 protein NP_068710 (SEQ ID NO: 71), FXYD3_HUMAN (SEQ ID NO: 70), NP_005962 and Q6IB59_HUMAN (SEQ ID NO: 70). FIG. 49E shows a comparative alignment of the R31375_P33 protein against the known FXYD3 protein NP_068710 (SEQ ID NO: 71), FXYD3_HUMAN (SEQ ID NO: 70), NP_005962 and Q6IB59_HUMAN (SEQ ID NO: 70). FIG. 50 shows an FXYD3 domain containing ion transport regulator 3 R31375 detectable in the normal and cancerous ovarian tissue by the amplicon shown as SEQ ID NO: R31375_junc30-33 (SEQ ID NO: 247). 1 represents a histogram showing expression of transcripts. FIG. 51 shows an FXYD3 domain containing ion transport regulator 3 R31375 transcript that is detectable by the amplicon shown in SEQ ID NO: R31375_junc30-33 (SEQ ID NO: 247) in various normal tissues. The histogram which shows the expression of is represented. FIG. 52 shows FXYD3 domain containing ion transport regulator 3 R31375, which is detectable in normal and cancerous ovarian tissues by the amplicon represented by SEQ ID R31375_seg33junc34-37 (SEQ ID NO: 250). 1 represents a histogram showing expression of transcripts. FIG. 53 shows the FXYD3 domain-containing ion transport regulator 3 R31375 transcript detectable in various normal tissues by the amplicon represented by the sequence name R31375_seg33junc34-37 (SEQ ID NO: 250). The histogram which shows the expression of is represented. FIG. 54 shows an FXYD3 domain-containing ion transport regulator 3 (FXYD3 domain containing ion transport regulator 3) that can be detected by the amplicon represented by SEQ ID NO: R31375_junc20-22seg30F6R6 (SEQ ID NO: 253) in normal and cancerous ovarian tissues. 1 represents a histogram showing expression of transcripts. FIG. 55 shows an FXYD3 domain-containing ion transport regulator 3R3 product that can be detected by an amplicon represented by the sequence name R31375_junc20-22seg30F6R6 (SEQ ID NO: 253) in various normal tissues. The histogram which shows the expression of is represented. FIG. 56A represents the nucleotide sequence of the recombinant full length EGFP ORF. Gene-specific sequences corresponding to candidate full-length sequences are shown in bold, EGFP sequences are italicized, not bold, and known SNP / silence mutations are underlined. FIG. 56A represents the full length_EGFP ORF nucleic acid sequence of FXYD3_T0_P0_EGFP DNA (996 bp) (SEQ ID NO: 77). FIG. 56B represents the nucleotide sequence of the recombinant full length EGFP ORF. Gene-specific sequences corresponding to candidate full-length sequences are shown in bold, EGFP sequences are italicized, not bold, and known SNP / silence mutations are underlined. FIG. 56B represents the full length EGFP ORF nucleic acid sequence of FXYD3_T25_P14_EGFP DNA (1083 bp) (SEQ ID NO: 78). FIG. 56C represents the nucleotide sequence of the recombinant full length EGFP ORF. Gene-specific sequences corresponding to candidate full-length sequences are shown in bold, EGFP sequences are italicized, not bold, and known SNP / silence mutations are underlined. FIG. 56C represents the full length EGFP ORF nucleic acid sequence of AI216611_T0_P0_EGFP DNA (1371 bp) (SEQ ID NO: 79). FIG. 56D represents the nucleotide sequence of the recombinant full length EGFP ORF. Gene-specific sequences corresponding to candidate full-length sequences are shown in bold, EGFP sequences are italicized, not bold, and known SNP / silence mutations are underlined. FIG. 56D represents the full length EGFP ORF nucleic acid sequence of AI216611_T1_P1_EGFP DNA (1332 bp) (SEQ ID NO: 80). FIG. 56E represents the nucleotide sequence of the recombinant full length EGFP ORF. Gene-specific sequences corresponding to candidate full-length sequences are shown in bold, EGFP sequences are italicized, not bold, and known SNP / silence mutations are underlined. FIG. 56E represents the full length EGFP ORF nucleic acid sequence of C1ORF32_T8_P8_EGFP DNA (1533 bp) (SEQ ID NO: 81). FIG. 56F represents the nucleotide sequence of the recombinant full length EGFP ORF. Gene-specific sequences corresponding to candidate full-length sequences are shown in bold, EGFP sequences are italicized, not bold, and known SNP / silence mutations are underlined. FIG. 56F represents the full length EGFP ORF nucleic acid sequence of LOC253012_T4_P5_EGFP DNA (2085 bp) (SEQ ID NO: 82). Figures 56A-56J represent the nucleotide sequence of the recombinant full length EGFP ORF. Gene-specific sequences corresponding to candidate full-length sequences are shown in bold, EGFP sequences are italicized, not bold, and known SNP / silence mutations are underlined. FIG. 56G represents the full length EGFP ORF nucleic acid sequence of ILDR1_T0_P3_EGFP DNA (2373 bp) (SEQ ID NO: 83). FIG. 56H represents the nucleotide sequence of the recombinant full length EGFP ORF. Gene-specific sequences corresponding to candidate full-length sequences are shown in bold, EGFP sequences are italicized, not bold, and known SNP / silence mutations are underlined. FIG. 56H represents the full length EGFP ORF nucleic acid sequence of ILDR1_T2_P5_EGFP DNA (2241 bp) (SEQ ID NO: 84). FIG. 56I represents the nucleotide sequence of the recombinant full length EGFP ORF. Gene-specific sequences corresponding to candidate full-length sequences are shown in bold, EGFP sequences are italicized, not bold, and known SNP / silence mutations are underlined. FIG. 56I represents the full length EGFP ORF nucleic acid sequence of VSIG1_T6_P5_EGFP DNA (2082 bp) (SEQ ID NO: 85). FIG. 56J represents the full-length EGFP ORF nucleic acid sequence of VSIG1_T5_P4_EGFP DNA (2004 bp) (SEQ ID NO: 86). FIG. 56J represents the nucleotide sequence of the recombinant full length EGFP ORF. Gene-specific sequences corresponding to candidate full-length sequences are shown in bold, EGFP sequences are italicized, not bold, and known SNP / silence mutations are underlined. FIG. 56J represents the full-length EGFP ORF nucleic acid sequence of VSIG1_T5_P4_EGFP DNA (2004 bp) (SEQ ID NO: 86). FIG. 57A represents the sequence of the full length EGFP fusion protein of the invention. The candidate unique sequence corresponding to the full-length protein sequence is shown in bold, the EGFP sequence is italicized, not bold, and amino acids modified by known SNPs are underlined. FIG. 57A represents the full-length EGFP ORF amino acid sequence of FXYD3_P0_EGFP protein (331aa) (SEQ ID NO: 87). FIG. 57B represents the sequence of the full-length EGFP fusion protein of the invention. The candidate unique sequence corresponding to the full-length protein sequence is shown in bold, the EGFP sequence is italicized, not bold, and amino acids modified by known SNPs are underlined. FIG. 57B represents the full-length EGFP ORF amino acid sequence of FXYD3_P14_EGFP protein (360aa) (SEQ ID NO: 88). FIG. 57C represents the sequence of the full length EGFP fusion protein of the invention. The candidate unique sequence corresponding to the full-length protein sequence is shown in bold, the EGFP sequence is italicized, not bold, and amino acids modified by known SNPs are underlined. FIG. 57C represents the full length EGFP ORF amino acid sequence of AI216611_P0_EGFP protein (456aa) (SEQ ID NO: 89). FIG. 57D represents the sequence of the full length EGFP fusion protein of the invention. The candidate unique sequence corresponding to the full-length protein sequence is shown in bold, the EGFP sequence is italicized, not bold, and amino acids modified by known SNPs are underlined. FIG. 57D represents the full length EGFP ORF amino acid sequence of AI216611_P1_EGFP protein (443aa) (SEQ ID NO: 90). FIG. 57E represents the sequence of the full length EGFP fusion protein of the invention. The candidate unique sequence corresponding to the full-length protein sequence is shown in bold, the EGFP sequence is italicized, not bold, and amino acids modified by known SNPs are underlined. FIG. 57E represents the full length EGFP ORF amino acid sequence of the C1ORF32_P8_EGFP protein (510aa) (SEQ ID NO: 91). FIG. 57F represents the sequence of the full-length EGFP fusion protein of the invention. The candidate unique sequence corresponding to the full-length protein sequence is shown in bold, the EGFP sequence is italicized, not bold, and amino acids modified by known SNPs are underlined. FIG. 57F represents the full-length EGFP ORF amino acid sequence of LOC253012_P5_EGFP protein (694aa) (SEQ ID NO: 92). FIG. 57G represents the sequence of the full length EGFP fusion protein of the invention. The candidate unique sequence corresponding to the full-length protein sequence is shown in bold, the EGFP sequence is italicized, not bold, and amino acids modified by known SNPs are underlined. FIG. 57G represents the full length EGFP ORF amino acid sequence of ILDR1_P3_EGFP protein (790aa) (SEQ ID NO: 93). FIG. 57H represents the sequence of the full-length EGFP fusion protein of the invention. The candidate unique sequence corresponding to the full-length protein sequence is shown in bold, the EGFP sequence is italicized, not bold, and amino acids modified by known SNPs are underlined. FIG. 57H represents the full-length EGFP ORF amino acid sequence of ILDR1_P5_EGFP protein (746aa) (SEQ ID NO: 94). FIG. 57I represents the sequence of the full length EGFP fusion protein of the invention. The candidate unique sequence corresponding to the full-length protein sequence is shown in bold, the EGFP sequence is italicized, not bold, and amino acids modified by known SNPs are underlined. FIG. 57I represents the full length EGFP ORF amino acid sequence of the VSIG1_P5_EGFP protein (693aa) (SEQ ID NO: 95). FIG. 57J represents the sequence of the full-length EGFP fusion protein of the invention. The candidate unique sequence corresponding to the full-length protein sequence is shown in bold, the EGFP sequence is italicized, not bold, and amino acids modified by known SNPs are underlined. FIG. 57J represents the full length EGFP ORF amino acid sequence of the VSIG1_P4_EGFP protein (667aa) (SEQ ID NO: 96). FIG. 58A shows the localization of the inventive protein to the cell membrane. FIG. 58A shows the subcellular localization of AI216611-EGFP_T0_P0 and AI216611-EGFP_T1_P1 proteins. FIG. 58D shows the subcellular localization of LOC253012-EGFP_T4_P5 protein. FIG. 58E shows the intracellular localization of VSIG1-EGFP_T6_P5 and VSIG1-EGFP_T5_P4 proteins. FIG. 58F shows the intracellular localization of ILDR1-EGFP_T0_P3 and ILDR1-EGFP_T2_P5 proteins. All images were obtained using a 40x (4Ox) object of a confocal microscope. FIG. 58B shows the localization of the inventive protein to the cell membrane. FIG. 58B shows the subcellular localization of FXYD3-EGFP_T0_P0 and FXYD3-EGFP_T25_P14 proteins. All images were obtained using a 40x (4Ox) object of a confocal microscope. FIG. 58C shows the localization of the protein of the invention to the cell membrane. FIG. 58C shows the intracellular localization of C1ORF32-EGFP_T8_P8 protein. All images were obtained using a 40x (4Ox) object of a confocal microscope. FIG. 58D shows the localization of the protein of the invention to the cell membrane. FIG. 58D shows the subcellular localization of LOC253012-EGFP_T4_P5 protein. All images were obtained using a 40x (4Ox) object of a confocal microscope. FIG. 58E shows the localization of the inventive protein to the cell membrane. FIG. 58E shows the intracellular localization of VSIG1-EGFP_T6_P5 and VSIG1-EGFP_T5_P4 proteins. All images were obtained using a 40x (4Ox) object of a confocal microscope. FIG. 58F shows the localization of the inventive protein to the cell membrane. FIG. 58F shows the intracellular localization of ILDR1-EGFP_T0_P3 and ILDR1-EGFP_T2_P5 proteins. All images were obtained using a 40x (4Ox) object of a confocal microscope. FIG. 59A represents the nucleotide sequence of the extracellular region of a candidate protein of the invention fused to a mouse Fc: ECD_mFc ORF. Candidate protein-specific sequences corresponding to ECD sequences are shown in bold, TEV cleavage site sequences are underlined, mFc sequences are italicized in bold, and IL6sp sequences are italicized in bold. FIG. 59A shows the FXYD3_T25_P14_ECD-mFc DNA sequence (924 bp) (SEQ ID NO: 97). FIG. 59B represents the nucleotide sequence of the extracellular region of the candidate protein of the invention fused to the mouse Fc: ECD_mFc ORF. Candidate protein-specific sequences corresponding to ECD sequences are shown in bold, TEV cleavage site sequences are underlined, mFc sequences are italicized in bold, and IL6sp sequences are italicized in bold. FIG. 59B shows the AI216611_T0_P0_ECD_mFc DNA sequence (1170 bp) (SEQ ID NO: 98). FIG. 59C represents the nucleotide sequence of the extracellular region of the candidate protein of the invention fused to the mouse Fc: ECD_mFc ORF. Candidate protein-specific sequences corresponding to ECD sequences are shown in bold, TEV cleavage site sequences are underlined, mFc sequences are italicized in bold, and IL6sp sequences are italicized in bold. FIG. 59C shows the C1ORF32_T8_P8_ECD_mFc DNA sequence (1287 bp) (SEQ ID NO: 99). FIG. 59D represents the nucleotide sequence of the extracellular region of the candidate protein of the invention fused to the mouse Fc: ECD_mFc ORF. Candidate protein-specific sequences corresponding to ECD sequences are shown in bold, TEV cleavage site sequences are underlined, mFc sequences are italicized in bold, and IL6sp sequences are italicized in bold. FIG. 59D shows the LOC253012_T4_P5_ECD_mFc DNA sequence (1740 bp) (SEQ ID NO: 100). FIG. 59E represents the nucleotide sequence of the extracellular region of the candidate protein of the invention fused to the mouse Fc: ECD_mFc ORF. Candidate protein-specific sequences corresponding to ECD sequences are shown in bold, TEV cleavage site sequences are underlined, mFc sequences are italicized in bold, and IL6sp sequences are italicized in bold. FIG. 59E shows the ILDR1_T0_P3_ECD_mFc DNA sequence (1167 bp) (SEQ ID NO: 101). FIG. 59F represents the nucleotide sequence of the extracellular region of the candidate protein of the invention fused to the mouse Fc: ECD_mFc ORF. Candidate protein-specific sequences corresponding to ECD sequences are shown in bold, TEV cleavage site sequences are underlined, mFc sequences are italicized in bold, and IL6sp sequences are italicized in bold. FIG. 59F shows the VSIG1_T6_P5_ECD_mFc DNA sequence (1641 bp) (SEQ ID NO: 102). FIG. 60A shows the amino acid sequence of the ECD_mFc fusion protein. The unique sequence of the candidate protein corresponding to the ECD sequence is shown in bold, the TEV cleavage site sequence is underlined, the mFc sequence is non-bold italic, and the IL6sp sequence is bold italic. FIG. 60A shows the FXYD3_T25_P14_ECD-mFc amino acid sequence (307aa) (SEQ ID NO: 103). FIG. 60B shows the amino acid sequence of the ECD_mFc fusion protein. The unique sequence of the candidate protein corresponding to the ECD sequence is shown in bold, the TEV cleavage site sequence is underlined, the mFc sequence is non-bold italic, and the IL6sp sequence is bold italic. FIG. 60B shows the AI216611_T0_P0_ECD_mFc amino acid sequence (389aa) (SEQ ID NO: 104). FIG. 60C shows the amino acid sequence of the ECD_mFc fusion protein. The unique sequence of the candidate protein corresponding to the ECD sequence is shown in bold, the TEV cleavage site sequence is underlined, the mFc sequence is non-bold italic, and the IL6sp sequence is bold italic. FIG. 60C shows the C1ORF32_T8_P8_ECD_mFc amino acid sequence (428aa) (SEQ ID NO: 105). FIG. 60D shows the amino acid sequence of the ECD_mFc fusion protein. The unique sequence of the candidate protein corresponding to the ECD sequence is shown in bold, the TEV cleavage site sequence is underlined, the mFc sequence is non-bold italic, and the IL6sp sequence is bold italic. FIG. 60D shows the LOC253012_T4_P5_ECD_mFc amino acid sequence (579aa) (SEQ ID NO: 106). FIG. 60E shows the amino acid sequence of the ECD_mFc fusion protein. The unique sequence of the candidate protein corresponding to the ECD sequence is shown in bold, the TEV cleavage site sequence is underlined, the mFc sequence is non-bold italic, and the IL6sp sequence is bold italic. FIG. 60E shows the ILDR1_T0_P3_ECD_mFc amino acid sequence (388aa) (SEQ ID NO: 107). FIG. 60F shows the amino acid sequence of the ECD_mFc fusion protein. The unique sequence of the candidate protein corresponding to the ECD sequence is shown in bold, the TEV cleavage site sequence is underlined, the mFc sequence is non-bold italic, and the IL6sp sequence is bold italic. FIG. 60F shows the VSIG1_T6_P5_ECD_mFc amino acid sequence (546aa) (SEQ ID NO: 108). FIG. 61 shows expressed FXYD3_ECD_mFc (SEQ ID NO: 103), AI216611 ECD_mFc (SEQ ID NO: 104), C1ORF32_ECD_mFc (SEQ ID NO: 105), LOC253012_ECD_mFc (SEQ ID NO: 106), ILDR1_ECD_mFc (SEQ ID NO: 107), VSIG1_CD The result of a Western blot analysis is shown. The lanes are as follows. Lane 1 molecular weight markers (Amersham, full range ranbow, catalog number RPN800), lane 2 -LOC253012_ECD_mFc, lane 3 -FXYD3_ECD_mFc, lane 4-AI216611 ECD_mFc, lane 5-C1ORF32_ECD_mFcE1, lane 6 -ILDRFcE FIG. 62A represents binding of Fc fused B7-like protein ECD to resting T cells or T cells activated with ConA for various times. FIG. 62A shows the binding result of VSIG1 ECD fused with Fc. FIG. 62B depicts binding of Bc-like protein ECD fused to Fc to resting T cells or T cells activated with ConA for various times. FIG. 62B shows the binding result of LOC253012 fused with Fc. FIG. 62C represents binding of Bc-like protein ECD fused to Fc to resting T cells or T cells activated with ConA for various times. FIG. 62C shows the binding result of C1ORF32 ECD fused with Fc. FIG. 62D represents binding of Bc-like protein ECD fused to Fc to resting T cells or T cells activated with ConA for various times. FIG. 62D shows the binding result of AI216611 ECD fused with Fc. FIG. 62E represents binding of Fc-fused B7-like protein ECD to resting T cells, or T cells activated with ConA for various times. FIG. 62E shows the binding result of FXYD3 ECD fused with Fc. FIG. 63 represents the dose response of binding of the F7 fused B7-like protein ECD to activated T cells. Purified T cells were cultured for 48 hours. ConA was added for the last 24 hours. Cells were then harvested and stained with increasing concentrations of Fc-fused VSIG1, LOC253012, C1ORF32, AI216611, ILDR1 or FXYD3 ECD (3, 6, 12, 25 and 50 μg / ml). As a negative control, mouse IgG2a was used at the same concentration. FIG. 64A represents the effect of ECD-Fc fusion protein on T cell proliferation or IL-2 secretion when activated with anti-CD3 Ab. FIG. 64A shows the level of BrdU incorporation. FIG. 64B represents the effect of ECD-Fc fusion protein on T cell proliferation or IL-2 secretion when activated with anti-CD3 Ab. FIG. 64B shows the level of IL-2 secretion. FIG. 65A illustrates binding of ECD to lymphocytes fused with Fc of VSIG1, ILDR1, LOC253012, AI216611, FXYD3 or C1ORF32. FIG. 65B illustrates binding of ECD fused to lymphocytes of VSIG1, ILDR1, LOC253012, AI216611, FXYD3 or C1ORF32 Fc. FIG. 66A illustrates the binding of ECD fused to ILDR1, C1ORF32 and AI216611 Fc to CD4 + T cells. FIG. 66B illustrates the binding of ECD fused to ILDR1, C1ORF32 and AI216611 Fc to CD4 + T cells. FIG. 67 shows the effect of B7-like protein on T cell activation. “CD3” means only CD3 without the presence of costimulatory molecules. “CD3 + B7.2” means CD3 + known B7 stimulation control, B7.2. “CD3 + B7H4” means CD3 and B7H4, known B7 inhibitory controls. “CD3 + B7H3” means CD3 and B7H3, known B7 stimulating proteins. “CD3 + 702” refers to the CD3 + LOC253012-ECD-Fc fusion (SEQ ID NO: 106). “CD3 + 721” means the CD3 + AI216611-ECD-Fc fusion (SEQ ID NO: 104). “CD3 + 754” means a CD3 + C1ORF32-ECD-Fc fusion (SEQ ID NO: 105). “CD3 + 768” means a CD3 + VSIG1-ECD-Fc fusion (SEQ ID NO: 108). “CD3 + 770” means a CD3 + ILDR1-ECD-Fc fusion (SEQ ID NO: 107). “CD3 + 789” means a CD3 + FXYD3-ECD-Fc fusion (SEQ ID NO: 103). 67A, B and C represent three different experiments with three different donors. FIG. 67 shows the effect of B7-like protein on T cell activation. “CD3” means only CD3 without the presence of costimulatory molecules. “CD3 + B7.2” means CD3 + known B7 stimulation control, B7.2. “CD3 + B7H4” means CD3 and B7H4, known B7 inhibitory controls. “CD3 + B7H3” means CD3 and B7H3, known B7 stimulating proteins. “CD3 + 702” refers to the CD3 + LOC253012-ECD-Fc fusion (SEQ ID NO: 106). “CD3 + 721” means the CD3 + AI216611-ECD-Fc fusion (SEQ ID NO: 104). “CD3 + 754” means a CD3 + C1ORF32-ECD-Fc fusion (SEQ ID NO: 105). “CD3 + 768” means a CD3 + VSIG1-ECD-Fc fusion (SEQ ID NO: 108). “CD3 + 770” means a CD3 + ILDR1-ECD-Fc fusion (SEQ ID NO: 107). “CD3 + 789” means a CD3 + FXYD3-ECD-Fc fusion (SEQ ID NO: 103). 67A, B and C represent three different experiments with three different donors. FIG. 67 shows the effect of B7-like protein on T cell activation. “CD3” means only CD3 without the presence of costimulatory molecules. “CD3 + B7.2” means CD3 + known B7 stimulation control, B7.2. “CD3 + B7H4” means CD3 and B7H4, known B7 inhibitory controls. “CD3 + B7H3” means CD3 and B7H3, known B7 stimulating proteins. “CD3 + 702” refers to the CD3 + LOC253012-ECD-Fc fusion (SEQ ID NO: 106). “CD3 + 721” means the CD3 + AI216611-ECD-Fc fusion (SEQ ID NO: 104). “CD3 + 754” means a CD3 + C1ORF32-ECD-Fc fusion (SEQ ID NO: 105). “CD3 + 768” means a CD3 + VSIG1-ECD-Fc fusion (SEQ ID NO: 108). “CD3 + 770” means a CD3 + ILDR1-ECD-Fc fusion (SEQ ID NO: 107). “CD3 + 789” means a CD3 + FXYD3-ECD-Fc fusion (SEQ ID NO: 103). 67A, B and C represent three different experiments with three different donors. 68A shows ILDR1-ECD-Fc (SEQ ID NO: 107), C1ORF32-ECD-Fc (SEQ ID NO: 105), AI216611-ECD-Fc (SEQ ID NO: 104), LOC253012-ECD-Fc (SEQ ID NO: 106), FXYD3- FIG. 6 represents FACS results of binding of ECD-Fc (SEQ ID NO: 103) and VSIG1-ECD-Fc (SEQ ID NO: 108) to resting B cells. 68B shows ILDR1-ECD-Fc (SEQ ID NO: 107), C1ORF32-ECD-Fc (SEQ ID NO: 105), AI216611-ECD-Fc (SEQ ID NO: 104), LOC253012-ECD-Fc (SEQ ID NO: 106), FXYD3- FIG. 6 represents FACS results of binding of ECD-Fc (SEQ ID NO: 103) and VSIG1-ECD-Fc (SEQ ID NO: 108) to activated B cells. 68C shows ILDR1-ECD-Fc (SEQ ID NO: 107), C1ORF32-ECD-Fc (SEQ ID NO: 105), AI216611-ECD-Fc (SEQ ID NO: 104), LOC253012-ECD-Fc (SEQ ID NO: 106), FXYD3- FIG. 5 represents FACS results of binding of ECD-Fc (SEQ ID NO: 103) and VSIG1-ECD-Fc (SEQ ID NO: 108) to B lymphoma cell lines. FIG. 69 shows BIACORE results showing the interaction between AI216611 and B7H4.

  The present invention relates to any one of the antigens referred to as VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3, its corresponding nucleic acid sequence and part and variants and containing conjugates thereof, and its as a therapeutic or diagnostic target Regarding usage. In particular, the present invention uses this antigen and its individual parts as drug targets for therapeutic small molecules, peptides, antibodies, antisense RNAs, siRNA, ribozymes and the like. More specifically, the present invention relates to diagnostic and therapeutic polyclonal and monoclonal antibodies and fragments thereof, particularly extracellular regions or portions or portions thereof, that bind to VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 and portions thereof and variants thereof. Those targeting mutants, in particular, antigens AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 15) 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839 1_P11 (SEQ ID NO: 24), H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P11 (PID No. 40) (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48), H19011_1_P9 (SEQ ID NO: 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73), R31375_P33 ( SEQ ID NO: 74) and VSIG1, ILDR1, LOC253012, A that specifically bind to variants thereof 216,611, C1ORF32, FXYD3 including those that promote or inhibit the activity triggered by, including those relating to the regulation of immune costimulatory, e.g. B7 related costimulation, relates to a human or chimeric monoclonal antibodies.

  In certain embodiments, the antibodies of the invention are derived from specific heavy and light chain germline sequences and / or include specific structural features such as CDR regions comprising specific amino acid sequences. The invention relates to isolated antibodies, methods of making such antibodies, immunoconjugates and bispecific molecules comprising such antibodies, and antibodies, immunoconjugates or bispecific molecules of the invention Pharmaceutical and diagnostic compositions comprising

  The present invention also provides for the expression of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3, for detecting VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3, as well as malignant tumors that differentially express VSIG1. It relates to in vitro and in vivo methods using antibodies and fragments for treating related diseases. The present invention further relates to methods for treating autoimmune deficiency diseases and living transplantation and graft body host diseases using antibodies and fragments specific for VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3. Thus, the present invention also uses the anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, anti-FXYD3 antibodies of the present invention and other agents that modulate VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3, malignant A method for treating a tumor is provided. For example, but not limited to, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, breast, prostate, spleen, kidney, Bladder, head and neck, uterus, testis, stomach, cervix, liver, bone, skin, pancreas, brain cancer, including cancer of the lung, ovarian cancer, colon cancer, non-solid, including cancer of the brain And treatment of solid tumors, sarcomas, hematological malignancies, etc. The invention modulates VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3, uses, but is not limited to, anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, anti-FXYD3 antibodies and other agents of the invention Further provides methods for treating non-malignant disorders such as immunodeficiency diseases, including autoimmune deficiency diseases, transplant rejection and graft host disease. Preferably, these antibodies will have ADCC or CDC activity against target cells such as cancer cells.

  The present invention also includes a VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 antigen and a soluble polypeptide containing the extracellular region of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 for use in immunotherapy. It relates to a part of the antigen including the union and / or the corresponding DNA or vector or cells expressing it. Furthermore, the present invention provides vectors, containing cells and uses thereof for the expression of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 antigen and individual parts and variants thereof. The invention is also based on non-antibodies such as peptides, antisense RNA, siRNAs, carbohydrates and other small molecules that specifically bind and / or modulate VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3-related activity. VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, and FXYD3 modulators are provided.

  In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

  The term VSIG1 is referred to herein as AI581519_T0 (SEQ ID NO: 1), AI581519_T1 (SEQ ID NO: 2), AI581519_T2 (SEQ ID NO: 3), AI581519_T3 (SEQ ID NO: 4), AI581519_T4 (SEQ ID NO: 5), AI581519_T5 ( SEQ ID NO: 6), AI581519_T6 (SEQ ID NO: 7), AI581519_T8 (SEQ ID NO: 8), AI581519_T10 (SEQ ID NO: 9), AI581519_T11 (SEQ ID NO: 10) refers to the protein encoded by the transcript, in particular AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13), AI581519_P7 (SEQ ID NO: 14), AI5815 9_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), for example, cancers such as lung cancer and ovarian cancer, which may be non-metastatic, invasive or metastatic, and limitations Although not, it refers to proteins and variants thereof that are differentially expressed in non-malignant disorders such as autoimmune diseases, transplantation rejection and immunodeficiency diseases including graft host disease.

  The term ILDR1 is encoded by any one of the AA424839_T0 (SEQ ID NO: 17), AA424839_T2 (SEQ ID NO: 18), AA424839_T4 (SEQ ID NO: 19), AA424839_1_T7 (SEQ ID NO: 20) transcripts reported herein. In particular AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23), AA424839_1_P11 (SEQ ID NO: 24), for example, non-metastatic, invasion Differences in cancers such as colon cancer and ovarian cancer, which can be sexual or metastatic, and non-malignant disorders such as, but not limited to, autoimmune diseases, immunodeficiency diseases including transplant rejection and graft body host disease Secondly expressed tongue It refers to click cytoplasm and its variants.

  The term LOC253012 is referred to herein as H68654_1_T0 (SEQ ID NO: 25), H68654_1_T4 (SEQ ID NO: 26), H68654_1_T5 (SEQ ID NO: 27), H68654_1_T8 (SEQ ID NO: 28), H68654_1_T15 (SEQ ID NO: 29), H68654_ SEQ ID NO: 30), refers to a protein encoded by any one of H68654_1_T17 (SEQ ID NO. 31), H68654_1_T18 (SEQ ID NO: 32), H68654_1_T19 (SEQ ID NO: 33), H68654_1_T20 (SEQ ID NO: 34) transcripts, and in particular H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P7 (SEQ ID NO: 37), H6865 _1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40) according to any one of, for example, lung cancer, in particular small cells, which may be non-metastatic, invasive or metastatic Refers to proteins and variants thereof that are differentially expressed in non-malignant disorders such as cancers such as lung cancer, and, but are not limited to, autoimmune diseases, transplant rejection and immunodeficiency diseases including graft host disease .

  The term AI216611 refers to the protein encoded by any one of the AI216611_T0 (SEQ ID NO: 41), AI216611_T1 (SEQ ID NO: 42) transcripts reported herein, in particular AI216611_PO (SEQ ID NO: 43), AI216166_P1 (SEQ ID NO. 44) described in any one of, for example, but not limited to, may be non-metastatic, invasive, or metastatic, but not limited to acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid Leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, breast, prostate, lung, ovary, colon, spleen, kidney, bladder, head and neck, uterus, testis, stomach, cervix, liver, bone, Non-solid and solid tumors, including cancers of skin, pancreas and brain, sarcomas, hematological malignancies, etc. And, without limitation, refer to autoimmune diseases, proteins and variants thereof differentially expressed in non-malignant disorders such as immunodeficiency diseases, including transplant rejection and graft versus host disease.

  The term C1ORF32 refers to the protein encoded by any one of the H19011_1_T8 (SEQ ID NO: 45), H19011_1_T9 (SEQ ID NO: 46) transcripts reported herein, in particular H19011_1_P8 (SEQ ID NO: 48), A cancer, such as, but not limited to, lung cancer, particularly small cell lung cancer, as described in any one of H19011_1_P9 (SEQ ID NO: 50), which may be, for example, non-metastatic, invasive or metastatic. It refers to proteins and variants thereof that are differentially expressed in non-malignant disorders such as diseases, transplant rejection and immunodeficiency diseases including graft host disease.

  The term FXYD3 is reported herein as R31375_T0 (SEQ ID NO: 51); R31375_T1 (SEQ ID NO: 52); R31375_T10 (SEQ ID NO: 61); R31375_T11 (SEQ ID NO: 62); R31375_T12 (SEQ ID NO: 63); R31375_T13 ( R31375_T2 (SEQ ID NO: 53); R31375_T3 (SEQ ID NO: 55); R31375_T5 (SEQ ID NO: 56); R31375_T6 (SEQ ID NO: 57); R31375_T7 (SEQ ID NO: 58); R31375_T8 (SEQ ID NO: 58) R31375_T9 (SEQ ID NO: 60): R31375_T19 (SEQ ID NO: 65); R31375_T25 (SEQ ID NO: 66); R31375_T26 (SEQ ID NO: 59); 67); R31375_T29 (SEQ ID NO: 68); R31375_T39 (SEQ ID NO: 69) refers to the protein encoded by any one of the transcripts, in particular R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 ( SEQ ID NO: 73), R31375_P31 (SEQ ID NO: 73) according to any one of, for example, ovarian cancer, which may be non-metastatic, invasive or metastatic, as well as but not limited to acute lymphocytic leukemia Chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, breast, prostate, lung, ovary, colon, spleen, kidney, bladder, head and neck, uterus, Non-solid and non-solid, including cancer of the testicles, stomach, neck, liver, bones, skin, pancreas and brain Solid refers tumors, sarcomas, proteins and variants thereof differentially expressed in cancer, such as hematological malignancies.

  Preferably, such a variant of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 has at least 80% sequence identity therewith, more preferably at least 90% sequence identity and even more preferably at least 95% Will have the same sequence identity.

  Any one of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 protein is involved in, for example, B7 immune co-stimulation, including T cell responses elicited against cancer cells, and autoimmunity based on the structure of the region It is expected to be an immune co-stimulatory protein that elicits an immune effect, such as causing an effect, such as a B7 protein family member.

  The term “soluble extracellular region (ECD)” or “extracellular region” of VSIG1 refers to the following polypeptide sequence or the corresponding nucleic acid sequence (not including the signal peptide and TM of the VSIG1 protein).

  > AI581519_P3 (SEQ ID NO: 11) residues 23 to 234 (SEQ ID NO: 138)

  QVTIPDGFVNVTVGSNVTLICIYTTTVASREQLSIQWSFFHKKEMEPISIYFSQGGQAVAIGQFKDRITGSNDPGNASITISHMQPADSGIYICDVNNPPDFLGQNQGILNVSVLVKPSKPLCSVQGRPETGHTISLSCLSALGTPSPVYYWHKLEGRDIVPVKENFNPTTGILVIGNLTNFEQGYYQCTAINRLGNSSCEIDLTSSHPEVG

  > AI581519_P4 (SEQ ID NO: 12) residues 23 to 270 (SEQ ID NO: 139)

  QVTIPDGFVNVTVGSNVTLICIYTTTVASREQLSIQWSFFHKKEMEPISHSSCLSTEGMEEKAVGQCLKMTHVRDARGRCSWTSEIYFSQGGQAVAIGQFKDRITGSNDPGNASITISHMQPADSGIYICDVNNPPDFLGQNQGILNVSVLVKPSKPLCSVQGRPETGHTISLSCLSALGTPSPVYYWHKLEGRDIVPVKENFNPTTGILVIGNLTNFEQGYYQCTAINRLGNSSCEIDLTSSHPEVG

  > AI581519_P5 (SEQ ID NO: 13) residues 23 to 296 (SEQ ID NO: 140)

  QVTIPDGFVNVTVGSNVTLICIYTTTVASREQLSIQWSFFHKKEMEPISHSSCLSTEGMEEKAVGQCLKMTHVRDARGRCSWTSESPWEEGKWPDVEAVKGTLDGQQAELQIYFSQGGQAVAIGQFKDRITGSNDPGNASITISHMQPADSGIYICDVNNPPDFLGQNQGILNVSVLVKPSKPLCSVQGRPETGHTISLSCLSALGTPSPVYYWHKLEGRDIVPVKENFNPTTGILVIGNLTNFEQGYYQCTAINRLGNSSCEIDLTSSHPEVG

  > AI581519_P7 (SEQ ID NO: 14) residues 23 to 193 (SEQ ID NO: 141)

  QVTIPDGFVNVTVGSNVTLICIYTTTVASREQLSIQWSFFHKKEMEPISIYFSQGGQAVAIGQFKDRITGSNDVPNGPSYHKLSNFLGQLDPNG

  > AI581519_P9 (SEQ ID NO: 15) residues 23 to 203 (SEQ ID NO: 142)

  QVTIPDGFVNVTVGSNVTLICIYTTTVASREQLSIQWSFFHKKEMEPISIYFSQGGQAVAIGG

  > AI581519_P10 (SEQ ID NO: 16) residues 23 to 231 (SEQ ID NO: 143)

  QVTIPDGFVNVTVGSNVTLICIYTTTVASREQLSIQWSFFHKKEMEPISIYFSQGGQAVAIGQFKDRITGSNDPGNASITISHMQPADSGIYICDVNNPPDFLGQNQGILNVSVLVKPSKPLCSVQGRPETGHTISLSCLSALGTPSPVYYWHKLEGRDIVPVKENFNPTTGILVIGNLTNFEQGYYQCTAINRLGNSSCEIDLTSSRQ,

  > AI581519_P5 (SEQ ID NO: 13) residues 26 to 293 (SEQ ID NO: 302)

  IPDGFVNVTVGSNVTLICIYTTTVASREQLSIQWSFFHKKEMEPISHSSCLSTEGMEEKAVSQCLKMTHARDARGRCSWTSESPWEEGKWPDVEAVKGTLDGQQAELQIYFSQGGQAVAIGQFKDRITGSNDPGNASITISHMQPADSGIYICDVNNPPDFLGQNQGILNVSVLVKPSKPLCSVQGRPETGHTISLSCLSALGTPSPVYYWHKLEGRDIVPVKENFNPTTGILVIGNLTNFEQGYYQCTAINRLGNSSCEIDLTSSHP,

  As well as variants having at least 80% sequence identity thereto, more preferably at least 90% sequence identity thereto, and more preferably at least 95, 96, 97, 98 or 99% sequence identity thereto.

  The terms “soluble extracellular region (ECD)” or “extracellular region” of ILDR1 refer to the following polypeptide sequences (not including the signal peptide and TM of ILDR1 protein) or corresponding nucleic acid sequences:

  Residues 24 to 105 of AA424839_1_P11 (SEQ ID NO: 24): SEQ ID NO: 296

  ALSLGQDPSNDCNDNQREVRIVAQRRGQNEPVLGVDYRQRQITIQNRADVINEVMMWWDHGVYYCTIEAPGDTSGDDPEVVK (SEQ ID NO: 296);

  Residues 24 to 162 of AA424839_P3 (SEQ ID NO: 22) and AA424839_P5 (SEQ ID NO: 21): SEQ ID NO: 75

  LLVTVQHTERYVTLFASIILKCDYTTSAQLQDVVVTWRFCKSFCCKDPIFDYYSASYQAALSLGQDPSNDCNDQREVRIVAQRRGQNEPVLGVDYRRQRKIVDVMVTWTGVPG

  Residues 24 to 457 of AA424839_P7 (SEQ ID NO: 23): SEQ ID NO: 76

  LLVTVQHTERYVTLFASIILKCDYTTSAQLQDVVVTWRFKSFCKDPIFDYYSASYQAALSLGQDPSNDCNDNQREVRIVAQRRGQNEPVLGVDYRQRKITIQNPLARHRYMKQAQALGPQMMGKPLYWGADRSSQVSSYPMHPLLQRDLSLPSSLPQMPMTQTTNQPPIANGVLEYLEKELRNLNLAQPLPPDLKGRFGHPCSMLSSLGSEVVERRIIHLPPLIRDLSSSRRTSDSLHQQWLTPIPSRPWDLREGRSHHHYPDFHQELQDRGPKSWALERRELDPSWSGRHRSSRLNGSPIHWSDRDSLSDVPSSSEARWRPSHPPFRSRCQE PRRPSPRESTQRHGRRRRHRSYSPPLPSGLSSWSSEEDKERQPQSWRAHRRGSHSPHWPEEKPPSYRSLDITPGKNSRKKGSVERRSEKDSSHSGRSVVI;

  Residues 50 to 160 of AA424839_P3 (SEQ ID NO: 36): SEQ ID NO: 301

  AQLQDVVVTRWRFKSFCCKDPIFDYYYSASYQAALSLGQDPSNDCNDNQREVRIVAQRRGQNEPVLGVDYRQRKITIQNRADLVINEVMMWWDHGVYCTIEAPPGTSDGPDKE,

  As well as variants having at least 80% sequence identity thereto, more preferably at least 90% sequence identity thereto, and more preferably at least 95, 96, 97, 98 or 99% sequence identity thereto.

  The term “soluble extracellular region (ECD)” or “extracellular region” of LOC253012 refers to the polypeptide sequence or corresponding nucleic acid sequence below (not including the signal peptide and TM of LOC253012 protein).

  H68654_1_P2 (SEQ ID NO: 35) residues 38 to 349: (SEQ ID NO: 144)

  SHTVHGVRGQALYLPVHYGFHTPASDIQIIWLFERPHTMPKYLLGSVNKSVVPDLEYQHKFTMMPPNASLLINPLQFPDEGNYIVKVNIQGNGTLSASQKIQVTVDDPVTKPVVQIHPPSGAVEYVGNMTLTCHVEGGTRLAYQWLKNGRPVHTSSTYSFSPQNNTLHIAPVTKEDIGNYSCLVRNPVSEMESDIIMPIIYYGPYGLQVNSDKGLKVGEVFTVDLGEAILFDCSADSHPPNTYSWIRRTDNTTYIIKHGPRLEVASEKVAQKTMDYVCCAYNNITGRQDETHFTVIITSVGLEKLAQKGKSL;

  H68654_1_P5 (SEQ ID NO: 36), H68654_1_P7 (SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40) from residues 19 337 :( SEQ ID NO: 145) JierukeibuitibuiPiesueichitibuieichijibuiarujikyueieruwaieruPibuieichiwaijiefueichitiPieiesudiaikyuaiaidaburyueruefuiaruPieichitiemuPikeiwaieruerujiesubuienukeiesubuibuiPidieruiwaikyueichikeiefutiemuemuPiPienueiesuerueruaienuPierukyuefuPidiijienuwaiaibuikeibuienuaikyujienujitieruesueiesukyukeiaikyubuitibuididiPibuitikeiPibuibuikyuaieichiPiPiesujieibuiiwaibuijienuemutierutishieichibuiijijitiaruerueiwaikyudaburyuerukeienujiaruPibuieichitiesuesutiwaiesuefuesuPikyuenuenutierueichiaieiPibuitikeiidiaijienuYSCLVRNPVSEMESDIIMPIIYYGP GLQVNSDKGLKVGEVFTVDLGEAILFDCSADSHPPNTYSWIRRTDNTTYIIKHGPRLEVASEKVAQKTMDYVCCAYNNITGRQDETHFTVIITSVGLEKLAQKGKSL

  H68654_1_P5 (SEQ ID NO: 36) residues 1 to 335 (SEQ ID NO 300): MWLKVFTTFLSFATGACSGLKVTVPSHTVHGVRGQALYLPVHYGFHTPASDIQIIWLFERPHTMPKYLLGSVNKSVVPDLEYQHKFTMMPPNASLLINPLQFPDEGNYIVKVNIQGNGTLSASQKIQVTVDDPVTKPVVQIHPPSGAVEYVGNMTLTCHVEGGTRLAYQWLKNGRPVHTSSTYSFSPQNNTLHIAPVTKEDIGNYSCLVRNPVSEMESDIIMPIIYYGPYGLQVNSDKGLKVGEVFTVDLGEAILFDCSADSHPPNTYSWIRRTDNTTYIIKHGPRLEVASEKVAQ TMDYVCCAYNNITGRQDETHFTVIITSVGLEKLAQKGK,

  As well as variants having at least 80% sequence identity thereto, more preferably at least 90% sequence identity thereto, and more preferably at least 95, 96, 97, 98 or 99% sequence identity thereto.

  The term “soluble extracellular region (ECD)” or “extracellular region” of AI216611 refers to the following polypeptide sequence or the corresponding nucleic acid sequence (not including the AI216611 protein signal peptide and TM):

  > AI216611_P0 (SEQ ID NO: 43) 29 to 147 (SEQ ID NO: 146)

  LQSQGVSLYIPQATINATVKEDILLSVEYSCHGVPTIEWTYSSSNWGTQKIVEEWKPGTQANISQSHKDRVCTFDNGSIQLFSVVGRDGSGYYVITVTERLGSSQFGTIVLHVSEILYEDLH

  > AI216611_P0 (SEQ ID NO: 43) 1 to 145 (SEQ ID NO: 298)

MRPLPSGRRKTRGISLGLFALCLAAARCLQSQGVSLYIPQATINATVKED
ILLSVEYSCHGVPTIEWTYSSNWGTQKIVEEWKPGTQANISQSHKDRVCTFDNGSIQ
LFSVGVRDSGYYVITVTERLGSQFGTIVVLHVSEILYED,

  As well as variants having at least 80% sequence identity thereto, more preferably at least 90% sequence identity thereto, and more preferably at least 95, 96, 97, 98 or 99% sequence identity thereto.

  The term “soluble extracellular region (ECD)” or “extracellular region” of C1ORF32 refers to the following polypeptide sequence or corresponding nucleic acid sequence (not including the signal peptide and TM of C1ORF32 protein).

  > H19011_1_P8 (SEQ ID NO: 48) residues 21 to 186 (SEQ ID NO: 147)

  LQVTVPDKKKKVAMLFQPTFLRCHFSTSSSHQPAVVQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLEWEDPYLDDCLDSRRTVRWASKQGSTVTLGDFYRGREITVLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGLGGL

  > H19011_1_P9 (SEQ ID NO: 50) residues 21 to 169 (SEQ ID NO: 148)

  Q

  > H19011_1_P8 (SEQ ID NO: 48) residues 1 to 184 (SEQ ID NO: 299)

  MDRVLLRWISLFWLTAMVEGLQVTVPDKKKKVAMLMFQPTVLRCHFSTSSHQPAVVQWKFKSYCQDRMGESLGLGTDIGRTMGLDGLDVTGLDGLGTDIV

  As well as variants having at least 80% sequence identity thereto, more preferably at least 90% sequence identity thereto, and more preferably at least 95, 96, 97, 98 or 99% sequence identity thereto.

  The term “soluble extracellular region (ECD)” or “extracellular region” of FXYD3 refers to the following polypeptide sequence or the corresponding nucleic acid sequence (not including the signal peptide and TM of FXYD3 protein).

  > R31375_P0 (SEQ ID NO: 70), R31375_P31 (SEQ ID NO: 73) 21 to 36 (SEQ ID NO: 149)

  NDLEDKNSPFYYDWHS

  > R31375_P14 (SEQ ID NO: 72) 21 to 65 (SEQ ID NO: 150)

  NDLEDKNSPFYYGAPYIFVKRMGGGQMKRTQAGTEVPSTFLLDWHS

  > R31375_P33 (SEQ ID NO: 74) 21 to 25 (SEQ ID NO: 151)

  NDLED

  > R31375_P14 (SEQ ID NO: 72) 1 to 63 (SEQ ID NO: 297)

  MQKVTLGLVFLAGFPVLDANDLEDKNSPFYYGAPYIFVKRMGGGQMKRTQAGTEVPSTFLLDW,

  As well as variants having at least 80% sequence identity thereto, more preferably at least 90% sequence identity thereto, and more preferably at least 95, 96, 97, 98 or 99% sequence identity thereto.

  The term “immune response” is, for example, soluble (including antibodies, cytokines and complement) produced by lymphocytes, antigen presenting cells, phagocytic cells, granulocytes and the above cells, or cells made by the liver or spleen. Refers to the action of macromolecules, resulting in selective damage, destruction, against invading pathogens, cells or tissues infected with pathogens, cancerous cells, or in the case of autoimmunity or pathological inflammation, normal human cells or tissues, Or removal from the human body.

  “Signal transduction pathway” refers to a biochemical relationship between various signaling molecules involved in the transmission of signals from one part of a cell to another.

  As used herein, the phrase “cell surface receptor” includes, for example, a molecule or complex of molecules that can receive a signal and transmit that signal across a cell membrane.

  As used herein, the term “antibody” includes whole polyclonal and monoclonal antibodies, as well as any antigen-binding fragment (ie, “antigen-binding portion”) or a single chain thereof. “Antibody” refers to a glycoprotein comprising at least two heavy chains (H) and light chains (L) interconnected by disulfide bonds or antigen binding portions thereof. Each heavy chain is composed of a heavy chain variable region (hereinafter abbreviated as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three regions, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (hereinafter abbreviated as VL) and a light chain constant region. The light chain constant region is composed of one region CL. VH and VL regions are further classified into hypervariable regions called complementarity determining regions and regions conserved with more conserved regions called framework regions. Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region of the heavy and light chains contains a binding region that interacts with an antigen. The constant region of an antibody can mediate binding of immunoglobulins to host tissues or factors, including cells of various immune systems (eg, effector cells) and the first component of the classical complement system (C1q).

  In the present invention, the term “antigen-binding portion” of an antibody (or simply “antibody portion”) refers to an antigen (eg, VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 protein, or VSIG1, ILDR1, LOC253012, AI216611). , C1ORF32 or FXYD3) refers to one or more fragments of an antibody that retain the ability to specifically bind. It has been shown that the antigen-binding function of an antibody is performed by fragments of a full-length antibody. Examples included in the term “antigen-binding portion” of an antibody include: (i) a monovalent fragment consisting of a Fab fragment, VLight, VHeavy, Constant light (CL) and CH1 region; (ii) an F (ab ′) 2 fragment A bivalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of the VH and CH1 regions, (iv) from the VL and VH regions of a single arm of the antibody Fv fragment, (v) dAb fragment consisting of VH region (Ward et al., (1989) Nature 341: 544-546) and (vi) isolated complementarity determining region (CDR). Furthermore, the two regions of the Fv fragment, VL and VH, are encoded by different genes, but the VL and VH regions pair to form a monovalent molecule (single chain Fv Can be ligated using recombinant methods, with an artificial linker that can be made (known as (scFv)). (See, eg, Bird et al. (1988) Science 242: 423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Antibodies are also intended to be included in the term “antigen-binding portion” of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are separated for utility in the same manner as complete antibodies.

  As used herein, “isolated antibody” is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities. (For example, VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 protein, or isolated antibodies that specifically bind to VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 are VSIG1, ILDR1, LOC216611, LOC215611, respectively. , C1ORF32, FXYD3 protein or VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 but substantially free of antibodies that specifically bind to antigens.) However, VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3, FXYD3, FXYD3 Or VSIG1, ILDR1, LOC253012 Isolated antibodies that specifically bind to AI216611, C1ORF32 or FXYD3 are VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 protein, or VSIG1, ILDR1, LOC253012, AI21661, C1ORF3F, X1ODF32F And may have cross-reactivity to other antigens. In addition, an isolated antibody may be substantially free of other cellular material and / or chemicals.

  As used herein, the term “monoclonal antibody” or “monoclonal antibody composition” refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

  As used herein, the term “human antibody” is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody includes a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the invention may contain amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, randomly or in vitro introduced mutations or somatic mutations in vivo). However, as used herein, the term “human antibody” is not intended to include antibodies in which CDR sequences from germ cells of other mammalian species such as mice are conjugated onto human framework sequences.

  The term “human monoclonal antibody” refers to an antibody that exhibits a single binding specificity, with both the framework and CDR regions having variable regions derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is obtained from a transgenic non-human animal, eg, a transgenic mouse, has a genome comprising a human heavy chain light gene and a light chain transgene, and is fused to an immortalized cell. Produced by hybridomas containing B cells.

  As used herein, the term “recombinant human antibody” refers to (a) an animal (eg, a mouse) that is transgenic or transchromosomal to a human immunoglobulin gene, or a hybridoma prepared therefrom (further An antibody isolated from (described below), (b) an antibody isolated from a host cell transformed to express the antibody, eg, a transfectoma, (c) a recombinant combinatorial Such as antibodies isolated from human antibody libraries, and (d) antibodies prepared, expressed, produced or isolated by other recombinant means including splicing of human immunoglobulin gene sequences to other DNA sequences By recombination means Te, including preparation, expression, all human antibodies created or isolated. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when animal transgenics are used for human Ig sequences). be able to. Thus, the amino acid sequences of the VH and VL regions of a recombinant antibody are sequences that may be derived from and related to the human germline VH and VL sequences while not naturally occurring in the in vivo human antibody germline repertoire. is there.

  As used herein, “isotype” refers to the antibody class (eg, IgM or IgG1) that is encoded by heavy chain constant region genes.

  In the present specification, the phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably with “an antibody which binds specifically to an antigen”.

As used herein, antibodies that “specifically bind to human VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 protein, or VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3” are human VSIG1, ILDR12, LOC, respectively. , AI216611, C1ORF32, FXYD3 proteins, or at VSIGl, ILDRl, LOC253012, AI216611, C1ORF32 antibody binding or to FXYD3,, preferably, 5X10 ^-8 M or less, more preferably less and more preferably 3X10 ^-8 M 1X10 ^- It is intended to refer to those with a KD of ⁹ M or less.

  As used herein, the terms “K-assoc” or “Ka” are intended to refer to the rate of association of a particular antibody-antigen interaction, while the terms “Kdis” or “Kd” are used herein. Is intended to refer to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term “KD” is intended to refer to the dissociation constant obtained from the ratio of Kd to Ka (ie, Kd / Ka) and expressed as molar concentration (M). The KD value for an antibody can be measured using methods established in the art. A preferred method for measuring the KD of the antibody is to use surface plasmon resonance, preferably Biacore RTM. By using a biosensor system such as a system.

As used herein, the term “high affinity” for an IgG antibody has a ^KD of 10 ⁻⁸ M or less, more preferably 10 ⁻⁹ M or less, and even more preferably 10 ⁻¹⁰ M or less for a target antigen. Refers to an antibody. However, “high affinity” binding can vary for other antibody isotypes. For example, “high affinity” binding for an IgM isotype refers to an antibody having a KD of 10 ⁻⁷ M or less, more preferably 10 ⁻⁸ M or less.

  As used herein, the term “subject” includes any human or non-human animal. The term “non-human animals” includes all vertebrates, eg mammals and non-mammals such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles.

  As used herein, the term “tail” refers to a peptide sequence at the end of an amino acid sequence that is unique to a splice variant according to the present invention. Thus, a splice variant with such a tail has at least the first half of the splice variant typically highly homologous (often 100% identical) to the corresponding known protein portion, Can optionally be considered as a chimera in that the portion of contains a tail.

  As used herein, the term “head” refers to a peptide sequence at the end of an amino acid sequence that is unique to a splice variant according to the present invention. Thus, a splice variant having such a head is such that the first half of the splice variant contains the head and at least the second half is typically highly enriched in the corresponding portion of the known protein. Can optionally be regarded as a chimera in that they are homologous to (often 100% identical).

  As used herein, “edge portion” refers to a connection between two portions of a splice variant according to the present invention that was not connected by a wild-type or known protein. The edge can optionally arise, for example, by binding between the “known protein” portion of the variant and the tail, and / or the inner portion of the wild-type sequence no longer exists In some cases, the two parts of the sequence may occur to be contiguous in the splice variant, but not contiguous in the known protein. A “bridge” may optionally be the edge portion, a linkage between the head and the “known protein” portion of the mutant, or a tail and a mutation. It may include a linkage between the “known protein” portions of the body, or a linkage between the insert and the “known protein” portion of the variant.

  In some embodiments, the bridge between the tail or head or the unique insert and the “known protein” portion of the variant is such that at least one amino acid tail / head ( head) / insert and at least about 10 amino acids, or in some embodiments at least about 20 amino acids, or some embodiments, wherein at least one amino acid is from the “known protein” portion of the variant. In which at least about 30 amino acids, or in some embodiments, at least about 40 amino acids. In some embodiments, the bridge is any number of amino acids from about 10 to about 40 (eg, length 10, 11, 12, 13, ... 37, 38, 39, 40 amino acids or between Any number of amino acids).

  It should be noted that a bridge cannot be extended beyond the array length in either direction, and the description of each bridge is that the length of the bridge is an array. It should be taken for granted that it is understood that it does not extend beyond itself.

  Further, in certain contexts below, bridges are described with respect to a sliding window. For example, one description of bridges features the following pattern: a bridge between two edges (a portion of a known protein is not present in a variant) is optionally Can be described as follows: a bridge portion of CONTIG-NAME_P1 (representing the protein name) having a polypeptide with length “n”, where n is at least about 10 in length Amino acids, optionally at least about 20 amino acids, preferably at least about 30 amino acids, more preferably at least about 40 amino acids and most preferably at least about 50 amino acids in length At least two amino acids are XX (the two amino acids in the middle of the bridge) Acid, one from each end of the edge) and having the following structure (numbering according to the sequence of CONTIG-NAME_Pl): starting from any amino acid number (eg) 49-x to 49, A sequence ending with any amino acid number (eg) 50 + ((n−2) −x), where x varies from 0 to n−2. This example should also be construed as including bridges, which are any number of amino acids between 10-50 amino acids in length. Furthermore, a bridge polypeptide cannot extend beyond the sequence, so (e.g.) 49-x is not shorter than 1, and (e.g.) 50 <+> ((n-2) -x) is Should not be construed as being longer than the total sequence length.

  Various aspects of the invention are described in further detail in the following subsections.

Nucleic acid “Nucleic acid fragment” or “oligonucleotide” or “polynucleotide” are used interchangeably herein to refer to a polymer of nucleic acid residues. The polynucleotide sequences of the present invention are isolated and provided in the form of RNA sequences, complementary polynucleotide sequences (cDNA), genomic polynucleotide sequences and / or composite polynucleotide sequences (eg, combinations of the above). Or a single-stranded or double-stranded nucleic acid sequence.

  Thus, the present invention relates to a nucleic acid sequence as described above, a fragment thereof, a sequence hybridizable thereto, a sequence homologous thereto (eg, at least 90%, at least 95, 96, 97, 98 or a nucleic acid sequence described herein). 99% or more identical), sequences encoding the same polypeptide with different codon usage, such as deletion, insertion or substitution of one or more nucleotides in a natural or artificial, random or targeted manner, etc. Includes modified sequences characterized by mutations. The present invention also includes homologous nucleic acid sequences that contain sequence regions unique to the polynucleotides of the invention (ie, sequences that form part of the polynucleotide sequences of the invention).

  Where the polynucleotide sequence of the present invention encodes a polypeptide not previously identified, the present invention also includes novel polypeptides encoded by the isolated polynucleotides described above and the corresponding nucleic acid fragments thereof. Including peptides or portions thereof.

  Thus, the present invention also includes a polypeptide encoded by a polynucleotide sequence of the present invention. The invention also includes homologues of these polypeptides, which can be at least 90%, at least 95, 96, 97, 98 or 99% or more homologous to the amino acid sequences described below. It can be determined using the BlastP software of the National Center of Biotechnology Information (NCBI), with initial parameters. Finally, the present invention also provides for the above polypeptides and polypeptides having mutations such as deletion, insertion or substitution of one or more nucleotides in a natural or artificially induced, random or targeted manner. Including fragments.

  As mentioned above, the biomolecular sequences of the present invention can be used efficiently as tissue or pathological markers and as putative drugs or drug targets for treating or preventing diseases.

  Oligonucleotides designed to carry out the methods of the invention (designed as described above) for any of the sequences provided herein are known to those skilled in the art, such as enzymatic synthesis or solid phase synthesis. Can be produced according to any oligonucleotide synthesis method. Equipment and reagents for performing solid phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for the synthesis can be employed. The actual synthesis of the oligonucleotide is well within the ability of one skilled in the art.

  Oligonucleotides used in accordance with this aspect of the invention range from about 10 to about 200 bases, preferably from about 15 to about 150 bases, more preferably from about 20 to about 100 bases, most preferably from about 20 to about 50 bases. It has a length selected from

  The oligonucleotides of the invention may comprise heterocyclic nucleotides consisting of purine and pyrimidine bases linked by 3'-5 'phosphodiester bonds.

  Preferred oligonucleotides are those modified either generally in the backbone, internucleotide linkages or bases as described generally below. Such modifications can often facilitate oligonucleotide uptake and resistance to intracellular conditions.

  Specific examples of preferred oligonucleotides useful in accordance with this aspect of the invention include oligonucleotides containing modified backbones or non-natural internucleotide linkages. As oligonucleotides having a modified backbone, US Pat. Nos. 4,469,863, 4,476,301, 5,023,243, 5,177,196, 5,188,897 No. 5,264,423, No. 5,276,019, No. 5,278,302, No. 5,286,717, No. 5,321,131, No. 5,399,676, 5,405,939, 5,453,496, 5,455,233, 5,466,677, 5,476,925, 5,519,126, 5 No. 5,536,821, No. 5,541,306, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. 5,587,361, and No. 5, The phosphorus atom is retained in the skeleton disclosed in 625,050. Those that can be mentioned.

  For example, preferred modified oligonucleotide backbones include phosphorothioates, enantiomeric phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, 3'-alkylene phosphonates and methyl and other alkyls including enantiomeric phosphonates. Phosphonates, phosphinates, phosphoramidates, 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphoramidates, thionoalkylphosphotriesters And boranophosphates with normal 3′-5 ′ linkages, their 2′-5 ′ linked analogs, and adjacent sets of nucleotide units from 3′-5 ′ to 5′-3 ′ or 2 Reverse polarity from '-5' to 5'-2 ' It shall have the like. Various salts, mixed salts and free acid forms can also be used.

  Alternatively, a modified oligonucleotide backbone that does not contain a phosphorus atom is a short alkyl or cycloalkyl internucleotide linkage, a mixed heteroatom and alkyl or cycloalkyl internucleotide linkage, or one or more short heteroatoms or heterocyclic internucleotide linkages. It has a skeleton formed by These include U.S. Pat. Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235. No. 5,033, No. 5,264,562, No. 5,264,564, No. 5,405,938, No. 5,434,257, No. 5,466,677, No. 5,470,967. No. 5,489,677, No. 5,541,307, No. 5,561,225, No. 5,596,086, No. 5,602,240, No. 5,610,289, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 633, 360, 5,677,437, and 5,677. As disclosed in US Pat. No. 439, morpholino bonds (partially formed from the sugar moiety of the nucleoside), siloxane skeleton, sulfide, sulfoxide and sulfone skeleton, formacetyl and thioformacetyl skeleton, methyleneformacetyl and thioformacetyl skeleton Alkene-containing skeletons, sulfamate skeletons, methyleneimino and methylenehydrazino skeletons, sulfonate and sulfonamide skeletons, amide skeletons, and others with mixed N, O, S and CH2 moieties Can be mentioned.

  Other oligonucleotides that can be used in accordance with the present invention are those modified with sugars and intranucleotide linkages, i.e., the backbone of the nucleotide unit is replaced with a novel group. Base units are maintained for complementation with the appropriate polynucleotide target. An example of such an oligonucleotide mimetic is peptide nucleic acid (PNA). PNA oligonucleotide refers to an oligonucleotide in which the sugar backbone is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone. The base is retained and attached, either directly or indirectly, to the aza nitrogen atom of the backbone amide moiety. US patents that teach the preparation of PNA compounds include, but are not limited to, US Pat. Nos. 5,539,082, 5,714,331, and 5,719,262, each by reference. Incorporated herein. Other backbone modifications that can be used in the present invention are disclosed in US Pat. No. 6,303,374.

  The oligonucleotides of the invention can also contain base modifications or substitutions. As used herein, “unmodified” or “natural” bases include purine bases, adenine (A) and guanine (G), and pyrimidine bases, thymine (T), cytosine (C) and uracil (U). Modified bases include, but are not limited to, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, adenine and 6-methyl and other alkyl derivatives of guanine, 2-Propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudo Uracil), 4-thiouracil, 8-halo, 8-amino, 8-thio, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoro Other synthetic and natural such as til and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine A base. Additional bases include those disclosed in U.S. Pat. No. 3,687,808, The Concise Encyclopedia Of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. et al. L, ed. Disclosed in John Wiley & Sons, 1990, Englisch et al. , Angelwandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, Y. et al. S. , Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S .; T. T. et al. and Lebleu, B.B. , Ed. , CRC Press, 1993. Such bases are particularly useful because they increase the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-Methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6-1.2 ° C (Sangvi YS et al. (1993) Antisense Research and Applications, CRC Press, Boca Raton 276. -278), presently preferred base substitutions, especially when combined with 2'-O-methoxyethyl sugar modifications.

Another modification of the oligonucleotides of the present invention involves chemically linking one or more moieties or conjugates that enhance activity, cell distribution or cellular oligonucleotides and uptake to the oligonucleotide. Such moieties include, but are not limited to, cholesterol moieties, cholic acid, thioether (eg hexyl-S-trityl) as disclosed in US Pat. No. 6,303,374. Thiol), thiocholesterol, aliphatic chains (eg dodecanediol or undecyl residues), phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3- H-phosphonate, polyamine or polyethylene glycol chain,
Alternatively, lipid sites such as adamantaneacetic acid, palmityl sites (moiety), octadecylamine, or hexylamino-carbonyl-oxycholesterol sites (moiiety) can be mentioned.

  Not all positions of a given oligonucleotide molecule need be uniformly modified, and in fact more than one of the above modifications can be incorporated into one compound or even one nucleotide within an oligonucleotide.

Peptides The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein and refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are analogs or mimetics of natural amino acids, as do natural amino acid polymers. Polypeptides can be modified, for example, by the addition of carbohydrate residues to form glycoproteins. The terms “polypeptide”, “peptide” and “protein” include glycoproteins as well as non-glycoproteins.

  Polypeptide products can be synthesized biochemically, such as by using standard solid phase techniques. Such methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment concentration, and classical solution synthesis. These methods are preferably used when the peptides are relatively short (ie 10 kDa) and / or involve different chemistry that cannot be produced by recombinant techniques (ie not encoded by nucleic acid sequences).

  Solid phase polypeptide synthesis procedures are well known in the art and are further described in John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 198).

  Synthetic polypeptides can be purified by preparative high performance liquid chromatography (Creighton T. (1983) Proteins, structures and molecular principals. WH Freeman and Co. N.Y., composition). Can be verified through amino acid sequencing.

  If a large amount of polypeptide is desired, Bitter et al. , (1987) Methods in Enzymol. 153: 516-544, Study et al. (1990) Methods in Enzymol. 185: 60-89, Brisson et al. (1984) Nature 310: 511-514, Takamatsu et al. (1987) EMBO J. et al. 6: 307-311, Coruzzi et al. (1984) EMBO J. et al. 3: 1671-1680 and Brogli et al. (1984) Science 224: 838-843, Gurley et al. (1986) MoI. Cell. Biol. 6: 559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463. Can be produced using recombinant techniques such as those described in.

  Peptides identified in accordance with the teachings of the present invention are degradation products, synthetic peptides or recombinant peptides and peptide mimetics, typically synthetic peptides, and modifications that are more stable in the body or can be penetrated by cells, for example. It will be understood that peptoids and semipeptoids, which are peptide analogs that can have Such modifications include, but are not limited to, N-terminal modification, C-terminal modification, but not limited to CH2-NH, CH2-S, CH2-S = O, O = C-NH, CH2-O, CH2- Peptide bond modifications including CH2, S = C-NH, CH = CH or CF = CH, backbone modifications, and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art and are described, for example, in Quantitative Drug Design, CA. Ramsden Gd. , Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated herein by reference in its entirety. Further details regarding this are provided below.

  The peptide bond (—CO—NH—) in the peptide is an N-methylated bond (—N (CH 3) —CO—), an ester bond (—C (R) H—C—O—O—C (R) -N-), ketomethylene bond (-CO-CH2-), α-aza bond (-NH-N (R) -CO-), wherein R is any alkyl such as methyl, carba ) Bond (—CH 2 —NH—), hydroxyethylene (—CH (OH) —CH 2 —), thioamide bond (—CS—NH—), olefin double bond (—CH═CH—), retroamide bond (retro) amide bonds) (—NH—CO—), peptide derivatives (—N (R) —CH 2 —CO) (where R is a “normal” side chain, a naturally occurring carbon atom)).

  These modifications can occur in any of the bonds along the peptide chain and even some (2-3) at the same time.

  Natural aromatic amino acids, Trp, Tyr and Phe are synthetic unnatural acids such as phenylglycine, TIC, naphthylalanine (Nol), ring methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr Can be substituted.

  In addition to the above, the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (eg, fatty acids, complex carbohydrates, etc.).

  In this specification and in the claims section below, the term “amino acid” or “amino acids” refers to the 20 natural amino acids such as hydroxyproline, phosphoserine and phosphothreonine. It is understood to include amino acids that are often post-translationally modified in vivo, as well as other rare amino acids including, but not limited to, 2-aminoazibic acid, hydroxylysine, isodesmosine, norvaline, norleucine and ornithine. Furthermore, the term “amino acid” includes both D- and L-amino acids.

  The peptides of the present invention are preferably used in therapy where the peptide is required to be in an insoluble form. The peptides of the present invention preferably comprise one or more non-natural or naturally polar amino acids, including but not limited to serine and threonine, which can increase peptide solubility due to hydroxyl-containing side chains.

  The peptides of the present invention are preferably used in a linear form. It will be understood that cyclic forms of peptides can also be used if cyclization does not significantly interfere with the properties of the peptide.

  The peptides of the invention can be synthesized biochemically, such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment concentration, and classical solution synthesis. These methods are preferably used when the peptides are relatively short (ie 10 kDa) and / or cannot be produced by recombinant techniques (ie not encoded by nucleic acid sequences) and thus involve different chemistry.

  Solid phase polypeptide synthesis procedures are well known in the art and are further described in John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 198).

  Synthetic polypeptides can be purified by preparative high performance liquid chromatography and are described in Creighton T. et al. (1983) Proteins, structures and molecular principals. WH Freeman and Co. N. Y. The composition can be verified through amino acid sequencing.

  If a large amount of polypeptide is desired, Bitter et al. , (1987) Methods in Enzymol. 153: 516-544, Study et al. (1990) Methods in Enzymol. 185: 60-89, Brisson et al. (1984) Nature 310: 511-514, Takamatsu et al. (1987) EMBO J. et al. 6: 307-311, Coruzzi et al. (1984) EMBO J. et al. 3: 1671-1680 and Brogli et al. (1984) Science 224: 838-843, Gurley et al. (1986) MoI. Cell. Biol. 6: 559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463. Can be produced using recombinant techniques such as those described in.

Expression System In order to allow cellular expression of the polynucleotide of the present invention, the nucleic acid construct according to the present invention may be used. The nucleic acid construct includes at least one coding region of the nucleic acid sequence, and further includes at least one cis-acting regulator. As used herein, the phrase “cis-acting regulator” refers to a promoter that binds to a polynucleotide sequence, preferably a trans-acting regulator and controls transcription of a coding sequence located downstream thereof.

  Any suitable promoter sequence can be used by the nucleic acid construct of the present invention.

  Preferably, the promoter used by the nucleic acid construct of the invention is active in the particular cell population transformed. For example, as cell type specific and / or tissue specific promoters, albumin (Pinkert et al., (1987) Genes Dev. 1: 268-277) which is liver specific, lymph specific promoter (Callame et al.,). (1988) Adv. Immunol. 43: 235-275), in particular the T cell receptor promoter (Winoto et al., (1989) EMBO J. 8: 729-733) and immunoglobulin (Banerji et al. (1983) Cell. 33729-740), neuron-specific promoters such as neurofilament promoters (Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:54 3-5477), pancreas specific promoters (Edrunch et al. (1985) Science 230: 912-916), or mammary gland specific promoters such as whey promoters (US Pat. No. 4,873,316 and Promoters such as European Application Publication No. 264,166). The nucleic acid constructs of the invention can further include an enhancer that can be adjacent or remote from the promoter sequence and that functions in enhancing transcription therefrom.

  The nucleic acid construct of the present invention preferably further comprises a suitable selectable marker and / or origin of replication. Preferably, the nucleic acid construct used is E. coli. A shuttle vector that can be propagated with E. coli (constructs contain appropriate selectable markers and origins of replication) and can be adapted for growth in cells or integration in selected genes and tissues. The construct according to the invention can be, for example, a plasmid, bacmid, phagemid, cosmid, phage, virus or artificial chromosome.

  For example, suitable constructs include, but are not limited to, pcDNA3, pcDNA3.1 (+/−), pGL3, PzeoSV2 (+/−), pDisplay, pEF / myc / cyto, pCMV / myc / cyto, Each of Invitrogen Co. (Www.invitrogen.com). Examples of retroviral vectors and packaging systems include the Retro-X vectors pLNCX and pLXSN, including Clontech, San Diego, Calif. They are more commercially available, they can be cloned into multiple cloning sites and the transgene is transcribed from the CMV promoter. Vectors derived from Mo-MuLV also include pBabe, etc., where the transgene will be transcribed from the 5 'LTR promoter.

  Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs such as adenovirus, lentivirus, herpes simplex I virus or adeno-associated virus (AAV) and lipid based systems. Lipids useful in lipid-mediated transfer of genes are, for example, DOTMA, DOPE, and DC-Chol (Tonkinson et al., Cancer Investigation, 14 (1): 54-65 (1996)). The most preferred construct for use in gene therapy is a virus, most preferably an adenovirus, AAV, lentivirus or retrovirus. Viral constructs, such as retroviral constructs, contain at least one transcriptional promoter / enhancer or locus-defining elements, or other splicing, RNA nuclear export or post-translational modification of messengers. Includes other factors that control gene expression by means. Such vector constructs also include packaging signals, long terminal repeats (LTR) or parts thereof, and positive and negative that are appropriate for the virus used if not already present in the viral construct. and negative) contains a strand primer binding site. In addition, such constructs typically include a signal sequence for secretion of the peptide from the host cell in which it is located. Preferably, the signal sequence for this purpose is a mammalian signal sequence or a signal sequence of a polypeptide of the invention. Optionally, the construct may also include a signal that directs polyadenylation, as well as one or more restriction enzyme recognition sites and a translation stop sequence. By way of example, such constructs will typically include a 5 'LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3' LTR or a portion thereof. Other vectors that are non-viral, such as cationic lipids, polylysine and dendrimers can be used.

Recombinant expression vectors and host cells Another aspect of the invention relates to vectors, preferably nucleic acid encoding a protein of the invention, or an expression vector comprising a derivative, fragment, analog or homologue thereof. In this specification, the term “vector” refers to a nucleic acid molecule that transports another nucleic acid linked to it. One type of vector is a “plasmid” that refers to a circular double stranded DNA loop into which additional DNA fragments can be ligated. Another type of vector is a viral vector in which additional DNA fragments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced. (Eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors.) Other vectors (eg, non-episomal mammalian vectors) are introduced into the genome of a host cell when introduced into the host cell. Integrated and thereby replicated with the host genome. In addition, certain vectors can direct the expression of operably linked genes. Such vectors are referred to herein as “expression vectors”. In general, expression vectors used in recombinant DNA technology are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the present invention is intended to include other forms of expression vectors that perform equivalent functions, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses).

  The recombinant expression vector of the present invention comprises the nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell, which nucleic acid is selected and expressed based on the host cell in which the recombinant expression vector is used for expression. It is meant to include one or more regulatory sequences operably-linked to the sequence. “Operatively linked” within a recombinant expression vector means that the nucleotide sequence (eg, in an in vitro transcription / translation system or when the vector is introduced into the host cell) when the vector is introduced into the host cell. It is intended to mean that the nucleotide sequence of interest is linked to a regulatory sequence so that it can be expressed in a host cell when introduced.

  The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of nucleotide sequences in a variety of host cells, and those that direct expression of nucleotide sequences only in certain host cells (eg, tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on factors such as the choice of the host cell to be transformed, the level of expression of the desired protein, and the like. The expression vectors of the invention can be introduced into host cells, thereby producing a protein or peptide, such as a fusion protein or peptide encoded by the nucleic acids described herein.

  The recombinant expression vectors of the invention can be designed for the production of mutant proteins in prokaryotic or eukaryotic cells. For example, the proteins of the invention can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are further described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using Tl promoter regulatory sequences and T7 polymerase.

  Protein expression in prokaryotes is most often performed in Escherichia coli using a vector containing a constitutive or inducible promoter that directs the expression of either a fused or non-fused protein. Fusion vectors add many amino acids to the protein encoded therein, the amino or C-terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (i) increase the expression of the recombinant protein, (ii) increase the solubility of the recombinant protein, and (iii) act as a ligand in affinity purification. To assist in the purification of the recombinant protein. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion site and the recombinant protein, allowing separation of the recombinant protein from the fusion site following purification of the fusion protein. . Such enzymes and their cognate recognition sequences include blood coagulation factor Xa (Factor Xa), thrombin, PreScission, TEV and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) And pRIT5. .), Each of which fuses glutathione S-transferase (GST), maltose E binding protein or protein A to the target recombinant protein.

  Proper induction, non-fusion E. coli Examples of E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69: 301-315) and pET 11d (Studer et al., Gene Expression Technology: Methods in Enzymology 18, Genes in Enzymology 18 (1990) 60-89)-(inaccurate, pETlla-d has an N-terminal T7 tag).

  E. One strategy for maximizing recombinant protein expression in E. coli is to express the protein in the host bacteria with a recombinant protein that has an impaired ability to proteolytically cleave the recombinant protein. For example, Gottesman, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is that each codon for each amino acid is E. coli. It is to change the nucleic acid sequence of the nucleic acid inserted into the expression vector so that it is preferentially used in E. coli. (See, for example, Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118.) Such nucleic acid sequence modifications of the present invention may be performed by standard DNA synthesis techniques. Can do. Another strategy for resolving codon bias is the rare E. coli. By using a BL21-codon plus bacterial strain (Invitrogen), or Rosetta bacterial strain (Novagen), which contains an extra copy of the E. coli tRNA gene.

  In another embodiment, the expression vector encoding the protein of the present invention is a yeast expression vector. Examples of vectors for expression in the yeast Saccharomyces cerevisiae include pYepSecl (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 94: 394 943. et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

  Alternatively, the polypeptides of the present invention can be produced in insect cells using baculovirus expression vectors. Examples of baculovirus vectors that can be used for protein expression in cultured insect cells (eg, SF9 cells) include pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and pVL series ( Luclow and Summers, 1989. Virology 170: 31-39).

  In yet another embodiment, the nucleic acids of the invention are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195), pIRESpuro (Clontech), pUBen (Invitro) Examples thereof include pCEP4 (Invitrogen), pREP4 (Invitrogen), and pcDNA3 (Invitrogen). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma virus, adenovirus 2, cytomegalovirus, rous sarcoma virus and simian virus 40. For other suitable expression systems for prokaryotic and eukaryotic cells, see, for example, Sambrook, et al. , Molecular Cloning: A Laboratory Manual. 2nd ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N .; Y. 1989. See Chapters 16 and 17.

  In another embodiment, the recombinant mammalian expression vector can preferentially direct expression of the nucleic acid in a particular cell type (eg, tissue specific regulators are used to express the nucleic acid. Regulatory regulators are known in the art Non-limiting examples of suitable tissue specific promoters include the albumin promoter (liver specific, Pinkert, et al., 1987. Genes Dev. 1: 268-277. ), Lymphoid-specific promoters (Callame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular the T cell receptor promoter (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins. (Ban rji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (eg, neurofilament promoter, Byrne and Ruddle, 1989. Proc. Natl. Aci. Sci. USA 86: 5473-5477), pancreas specific promoter (Edrund, et al., 1985. Science 230: 912-916) and mammary gland specific promoter (eg, whey promoter, US Pat. No. 4,873). No. 316, European Application Publication No. 264, 166), for example, mouse Also included are developmentally regulated promoters such as the ox promoter (Kessel and Gross, 1990. Science 249: 374-379) and the alpha-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546). .

  The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned in the antisense orientation into the expression vector. That is, the DNA molecule is operably linked to regulatory sequences in a manner that allows expression of the RNA molecule that is antisense to the mRNA encoding the protein of the invention (by transcription of the DNA molecule). -Linked). Regulatory sequences that direct the constitutive expression of antisense RNA molecules operably linked to nucleic acids cloned in the antisense orientation are selected in a variety of cell types. be able to. For example, viral promoters and / or enhancer or regulatory sequences that direct tissue-specific or cell type-specific expression of constitutive antisense RNA can be selected. An antisense expression vector is a recombinant plasmid, phagemid or attenuated virus whose antisense nucleic acid is generated under the control of a highly efficient regulatory region and whose activity can be determined by the cell type into which the vector has been introduced. Can be in the form of For a discussion on the control of gene expression using antisense genes, see, for example, Weintraub, et al. "Antisense RNA as a molecular tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1 (1) See 1986.

  Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that it refers not only to a particular subject cell, but also to the progeny or potential progeny of such a cell. In fact, such progeny may not be identical to the parent cell, as certain modifications can occur in the next generation, either due to mutations or environmental effects, Included in.

  The host cell can be any prokaryotic or eukaryotic cell. For example, the protein of the present invention may be E. coli. It can be produced in bacterial cells such as E. coli, insect cells, yeast, plant or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS or 293 cells). Other suitable host cells are known to those skilled in the art.

  Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. In this specification, the terms “transformation” and “transfection” refer to foreign nucleic acids (eg, DNA), including calcium phosphate coprecipitation or calcium chloride, DEAE-dextran mediated transfection, lipofection or electroporation. It is intended to refer to a variety of art-recognized techniques for introduction into cells. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., and others.) .

  For stable gene transfer transfection of mammalian cells, depending on the expression vector and transfection technique used, it is known that only a few cells can integrate foreign DNA into their genome. In order to identify and select these uptakes, a gene encoding a selectable marker (eg, resistance to antibiotics) is generally introduced into the host cell along with the gene of interest. A variety of selectable markers include those that confer resistance to drugs such as G418, hygromycin, puromycin, blasticidin and methotrexate. The nucleic acid encoding the selectable marker can be introduced into the host cell on the same vector as the nucleic acid encoding the protein of the invention, or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection. (For example, cells that have incorporated the selectable marker gene will survive, while other cells will die.)

  A host cell of the invention in culture, such as a prokaryotic or eukaryotic host cell, can be used to produce (ie, express) a protein of the invention. Accordingly, the present invention further provides a method for producing the protein of the present invention using the host cell of the present invention. In one embodiment, the method comprises culturing a host cell of the invention (introduced with a recombinant expression vector encoding the protein of the invention) in a suitable medium such that the protein of the invention is produced. Including doing. In another embodiment, the method further comprises isolating the protein of the invention from the medium or the host cell.

  For efficient production of the protein, it is preferable to place the nucleotide sequence encoding the protein of the invention under the control of an expression control sequence optimized for expression in the desired host. For example, the sequence can include optimized transcriptional and / or translational regulatory sequences (such as modified Kozak sequences).

Protein Modification Fusion Proteins According to the present invention, fusion proteins can be prepared from the proteins of the present invention by fusion with a portion of an immunoglobulin containing the constant region of an immunoglobulin. More preferably, the immunoglobulin portion comprises a heavy chain constant region which is optionally and more preferably a human heavy chain constant region. The heavy chain constant region is most preferably an IgG heavy chain constant region, and is optionally and more preferably an Fc chain, most preferably an IgG Fc fragment comprising CH2 and CH3 regions. Any IgG subtype can be optionally used, but the IgG1 subtype is preferred. The Fc chain may optionally be a known or “wild-type” Fc chain, or may be mutated. Non-limiting, exemplary, typical types of mutations are described in US Patent Application No. 20060034852 (issued February 16, 2008), which is hereby incorporated by reference in its entirety. The term “Fc chain” also optionally includes any type of Fc fragment.

  In the IgG subclass, some of the specific amino acid residues that are important for the activity mediated by the antibody constant region have been identified. Thus, the inclusion, substitution or exclusion of these specific amino acids allows the inclusion or exclusion of specific immunoglobulin constant region mediated activities. Furthermore, certain changes can result in, for example, aglycosylation, and / or other desired changes to the Fc chain. As described in more detail below, at least some changes may optionally be made to interfere with Fc functions that are considered undesirable, such as undesirable immune system effects.

  Non-limiting, illustrative examples of mutations to Fc that can be made to modulate the activity of the fusion protein include the following changes (for Fac sequence nomenclature: Kabat EA et al: Sequences of Proteins of Immunological Inter. From US Department of Health and Human Services, NIH, 1991, according to Kabat): 220C-> S, 233-238ELLGPP-> EAEGAP-> 265D-> N, preferably A-34 297N-> A (eg to prevent N-glycosylation), 318-322EYKCK-> AYACA, 330-331AP-> SS, (See, for example, M. Clark, “Chemical Immunol and Antibody Engineering”, pp 1-31 for a description of these mutations and their effects). Optionally and preferably includes a combination of the CH2 and CH3 regions of the hinge region.

  The modification can optionally be performed to enhance desired properties or alternatively prevent undesirable properties. For example, deglycosylation of antibodies has been shown to cause the prevention of T cell depletion or the release of cytokines, which may be an undesirable function, while maintaining the desired binding functionality (M. Clark, Chemical See Immunol and Antibody Engineering, pp 1-31). Replacement of proline at position 331 with serine can interfere with the ability of activated complement, which can optionally be regarded as an undesirable function (see M. Clark, Chemical Immunol and Antibody Engineering, pp 1-31). Also, in conjunction with this change, changing alanine at position 330 to serine can enhance the desired effect of hindering the ability to activate complement.

  Residues 235 and 237, as described above, altering the inhibition of residues from 233-238 also allows antibody-dependent cells to be able to inhibit such activity if ADCC is considered an undesirable function. It has been shown to be involved in mediated cytotoxicity (ADCC).

  Residue 220 is usually the cysteine in Fc from IgG1 and is the site where the heavy chain forms a covalent bond with the light chain. Optionally, this residue can be changed to serine to avoid any type of covalent bond. (See M. Clark, “Chemical Immunol and Antibody Engineering”, pp 1-31.)

  Such changes to residues 265 and 434 can optionally interfere with undesirable functions of Fc associated with immune system functions that may be performed to reduce or inhibit binding to Fc receptors. (See “Binding site on Human IgGl for Fc Receptors”, Shields et al, VoI 276, pp 6591-6604, 2001.)

  The above changes are intended only as examples of selective changes and do not imply any limitation. Further, the above description is not meant to be bound by a single hypothesis, but is provided for convenience.

Addition of groups If the protein according to the invention is a linear molecule, various functional groups can be added at various points on the linear molecule that can accept or be suitable for chemical modification. Is possible. Functional groups can be added to the linear ends of the proteins of the invention. In some embodiments, functional groups include, but are not limited to, increased stability, penetration (through cell membranes and / or tissue barriers), tissue localization, efficiency, reduced clearance, reduced toxicity Enhances the activity of a protein with respect to one or more properties, including improved selectivity, improved resistance to excretion by cell pumps, and the like. For convenience and without wishing to be limited, one free N-terminus of the sequences included in the compositions of the present invention is referred to as the N-terminus of the composition, and sequence binding The free C-terminus will be considered the C-terminus of the composition. The C-terminus or N-terminus of the sequence, or both can be linked to a carboxylic acid functionality or an amine functionality, respectively.

  Non-limiting examples of suitable functional groups are described in Green and Wuts, “Protecting Groups in Organic Synthesis”, John Wiley and Sons, Chapters 5 and 7, 1991, the teachings of which are incorporated herein by reference. . Preferred protecting groups are those which facilitate the transport of the active ingredient added thereto to the cell, for example by reducing the hydrophilicity of the active ingredient and increasing the lipophilicity. These are examples of “sites for transport across cell membranes”.

These sites are optionally and preferably cleaved in vivo, either hydrolytically or enzymatically, within the cell. (Ditter et al., J. Pharm. Sci. 57: 783 (1968); Ditter et al., J. Pharm. Sci. 57: 828 (1968); Ditter et al., J. Pharm. Sci. 58: 557 (1969); King et al., Biochemistry 26: 2294 (1987); Lindberg et al., Drug Metabolism and Disposition 17: 311 (1989); and Tunek et al., Biochem. Anderson et al., Arch. Biochem. Biophys. 239: 538 (1985) and Singhal et al., FASEB J. et al. 1: 220 (1987)).
Hydroxyl protecting groups include carbonic acid and carbamate protecting groups. Examples of amine protecting groups include alkoxy and aryloxy carbonyl groups as described above for the N-terminal protecting group. Carboxylic acid protecting groups include aliphatic, benzyl, and aryl esters as described above for the C-terminal protecting group. In one embodiment, the side chain carboxylic acid group of one or more glutamic acid or aspartic acid in the composition of the present invention is preferably a methyl, ethyl, benzyl or substituted benzyl ester, more preferably a benzyl ester. Protected by.

  Non-limiting, illustrative examples of N-terminal protecting groups include acyl groups (CO—R 1) and alkoxycarbonyl or aryloxycarbonyl groups (CO—O—R 1), where R 1 is aliphatic, substituted aliphatic, benzyl , Substituted benzyl, aromatic, or substituted aromatic groups). Specific examples of acyl groups include, but are not limited to, acetyl, (ethyl) -CO-, n-propyl-CO-, iso-propyl-CO-, n-butyl-CO, sec-butyl-CO, tert-butyl-CO-, hexyl, lauroyl, palmitoyl, myristoyl, stearyl, CO-, including oleoylphenyl, substituted phenyl-CO-, benzyl-CO- and (substituted benzyl) -CO-. Examples of alkoxycarbonyl and aryloxycarbonyl groups include CH3-O-CO, (ethyl) -O-CO-, n-propyl-O-CO, isopropyl-O-CO-, n-butyl-O-CO-. , Sec-butyl-O-CO, t-butyl-O-CO-, substituted phenyl-O-CO, substituted phenyl-O-CO- or benzyl-O-CO,-(substituted benzyl-O-CO, adamantane, Naphthalene, myristolyl, toluene, biphenyl, cinnamoyl, nitrobenzoyl, toluoyl, furoyl, benzoyl, cyclohexane, norbornane, or Z-capron.To facilitate N-acylation, 1 to 4 glycine residues are It can be present at the N-terminus.

  The C-terminal carboxyl group of the compound may be, for example, without limitation, an amide (ie, the C-terminal hydroxyl group is replaced with —NH 2, —NHR 2 and —NR 2 R 3) or an ester (ie, a C-terminal hydroxyl group). Can be protected with a group comprising: R2 and R3 are optionally independently an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group. In addition, in conjunction with the nitrogen atom, R2 and R3 can optionally form a C4-C8 heterocycle with about 0 to 2 additional heteroatoms such as nitrogen, oxygen or sulfur. Non-limiting, suitable examples of suitable heterocycles include piperidinyl, pyrrolidinyl, morpholino, thiomorpholino or piperazinyl. Examples of C-terminal protecting groups include, but are not limited to, -NH2, -NHCH3, -N (CH3) 2, -NH (ethyl), -N (ethyl) 2, -N (methyl) (ethyl),- NH (benzyl), -N (C1-C4 alkyl) (benzyl), -NH (phenyl), -N (C1-C4 alkyl) (phenyl), -OCH3, -O- (ethyl)-, -O- ( n-propyl), -O- (n-butyl), -O- (isopropyl), -O- (sec-butyl), -O- (t-butyl), -O-benzyl, and -O-phenyl. Can be mentioned.

Substitution with Peptidomimetic Sites “Peptidomimetic organic moieties” can optionally be substituted as conservative and non-conservative substitutions for amino acid residues of the compositions of the invention. These sites are also referred to as “unnatural amino acids” and optionally serve as spacer groups within the peptide in place of amino acid residues, amino acid substitutions, or deletion amino acids. Peptidomimetic organic moieties optionally and preferably have the same conformational, electronic or configurational properties as the amino acid to be substituted, and such peptidomimetics are essential positions. Used for amino acid substitutions and is considered a conservative substitution. However, such similarity is not necessarily required. According to a preferred embodiment of the present invention, the one or more peptidomimetics are selected to at least substantially retain physiological activity compared to the native protein described in the present invention.

Peptidomimetics can optionally be used to inhibit the degradation of peptides by enzymatic or other degradation processes. Peptidomimetics can optionally and preferably be generated by organic synthesis techniques. Non-limiting examples of suitable peptidomimetics include the corresponding L amino acid, D amino acid,
Tetrazole (Zabrocki et al., J. Am. Chem. Soc. 110: 5875-5880 (1988)), an amide bond isostere (Jones et al, Tetrahedron Lett. 29: 3853-3856 (1988)), LL -3-amino-2-propenidone-6-carboxylic acid (LL-Acp) (Kemp et al., J. Org. Chem. 50: 5834-5838 (1985)). Similar analogs are described in Kemp et al. Tetrahedron Lett. 29: 5081-5082 (1988), and Kemp et al. Tetrahedron Lett. 29: 5057-5060 (1988), Kemp et al. Tetrahedron Lett. 29: 4935-4938 (1988) and Kemp et al. , J .; Org. Chem. 54: 109-115 (1987). Other suitable but typical peptidomimetics are described by Nagai and Sato, Tetrahedron Lett. 26: 647-650 (1985); Di Maio et al. , J .; Chem. Soc. Perkin Trans. 1687 (1985); Kahn et al. Tetrahedron Lett. 30: 2317 (1989); Olson et al. , J .; Am. Chem. Soc. 112: 323-333 (1990); Garvey et al. , J .; Org. Chem. 56: 436 (1990). In addition, suitable typical peptidomimetics include hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al., J. Takeda Res. Labs 43: 53-76 (1989). )), 1,2,3,4-tetrahydro-isoquinoline-3-carboxylate (Kazmierski et al., J. Am. Chem. Soc. 133: 2275-2283 (1991)), histidine isonaphthoquinonecarboxylic acid ( (HIC) (Zechel et al., Int. J. Pep. Protein Res. 43 (1991)), (2S, 3S) -methyl-phenylalanine, (2S, 3R) -methyl-phenylalanine), (2R, 3S) -Methyl-phenylalanine, and (2R, 3 ) - methyl - phenyl - alanine (. Kazmierski and Hruby, Tetrahedron Lett (1991)) and the like.

  Typical, descriptive, non-limiting unnatural amino acids are beta amino acids (beta 3 and beta 2), homoamino acids, cyclic amino acids, aromatic amino acids, Pro and Pyr derivatives, 3-substituted alanine derivatives, glycine derivatives, rings Substituted Phe and Tyr derivatives, linear core amino acids or diamino acids. For example, various suppliers such as Sigma-Aldrich (USA) are available.

Chemical Modification In the present invention, any part of the protein of the present invention can optionally be chemically modified, i.e. altered by the addition of functional groups. For example, side chain amino acid residues that appear in the native sequence can be optionally modified, although other parts of the protein can optionally be modified in addition to or in place of side chain amino acid residues as described below. Modifications can optionally be made during the synthesis of the molecule, for example, by adding chemically modified amino acids when the chemical synthesis process continues. However, chemical modifications of amino acids are also possible ("in situ" modifications) if already present in the molecule.

  Any amino acid in the sequence region of the molecule is optionally modified (in peptides that can be considered conceptually as “chemical modifications”), optionally by any one of the following typical types of modifications: be able to. Non-limiting, typical types of modifications include carboxymethylation, acylation, phosphorylation, glycosylation or fatty acylation. The ether linkage can optionally be used to link a serine or threonine hydroxy group to a sugar hydroxy group. An amide bond can optionally be used to link the carboxyl group of glutamic acid or aspartic acid to the amino group of the sugar. (Garg and Jeanloz, Advances in Carbohydrate Chemistry and Biochemistry, Vol. 43, Academic Press (1985); Kunz, Ang. Chem. Int. Ed. It can be formed between amino acids and carbohydrates. Fatty acyl derivatives can optionally be made, for example, by acylation of a free amino group (eg lysine). (Toth et al., Peptides: Chemistry, Structure and Biology, Riviera and Marshal, eds., ESCOM Publ., Leiden, 1078-1079 (1990)).

  As used herein, when referring to a protein or peptide described in the present invention, the term “chemical alteration” means that at least one amino acid residue is a natural process such as processing or other post-translational modification, or Refers to a protein or peptide that is modified by chemical modification techniques well known in the art. Examples of numerous known modifications include, but are not limited to, typically acetylation, acylation, amidation, ADP ribosylation, glycosylation, GPI anchor formation, covalent addition of lipids or lipid derivatives, methyl , Myristoylation, pegylation, prenylation, phosphorylation, ubiquitination or any similar process.

  Other types of modifications optionally include the addition of cycloalkanes to biomolecules such as proteins, as described in PCT application number WO2006 / 050262, which is hereby incorporated by reference in its entirety. Can be. These moieties are designed for use with biomolecules and can optionally be used to confer a variety of properties to the protein.

  Further, optionally, any part of the protein can be modified. For example, PEGylation of glycosylation sites on proteins can optionally be performed as described in PCT Application No. WO 2006/050247, which is incorporated herein by reference in its entirety. One or more polyethylene glycol (PEG) groups can optionally be added to O-linked and / or N-linked glycosylation. The PEG group can optionally be branched or linear. Optionally, any type of water soluble polymer can be added through a glycosyl linker to a glycosylation site on the protein.

Modified Glycosylation Proteins of the invention can be modified to have a modified glycosylation pattern (ie modified from the original or native glycosylation pattern). As used herein, “modified” means that one or more hydrocarbon sites are deleted and / or at least one glycosylation site is added to the original protein.

  Protein glycosylation is typically either N-linked or O-linked. N-linked refers to the addition of a hydrocarbon moiety to the side chain of an asparagine residue. The tripeptide sequences, asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for the enzymatic addition of hydrocarbons to the asparagine side chain. is there. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a site that can undergo glycosylation. O-linked glycosylation refers to the addition of one of the sugars of N-acetylgalactosamine, galactose or xylose to one of the hydroxy amino acids, most commonly serine or threonine. However, 5-hydroxyproline or 5-hydroxylysine may be used.

  Addition of glycosylation sites to the proteins of the present invention is conveniently done to include one or more of the above tripeptide sequences (relative to N-linked glycosylation sites) by altering the amino acid sequence of the protein. Achieved. Modifications can also be made by addition or substitution of one or more serine or threonine residues in the original protein sequence (relative to the O-linked glycosylation site). The amino acid sequence of a protein can also be altered by introducing changes at the DNA level.

  Another means of increasing the number of hydrocarbon moieties on the protein is by chemically or enzymatically linking glycosides to the amino acid residues of the protein. Depending on the coupling mode used, the sugar may be unbound (free) such as (a) arginine and histidine, (b) unbound (free) carboxyl group, (c) cysteine sulfhydryl group. ) A sulfhydryl group, (d) an unattached (free) hydroxyl group such as the hydroxyl group of serine, threonine or hydroxyproline (e) an aromatic residue such as a phenylalanine, tyrosine or tryptophan aromatic residue, or (f) It can be added to the amide group of glutamine. These methods are described in WO 87/05330 and Aplin and Wriston, CRC Crit. Rev. Biochem. 22: 259-306 (1981).

  Removal of any hydrocarbon present on the protein of the invention is accomplished chemically or enzymatically. Chemical deglycosylation requires exposure to trifluoromethanesulfonic acid or an equivalent compound. This treatment results in cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine) leaving the amino acid sequence intact.

  Chemical deglycosylation is described by Hakimuddin et al. , Arch. Biochem. Biophys. , 259: 52 (1987); and Edge et al. , Anal. Biochem. 118: 131 (1981). Enzymatic cleavage of hydrocarbon sites on proteins is described by Totakura et al. , Meth. Enzymol. 138: 350 (1987), which can be achieved by using various endoglycosidases and exoglycosidases.

Methods of Treatment As described above, VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 protein, or VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 protein and polypeptide of the present invention, or a nucleic acid sequence or fragment thereof, particularly , VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 protein extracellular region or secreted form and VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 protein and / or splice variant and / or VSIG1, ILDR1, LOC25 of other sites (moiies) 012, AI216611, C1ORF32, agents that agonize or antagonize binding to FXYD3 protein and / or splice variants, and / or modulate (activate or modulate) at least one VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3-related biological activity Antagonizing) agents (such agents include, by way of example, antibodies, small molecules, peptides, ribozymes, antisense molecules, siRNA, etc.), but are not limited to acute lymphocytic leukemia, chronic lymphocytic leukemia, Acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, breast, prostate, lung, ovary, colon, spleen, kidney, bladder, head and neck, uterus, testis, stomach, neck, liver , Including cancer of bone, skin, pancreas, brain It can be used to treat cancers including but not limited to non-solid and solid tumors, sarcomas, hematological malignancies and can be non-metastatic, invasive or metastatic.

  VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 protein or VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 protein and polypeptide of the present invention, or nucleic acid sequences or fragments thereof, in particular VSIG1, ILDR1, LOC21612, , Extracellular region or secreted form of FXYD3 protein, and VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, agents that specifically bind to FXYD3 protein and / or splice variants, and / or other sites, VSIG1, ILDR1 , LOC253012, AI216611, C1ORF32, FXY Agents that agonize or antagonize binding to three proteins and / or agents that modulate (actuate or antagonize) at least one VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3-related biological activity (examples of such agents include: As well as antibodies, small molecules, peptides, ribozymes, antisense molecules, siRNA, etc.) are further included, but are not limited to non-immune disorders such as autoimmune diseases, transplant rejection and transplanted host diseases. It can be used to treat malignant disorders and / or to inhibit or promote immune co-stimulation mediated by VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 polypeptides.

  Thus, according to additional aspects of the invention, but not limited to, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, Non solid and non-solid Cancers such as solid tumors, sarcomas, hematologic malignancies, and can include non-metastatic, invasive or metastatic cancers, and include but are not limited to autoimmune diseases, transplant rejection and graft host disease VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 to treat non-malignant disorders such as immunodeficiency diseases and / or in a subject Methods for inhibiting or promoting immune costimulation mediated by Ripepuchido is provided.

  The subject described in the present invention is a mammal and is preferably diagnosed with one of the above diseases, disorders or conditions, or alternatively, but not limited to acute lymphocytic leukemia, chronic lymphocytic leukemia Acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, breast, prostate, lung, ovary, colon, spleen, kidney, bladder, head and neck, uterus, testis, stomach, neck, Cancers including, but not limited to, non-solid and solid tumors, sarcomas, hematologic malignancies, including but not limited to liver, bone, skin, pancreas, brain cancer, and autoimmune diseases, transplant rejection and transplantation A human who is susceptible to at least one type of non-malignant disorder such as an immunodeficiency disorder including unilateral host disease.

  As used herein, the term “treat” prevents, corrects, converts, reduces, alleviates, minimizes, inhibits or stops the harmful effects of the above-mentioned diseases, disorders or conditions. Refers to that.

  According to the present invention, treatment can be performed in a subject by specifically enhancing the expression of at least one polypeptide of the present invention.

  Optionally, upregulation is performed by administering to the subject at least one of the (eg, recombinant or synthetic) polypeptides of the invention, or an active portion thereof, as described herein. obtain. However, since the bioavailability of large polypeptides can be relatively small due to the high degradation and low penetration rates, administration of polypeptides is preferably small peptide fragments (eg, about 100 Amino acids). The polypeptide or peptide can optionally be administered as part of a pharmaceutical composition, as described in more detail below.

  The treatment according to the invention of the above diseases can be combined with other treatment methods known to those skilled in the art (ie combination treatment). Thus, it will be appreciated that treatment of malignant tumors using the agents of the present invention can be combined with, for example, radiation therapy, antibody therapy, and / or chemotherapy.

  Alternatively or additionally, the method of upregulating optionally optionally specifically increasing at least one amount (optionally expressed) of the polypeptide of the invention or an active part thereof in the subject. Can be executed by

  As described above and in the Examples section that follows, the biomolecular sequence of this aspect of the invention is that the altered activity or expression of the wild-type gene product (known protein) is associated with the onset or progression of a disease, disorder or condition. Can be used as valuable therapeutic tools in the treatment of diseases, disorders or conditions known to contribute to For example, if the disease is caused by overexpression of a membrane-bound receptor, the soluble variant can be used as an antagonist. Antagonists compete with the receptor for ligand binding, thereby terminating signaling from the receptor.

Anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, anti-FXYD3 antibodies Specific germline sequences, homologous antibodies, antibodies with conservative modifications, processed and modified antibodies The antibodies of the invention comprising are characterized by unique properties or properties. For example, the antibody specifically binds to human VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3. Preferably, an antibody of the invention has a high affinity for the corresponding VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3, for example a KD of 10 ⁻⁸ M or less, a KD of 10 ⁻⁹ M or less, or 10 ⁻ Even KD below ¹⁰ M binds. The anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibody of the present invention preferably exhibits one or more of the following properties.

(I) binds to the corresponding human VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 with a KD of 5 × 10 ⁻⁸ M or less,

  (Ii) modulate (enhance or inhibit) and / or modulate related activities or functions such as B7 immune co-stimulation and anti-tumor and autoimmune T cell responses

  (Iii) binds to VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigens expressed by cancer cells including, for example, lung cancer, ovarian cancer, colon cancer, but does not substantially bind to normal cells. In addition, preferably these antibodies and conjugates thereof will be effective to induce selective killing of such cancer cells and to modulate autoimmunity and cancer related immune responses.

More preferably, the antibody, the corresponding human VSIGl, ILDRl, LOC253012, AI216611, C1ORF32 or FXYD3 antigen, at 3X10 ^-8 M following KD, or 1X10 ^-9 M or less of KD, 0.1Xl0 ^-9 M or less KD, or 0.05. It binds with a KD of X10 ⁻⁹ M or less, or with a KD between 1 × 10 ⁻⁹ and 1 × 10 ⁻¹¹ M.

  Standard assays for assessing the ability of an antibody to bind to VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 are known in the art and include, for example, ELISA, Western blot and RIA. Suitable assays are described in detail in the examples. The binding kinetics (eg, binding affinity) of an antibody can also be assessed by standard assays known in the art, such as by Biacore analysis.

  In generating anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibody, the sequence from the antibody can bind to VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3. VH and VL sequences can be “mixed and matched” to create other anti-VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 binding molecules of the invention. Such “mixed and matched” antibodies VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 binding can be tested using the above binding assays such as, for example, ELISA. Preferably, when the VH and VL chains are mixed and matched, the VH sequences from a particular VH / VL pair are replaced with structurally similar VH sequences. Similarly, preferably VL sequences from a particular VH / VL pair are replaced with structurally similar VL sequences. For example, the VH and VL sequences of homologous antibodies are particularly suitable for mixing and matching.

Antibodies with Specific Germline Sequences In certain embodiments, the antibodies of the present invention comprise light chain variable regions from specific germline heavy chain immunoglobulin genes and / or light chain from germline light chain immunoglobulin genes. Contains the chain variable region.

  As used herein, a human antibody is a heavy or light chain that is “product” or “derived from” a particular germline sequence when the variable region of the antibody is obtained from a system that uses human germline immunoglobulin genes. Includes variable regions. Such systems include immunizing transgenic mice carrying human immunoglobulin genes with the antigen of interest, or screening a human immunoglobulin gene library displayed on phage with the antigen of interest. A human antibody “derived from” or “derived from” a human germline immunoglobulin sequence is sequenced by comparing the amino acid sequence of the human antibody with the amino acid sequence of the human germline immunoglobulin, and in the sequence of the human antibody. And so on, such as by selecting the human germline immunoglobulin sequence that is closest (ie, the maximum% identity).

  A specific human germline immunoglobulin sequence "product" or "derived from" a human antibody may, for example, be a natural somatic mutant cell mutation or a site-specific mutation intention compared to a germline sequence. Including amino acid differences due to general introduction. However, the selected human antibody is typically at least 90% identical to the amino acid sequence encoded by the human germline immunoglobulin gene and the germline immunoglobulin amino acid sequence of other species ( For example, it includes amino acid residues that identify a human antibody as being human when compared to the mouse germline sequence. In certain cases, the human antibody has at least 95, 96, 97, 98 or 99% amino acid sequence, or at least 96%, 97%, 98%, or amino acid sequence encoded by a germline immunoglobulin gene. It can be 99% identical. Typically, a human antibody derived from a particular human germline sequence will show no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In some cases, human antibodies will show no more than 5 amino acid differences, or no more than 4, 3, 2, or 1 amino acid sequence from the amino acid sequence encoded by the human germline immunoglobulin gene.

Homologous antibodies In yet another embodiment, the antibody of the invention retains the desired functional properties of the parent anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibody. Amino acids homologous to the isolated anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 amino acid sequences of the preferred anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibody, respectively Includes heavy and light chain variable regions containing sequences.

  As used herein, the percent identity between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences. (Ie,% identity = number of identical positions / total number of positions × 100) (considering the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences). Comparison and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

  The percent identity between the two amino acid sequences was determined by the E.I. Meyers and W.M. Using the Miller algorithm, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be determined. (Comput. Appl. Biosci., 4: 11-17 (1988)) In addition, the percent identity between two amino acid sequences is Needleman and Wunsch, which is incorporated into the GAP program in the GCG software package (commercially available). (J. MoI. Biol. 48: 444-453 (1970)) algorithm, using the Blossum 62 matrix (matrix) or PAM250 matrix (matrix), and gap weights 16, 14, 12, 10, 8 , 6, or 4 and length weight 1, 2, 3, 4, 5, or 6.

  Additionally or alternatively, the protein sequences of the present invention can further be used as “query sequences” that perform, for example, public database searches to identify related sequences. Such a search is described in Altschul, et al. (1990) J MoI. Biol. 215: 403-10. The XBLAST program (version 2.0) can be used. The BLAST protein search is executed by the XBLAST program, score = 50, wordlength = 3, and an amino acid sequence homologous to the antibody of the present invention can be obtained. To obtain a gapped alignment for comparison purposes, Altschul et al. (1997) Nucleic Acids Res. 25 (17): 3389-3402, Gapped BLAST can be used. When using BLAST and Gapped BLAST programs, the default parameters of each program (eg, XBLAST and NBLAST) can be used.

Antibodies with Conservative Modifications In certain embodiments, an antibody of the invention comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, one These CDR sequences above have been isolated and generated using the methods of the present invention, preferred anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, or anti-FXYD3 antibodies, or conservative modifications thereof. Wherein the antibody is an anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXY of the present invention, respectively. Retain the desired functional properties of the D3 antibody.

  In various embodiments, the anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibody can be a human antibody, a humanized antibody or a chimeric antibody.

  As used herein, “conservative sequence modifications” are intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of antibodies, including amino acid sequences. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, Tyrosine, cysteine, tryptophan), nonpolar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta branched side chains (eg threonine, valine, isoleucine) and aromatic side chains (eg Amino acids having tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of the antibodies of the invention can be replaced with other amino acid residues from the same side chain family, and the modified antibody has a retained function. With respect to (ie, the functions described in (c) to (j) above), it can be tested using the functional assay described herein.

Antibodies that bind to the same epitope as the anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibodies of the present invention In another embodiment, the present invention may be used to modulate desired functions, such as modulation of B7 costimulation and related functions. Antibodies that bind to a preferred epitope on human VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 with functional properties are provided. Other antibodies with the desired epitope specificity will be selected and will have the ability to cross-compete with the desired antibody for binding to the VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen. .

Processed and Modified Antibodies The antibodies of the present invention may further be based on antibodies having one or more of VH and / or VL sequences derived from anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibodies. Can be used to process the modified antibody, which can have altered properties from the starting antibody. An antibody can be modified by modifying one or more residues in one or both variable regions (ie, VH and / or VL), eg, one or more CDR regions and / or one or more framework regions. Can be processed. In addition or alternatively, antibodies can be engineered, for example, by altering residues in the constant region to alter the effector functions of the antibody.

  One type of variable region processing that can be performed is CDR grafting. Antibodies interact with target antigens primarily through amino acid residues located in six heavy and light chain complementary regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Since CDR sequences are largely involved in antibody-antigen interactions, by constructing an expression vector containing CDR sequences from specific natural antibodies grafted onto framework sequences from different antibodies with different properties It is possible to express a recombinant antibody that mimics the properties of the antibody. (See, eg, Riechmann, L. et al. (1998) Nature 332: 323-327; Jones, P. et al. (1986) Nature 321: 522-525; Queen, C. et al. (1989) Proc. Natl. Acad. See USA 86: 10029-10033, US Patent No. 5,225,539 (Winter), and US Patent Nos. 5,530,101, 5,585,089, (See 5,693,762 and 6,180,370 (Queen et al.).)

  Suitable framework sequences can be obtained from public databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the “VBase” human germline sequence database (available on the Internet), as well as Kabat, E., et al. A. , Et al. (1991) Sequences of Proteins of Immunological Interest, First Edition, U.S.A. S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I .; M.M. , Et al. (1992) "The Repertoire of Human Germline VH Sequences Reviews About Fifty Groups of VH Segments with Different Hyperloops." MoI. Biol. 227: 776-798; and Cox, J. et al. P. L. et al. (1994) "A Directory of Human Germ-line VH Segments Reveals a Strong Bias in the Usage" Eur. J Immunol. 24: 827-836. The contents of each are specifically incorporated herein by reference.

  Another type of variable region modification is to mutate amino acid residues within the VH and / or VL CDR1, CDR2 and / or CDR3 regions, thereby altering one or more binding properties of the subject antibody (eg, affinity ). To introduce mutations, site-directed mutagenesis or PCR-mediated mutagenesis can be performed. The effect of antibody binding or other target functional properties can be assessed in a suitable in vitro or in vivo assay. Preferably, conservative modifications (above) are introduced. The mutation can be an amino acid substitution, addition or deletion, but is preferably a substitution. Furthermore, typically only 1, 2, 3, 4 or 5 residues are modified within the CDR regions.

  The engineered antibodies of the invention include modifications made to framework residues within VH and / or VL, for example, to improve antibody properties. Typically, such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach “backmutates” one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence derived from the antibody. Such residues can be identified by comparing antibody framework sequences to germline sequences derived from the antibody.

  Typically, serum half-life, complement binding, Fc receptor binding and / or antigen-dependent cytotoxicity to include modifications in the Fc region in addition to or in place of modifications made in the framework or CDR regions The antibodies of the invention can be engineered to modify the functional properties of one or more antibodies such as Furthermore, the antibodies of the invention can be chemically modified (eg, one or more chemical moieties can be added to the antibody) or altered glycosylation, one or more The functional properties of these antibodies can be modified again. Such embodiments are further described below. The numbering of residues in the Fc region is that of Kabat's EU index.

  In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered (eg, increased or decreased). This approach is further described in US Pat. No. 5,677,425 (by Bodmer et al). The number of cysteine residues in the CH1 hinge region is altered, for example, to facilitate assembly of light and heavy chains, or to increase or decrease antibody stability.

  In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half life of the antibody. More specifically, the Fc- such that one or more amino acid mutations have impaired SpA binding compared to the native Fc-hinge region Staphylococcal protein A (SpA) binding. Introduced in the CH2-CH3 domain interface region of the hinge fragment, this approach is described in more detail in US Patent No. 6,165,745 (by Ward et al).

  In another embodiment, the antibody is modified to increase its biological half life. Various approaches are possible. For example, one or more of the following mutations can be introduced as described in US Pat. No. 6,277,375 (Ward): T252L, T254S, T256F. Alternatively, to increase the biological half-life, the antibody may be an IgG Fc region, as described in US Pat. Nos. 5,869,046 and 6,121,022 (Presta et al.). Can be altered within the CH1 or CL region to include a salvage receptor that binds to an epitope taken from the two loops of the CH2 region.

  In yet another embodiment, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 have altered affinity for the antibody to the effector, but the parent antibody's It can be substituted with a different amino acid residue so as to retain the antigen binding ability. An effector ligand with altered affinity for it can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in US Pat. Nos. 5,624,821 and 5,648,260 (both by Winter et al).

  In another example, the one or more amino acids selected from amino acid residues 329, 331 and 322 such that the antibody has altered C1q binding and / or reduced or eliminated complement dependent cytotoxicity (CDC), Can be substituted with different amino acid residues. This approach is described in further detail in US Pat. No. 6,194,551 (by Idusogie et al.).

  In another example, one or more amino acid residues at amino acid positions 231 and 239 are altered, thereby altering complement binding ability. This approach is described in further detail in PCT Publication WO 94/29351 (by Bodmer et al.).

  In yet another example, the Fc region modifies one or more amino acids in the next position to increase the ability of the antibody to mediate antibody-dependent cytotoxicity (ADCC) and / or the affinity of the antibody for the Fey receptor. Modified by: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289 290,292,293,294,295,296,298,301,303,305,307,309,312,315,320,322,324,326,327,329,330,331,333,334,335 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 3 8,414,416,419,430,434,435,437,438 or 439. This approach is described in further detail in PCT Publication WO 00/42072 (by Presta). Furthermore, the binding sites on human IgG1 of Fc grammar, Fc gamma RII, Fc gamma RIII and FcRn have been mapped and variants with improved binding have been described. (See Shields, RL et al. (2001) J. Biol. Chem. 276: 6591-6604.) Certain mutations at positions 256, 290, 298, 333, 334 and 339 are directed to FcyRIII. It has been shown to improve the coupling. In addition, the following combination mutants have been shown to improve Fc gamma RIII binding: T256A / S298A, S298A / E333A, S298A / K224A and S298A / E333A / K334A.

  In yet another embodiment, the glycosylation of the antibody is altered. For example, non-glucosylated antibodies (ie, antibodies that lack glycosylation) can be made. Glycosylation can be altered, for example, to increase the affinity of the antibody for antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that would eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such non-glycosylated can increase the affinity of the antibody for antigen. Such an approach is described in further detail in US Pat. Nos. 5,714,350 and 6,350,861 (by Co et al.).

  Additionally or alternatively, antibodies with altered types of glycosylation can be made, such as low fucose antibodies with reduced amounts of fucose residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such hydrocarbon modification can be accomplished, for example, by expressing the antibody in a host cell that has a modified glycosylation mechanism. Cells with altered glycosylation mechanisms have been described in the art and can be used as host cells to express the recombinant antibodies of the invention and thereby produce antibodies with altered glycosylation. . For example, cell lines Ms704, Ms705, and Ms709 lack the fucose transferase gene, FUT8 (alpha (1,6) fucose transferase), and antibodies expressed in Ms704, Ms705, and Ms709 cell lines add fucose to their carbohydrates. Lack. The Ms704, Ms705 and Ms709 FUT8 − / − cell lines are created by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors. (See U.S. Patent Publication No. 20040110704 (by Yamane et al.) And Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87: 614-22). As another example, European Patent No. 1,176,195 (According to Hanai et al.) Describe a cell line with a FUT8 gene encoding a functionally disrupted fucose transferase. Antibodies expressed in such cell lines show hypofucosylation by reducing or eliminating alpha 1,6 binding related enzymes. Hanai et al. Also, for example, a cell line that has low or no enzyme activity to add fucose to N-acetylglucosamine that binds to the Fc region of an antibody, rat myeloma cell line YB2 / 0 (ATCC CRL 1662) Is described. PCT Publication WO 03/035835 (according to Presta) reduces the ability to add fucose to Asn (297) -linked carbohydrate, resulting in hypofucosylation of the antibody expressed in the host cell. , Lecl3 cells are described. (See also Shields, RL et al. (2002) J. Biol. Chem. 277: 26733-26740) PCT Publication WO 99/54342 (by Umana et al.) Is a glycoprotein modified sugar. Describes a cell line engineered to express a transferase (eg, beta (1,4) -N-acetylglucosamine transferase III (GnTIII)), and the antibody expressed in the engineered cell line is: It exhibits an increased branched GlcNac structure, resulting in increased ADCC activity of the antibody. (See also Umana et al. (1999) Nat. Biotech. 17: 176-180.) Alternatively, the fucose residues of the antibody can be cleaved using fucosidases. For example, the fucosidase alpha-L-fucosidase enzyme removes fucosyl residues from antibodies (Tarentino, AL et al. (1975) Biochem. 14: 5516-23).

  Another modification of the antibodies herein that is contemplated by the present invention is pegylation. An antibody can be PEGylated, for example, increasing the biological (eg, serum) half life of the antibody. To PEGylate an antibody, the antibody or fragment thereof is typically a polyethylene glycol, such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are added to the antibody or antibody fragment. Reacted with (PEG). Preferably, pegylation is carried out through an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a reactive water-soluble polymer analog). As used herein, the term “polyethylene glycol” refers to PEG that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. It is intended to include any form of In certain embodiments, the PEGylated antibody is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See, for example, European Patent No. 0154316 (by Nishimura et al.) And European Patent No. 0401384 (by Ishikawa et al.).

Methods for processing antibodies As described above, the anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibodies having VH and VK sequences disclosed in the present invention are VH and / or VL, respectively. By modifying the sequence or the constant region added to it, it can be used to create new anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibodies. Thus, in another aspect of the invention, the structural features of the anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibodies of the present invention include, for example, human VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 Or used to create structurally related anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibodies that retain at least one functional property of the antibodies of the invention, such as binding to each of FXYD3 It is done. For example, as described above, one VSIG1, ILDR1, LOC253012, to generate additional recombinant engineered anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibodies of the present invention. One or more CDR regions of an AI216611, C1ORF32 or FXYD3 antibody or variant thereof can be recombinantly combined with known framework regions and / or other CDRs. Other types of modifications include those described in previous sections. The starting material for the processing method is one or more VH and / or VK sequences prepared herein, or one or more CDR regions thereof. The need to actually prepare (ie, express as a protein) an antibody with one or more of the VH and / or VK sequences provided herein, or one or more of its CDR regions, to create a engineered antibody. There is no. Rather, the information contained in the sequence is used as a starting material for creating a “second generation” sequence derived from the original sequence.

  Standard molecular biology techniques can be varied and used to prepare and express antibody sequences.

  Preferably, the antibody encoded by the modified antibody sequence is produced by one, some or all of the methods given herein and has an anti-VSIG1, It retains the functional properties of each of ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibody. Its functional properties include binding to VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen and / or modulation of B7 costimulation and / or VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or below a specific KD level It includes selective binding to desired target cells such as lung cancer, ovarian cancer, colon cancer, etc. that express FXYD3 antigen.

  The functional properties of the antibody can be assessed using standard assays available in the art and / or described herein.

  In certain embodiments of the methods of processing an antibody of the invention, the mutation is random or all or part of the sequence encoding the anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, or anti-FXYD3 antibody. The resulting modified anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibody can be selectively screened for binding ability and / or other desired functional properties. Can be.

  Mutation methods have been described in the art. For example, PCT Publication WO 02/092780 (by Short) describes a method for creating and selecting antibody mutations using saturation mutagenesis, synthetic ligation assembly or combinations thereof. Instead, PCT Publication WO 03/074679 (by Lazar et al.) Describes a method for optimizing the physiochemical properties of antibodies using a computational sorting method.

Another aspect of the present invention relates to a nucleic acid molecule that encodes an antibody. The nucleic acid can be present in whole cells, cell lysates or in partially purified or substantially pure form. Nucleic acids may be produced by standard techniques such as, for example, alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art, “Isolated” or “rendered substantively pure” when purified to remove other impurities. F. Ausubel, et al. , Ed. (1987) Current Protocols in Molecular Biology, Green Publishing and Wiley Interscience, New York. The nucleic acids of the present invention can be, for example, DNA or RNA and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

  The nucleic acids of the invention can be obtained using standard molecular biology techniques. For an antibody expressed by a hybridoma (eg, a hybridoma prepared from a transgenic mouse containing a human immunoglobulin gene, as described further below), it encodes the light and heavy chains of the antibody produced by the hybridoma. cDNA can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (eg, using phage display technology), the nucleic acid encoding the antibody can be recovered from the library.

  Once DNA fragments encoding the VH and VL sections are obtained, these DNA fragments can be expressed by standard recombination, for example, to convert variable region genes to full-length antibody chain genes, Fab fragment genes or scFv genes. It can be further manipulated by DNA technology. In these manipulations, a DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein such as an antibody constant region or a flexible linker.

  As used in this context, the term “operably linked” means that the two DNA fragments are ligated so that the amino acid sequence encoded by the two DNA fragments remains in-frame. Is meant to mean that.

  The isolated DNA encoding the VH region is operably linked to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3). Can be converted into a full-length heavy chain gene. The sequence of the human heavy chain constant region gene is known in the art (see, for example, Kabat, EA, el al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department). (See Human Services, NIH Publication No. 91-3242), DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. However, most preferred is an IgG1 or IgG4 constant region. With respect to the Fab fragment heavy chain gene, the DNA encoding VH can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

  The isolated DNA encoding the VL region is ligated to another DNA molecule encoding the light chain constant region CL operably linked to the DNA encoding the VL (full length heavy). Can be converted to a chain gene (as well as a Fab light chain gene). The sequence of the human light chain constant region gene is known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Institute, Fifth Edition, US Departmention). (See Human Services, NIH Publication No. 91-3242), DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, most preferably a kappa constant region

  To create the scFv gene, DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker, eg, encoding the amino acid sequence (Gly4-Ser) 3. Operatively linked, VH and VL sequences can be expressed as a continuous single chain protein with VL and VH regions linked by a linker. (See, eg, Bird et al. (1988) Science 242: 423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al., (1990) Nature: (See 552-554)

Generation of Anti-VSIG1, Anti-ILDR1, Anti-LOC253012, Anti-AI216611, Anti-C1ORF32, or Anti-FXYD3 Monoclonal Antibodies of the Invention Monoclonal antibodies (mAbs) of the invention can be prepared, for example, by the standard of Kohler and Milstein (1975) Nature 256: 495. It can be produced by a variety of techniques including conventional monoclonal antibody methodology, such as somatic cell hybridization techniques. Somatic cell hybridization procedures are preferred in principle, but other techniques for generating monoclonal antibodies can be employed, such as virus or tumorigenic transformation of B lymphocytes.

  A preferred animal system for preparing hybridomas is the murine system. Hybridoma production in mice is a well established procedure. Immunization protocols and techniques for isolating spleen cells (immunized splenocytes) immunized for fusion are known in the art. Partners to fuse (eg, mouse myeloma cells) and fusion procedures are also known.

  The chimeric or humanized antibody of the present invention can be prepared based on the sequence of a mouse monoclonal antibody prepared as described above. DNA encoding heavy and light chain immunoglobulins is obtained from a subject mouse hybridoma using standard molecular biology techniques and processed to contain non-mouse (eg, human) immunoglobulin sequences. be able to. For example, to make a chimeric antibody, a mouse variable region can be linked to a human constant region using methods known in the art. (See, eg, US Pat. No. 4,816,567 (Cabilly et al.).) To make humanized antibodies, the murine CDR regions can be isolated from human frames using methods known in the art. Can be inserted into the work area. (For example, U.S. Pat. Nos. 5,225,539 (Winter) and U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370 ( (See Queen et al.).)

  In a preferred embodiment, the antibodies of the present invention are human monoclonal antibodies. Such human monoclonal antibodies against VSIG1 can be generated using transgenic or transchromosomal mice with part of the human immune system rather than the mouse system. These transgenic or transchromosomal mice are mice referred to herein as HuMAb Mouse RTM and KM Mouse RTM, respectively, and are collectively referred to herein as “human Ig mice”. Together with targeted mutations that inactivate endogenous mu and kappa chain loci (see, eg, Lonberg, et al. (1994) Nature 368 (6474): 856-859), HuMAb Mouse TM. (Medarex. Inc.) contains the human immunoglobulin gene minilocus that encodes unrearranged human heavy chain (mu and gamma) and kappa light chain immunoglobulin sequences. Thus, mice show reduced expression of murine IgM or kappa, and in response to immunization, the introduced human heavy and light chain transgenes undergo high-affinity humans via class switching and somatic mutation. Generate IgG kappa monoclonal. (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113: 49-101; Lonberg, N. ar. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. NY Acad. Sci. 764: 536-546). The preparation and utilization of HuMab Mouse RTM and the genomic modifications performed in the mouse are described in Taylor, L., et al. et al. (1992) Nucleic Acids Research 20: 6287-6295; Chen, J. et al. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3720-3724; Choi et al. (1993) Nature Genetics 4: 117-123; Chen, J. et al. et al. (1993) EMBO J. et al. 12: 821-830; Tuaillon et al. (1994) J. Org. Immunol. 152: 2912-2920; Taylor, L .; et al. (1994) International Immunology 6: 579-591; and Fishwild, D .; et al. (1996) Nature Biotechnology 14: 845-851, the entire contents of which are specifically incorporated herein by reference in their entirety. Further, U.S. Pat. Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397. No. 5,661,016, No. 5,814,318, No. 5,874,299 and No. 5,770,429 (all by Lonberg and Kay), US Pat. No. 5,545,807. (Surani et al.), PCT Publication Nos. WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884, and WO 99/45662 (all by Lonberg and Kay) and PCT Publication No. See WO01 / 14424 (Korman et al.).

  In another embodiment, human antibodies of the invention can be made using mice with human immunoglobulin sequences on the transgene and transchromosome. Such mice, referred to herein as “KM Mouse ™”, such as mice with human heavy chain transgenes and human light chain transchromosomes, are described in detail in PCT Publication WO 02/43478 (Ishida et al). ing.

  In addition, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to make anti-VSIG1 antibodies of the invention. For example, an alternative transgenic line called Xenomouse (Abgenix, Inc.) can be used, such as US Pat. Nos. 5,939,598, 6,075, 181, 6,114,598, 6,150,584 and 6,162,963 (Kucherlapati et al.).

  In addition, alternative transchromosomal animal systems that express human immunoglobulin genes are available in the art and can be used for anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibodies of the present invention. Can be used to create. For example, a mouse having both a human heavy chain transchromosome and a human light chain transchromosome, referred to as a “TC mouse,” can be used, and such a mouse is described in Tomizuka et al. (2000) Proc. Natl. Acad Sci. USA 97: 722-727. In addition, bovines with human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al. (2002) Nature Biotechnology 20: 889-894) to generate anti-VSIG1 antibodies of the invention. Can be used.

  Human monoclonal antibodies of the invention can also be prepared using phage display methods that screen libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are established in the art. For example, U.S. Pat. Nos. 5,223,409, 5,403,484, and 5,571,698 (Ladner et al.), U.S. Pat. Nos. 5,427,908 and 5,580, 717 (Dower et al.), US Pat. Nos. 5,969,108 and 6,172,197 (McCafferty et al.), And US Pat. Nos. 5,885,793, 6,521,404. No. 6,544,731, 6,555,313, 6,582,915 and 6,593,081 (Griffiths et al.).

  Human monoclonal antibodies of the invention can also be prepared using SCID mice in which human immune cells have been reconstituted so that a human antibody response can be generated upon immunization. Such mice are described, for example, in US Pat. Nos. 5,476,996 and 5,698,767 (Wilson et al.).

Immunization of human IG mice Lonberg, N .; et al. (1994) Nature 368 (6474): 856-859; Fishwild, D .; et al. (1996) Nature Biotechnology 14: 845-851; and PCT Publications WO 98/24884 and WO 01/14424, such as when human Ig mice are used to make human antibodies of the invention. Mice can be VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen, and / or recombinant VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3, or VSIG1, ILDR1, LOC253012, X2RF311F, X216RF1, C2 The prepared preparation can be used to immunize. Preferably, the mice are 6-16 weeks of age upon the first infusion. For example, purified or recombinant preparations (5-50 mu.g) of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen can be used to immunize human Ig mice intraperitoneally. .

Previous experience with various antigens by other people is that first IP immunization with antigen in complete Freund's adjuvant followed by IP immunization weekly with antigen in incomplete Freund's adjuvant. Transgenic mice have been shown to respond when inoculated (up to a total of 6). However, adjuvants other than Freund have also proven effective. Furthermore, it has been found that whole cells in the absence of adjuvant have high immunogenicity.
The immune response can be observed during an immunization protocol in which plasma samples are obtained by retroorbital bleeds. Plasma can be screened by ELISA (as described below) and mice with sufficient titration concentrations of anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 human immunoglobulin are used for fusion. Can be done. Mice can be boosted with antigen intravenously 3 days before sacrifice and removal of the spleen. It is anticipated that 2-3 fusions for each immunization may need to be performed. 6 to 24 mice are typically immunized for each antigen. Usually both HCo7 and HCol2 strains are used. Furthermore, HCo7 and HCol2 transgenes can be bred into a single mouse with two different human heavy chain transgenes (HCo7 / HCo12). Alternatively or additionally, the KM mouse RTM strain can be used.

Generation of hybridomas producing human monoclonal antibodies of the invention To produce hybridomas producing human monoclonal antibodies of the invention, splenocytes and / or lymph node cells can be isolated from immunized mice, It can be fused to a suitable immortal cell line such as a mouse myeloma cell line. The resulting hybridomas can be screened for production of antigen-specific antibodies. For example, a single cell suspension of splenic lymphocytes from an immunized mouse can contain one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) and 50% PEG Can be fused. Cells are seeded at approximately 2 × 10 ⁻⁵ in flat bottom microplates, followed by 20% fetal clonal serum, 18% “653” conditioned medium, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units / ml penicillin, 50 mg / ml streptomycin, 50 mg / ml gentamicin and IX HAT (Sigma, HAT added 24 hours after fusion) Incubate in selective medium containing 2 weeks. After about 2 weeks, the cells can be cultured in medium in which HAT is replaced with HT. Individual wells are then sorted by ELISA for human monoclonal IgM and IgG antibodies. Once vigorous hybridoma growth occurs, the medium can usually be observed after 10-14 days. Hybridomas that secrete antibodies can be reseeded and screened again. If still positive for human IgG, the monoclonal antibody can be subcloned by limiting dilution at least twice. Stable subclones can then be cultured in vitro, producing small amounts of antibody in tissue culture medium for characterization.

  To purify human monoclonal antibodies, selected hybridomas can be cultured in 2 liter spinner flasks for monoclonal antibody purification. The supernatant can be filtered and concentrated prior to affinity chromatography with protein A-Sepharose (Pharmacia, Piscataway, NJ). The eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to confirm purity. The buffer can be exchanged into PBS and the enrichment can be measured by OD280 using an extinction coefficient of 1.43. Monoclonal antibodies can be aliquoted and stored at -80 ° C.

Generation of Transfectomas that Produce Monoclonal Antibodies of the Invention The antibodies of the invention can also be used in host cells using, for example, a combination of recombinant DNA technology and gene transfection methods, as is well known in the art. It can be generated with a transfectoma. (For example, Morrison, S. (1985) Science 229: 1202).

  For example, to express an antibody or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (eg, PCR amplification using a hybridoma expressing the antibody of interest or cDNA cloning) and DNA can be inserted into an expression vector such that the gene is operably linked to transcriptional and translational regulatory sequences. In this context, the term “operably linked” means that the antibody gene has the intended function that the transcriptional and translational regulatory sequences in the vector control the transcription and translation of the antibody gene. Is meant to be bound to The expression vector and expression control sequences are chosen to suit the expression host cell used. Antibody light chain genes and antibody heavy chain genes can be inserted into separate vectors or more typically both genes in the same expression vector. The antibody gene is inserted into the expression vector by standard methods (eg, complementary restriction site ligation of antibody gene fragments and vector, or blunt end ligation if no restriction sites are present). The light and heavy chain variable regions of the antibodies described herein are operably linked to the VH segment to the CH segment in the vector, and the VK segment is the CL segment in the vector. A full-length antibody gene of any antibody isotype by inserting them into an expression vector that already encodes the heavy and light chain constant regions of the desired isotype so that they are operably linked to Can be used to create Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).

  In addition to antibody chain genes, the recombinant expression vectors of the invention have regulatory sequences that control the expression of antibody chain genes in host cells. The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). For example, one skilled in the art will appreciate that the design of an expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, etc. . Preferred regulatory sequences for mammalian host cell expression include cytomegalovirus (CMV), simian virus 40 (simian virus 40), adenovirus (eg, the adenovirus major rate promoter (AdMLP) and polyoma virus). Viral factors that direct high level protein expression in mammalian cells such as derived promoters and / or enhancers. Alternatively, non-viral regulatory sequences such as a ubiquitin promoter or beta globin promoter can be used. In addition, regulatory elements (Takebe, Y., et al.) Composed of sequences from different sources, such as the SRalpha promoter system, including sequences from the SV40 early promoter and the human T cell leukemia virus type 1 terminal repeat. et al. (1988) Mol. Cell. Biol. 8: 466-472).

  In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may have additional sequences such as sequences that regulate replication of the vector in host cells (eg, origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced. (Eg, US Pat. Nos. 4,399,216, 4,634,665, and 5,179,017 (all by Axel et al.)) For example, typically the selectable marker gene is A host cell into which the vector has been introduced confers resistance to drugs such as G418, hygromycin or methotrexate. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

  For light and heavy chain expression, expression vectors encoding heavy and light chains are transfected into host cells by standard techniques. Various forms of the term “transfection” include various techniques commonly used for introducing foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE dextran transfection, etc. Is intended to include While it is theoretically possible to express an antibody of the invention in either a prokaryotic or eukaryotic host cell, expression of the antibody in a eukaryotic cell, most preferably a mammalian host cell, is most preferred. This is because eukaryotic cells, and particularly mammalian cells, are more likely to fold and assemble and secrete immunologically active antibodies than prokaryotic cells. Prokaryotic expression of antibody genes has been reported to have no effect on high yield production of active antibodies (Boss, MA and Wood, CR (1985) Immunology Today. 6: 12-13).

  Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese hamster ovary cells (as described in R. J. Kaufman and PA Sharp (1982) MoI. Biol. 159: 601-621). CHO cells as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220), NSO myeloma cells, COS cells and SP2 cells. Another preferred expression system, particularly for use with NSO myeloma cells, is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When an antibody gene encoding a recombinant expression vector is introduced into a mammalian host cell, the antibody can be expressed in the host cell or, more preferably, by secretion of the antibody into the medium in which the host cell is cultured. Produced by culturing host cells for a sufficient period of time. Antibodies can be recovered from the culture medium using standard protein purification methods.

Characterization of Antibodies that Bind Antigen Antibodies of the invention can be tested for binding to VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3, for example, by standard ELISA. Briefly, microplates are 0.25. mu. Coat with g / ml of purified VSIG1 and then block with 5% bovine serum albumin in phosphate buffered saline. Antibody dilutions (eg, dilutions of plasma from mice immunized with VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3) are added to each well and incubated at 37 ° C. for 1-2 hours . The plate is washed with PBS / Tween and then at 37 ° C. using a second reagent conjugated to alkaline phosphatase (eg, a polyclonal antibody specific for human antibodies, goat anti-human IgG Fc). Incubate for 1 hour. After washing, the plate is developed with pNPP substrate (1 mg / ml) and analyzed at an OD of 405-650. Preferably, the mouse that develops the highest titer will be used for the fusion.

  The ELISA assay described above can also be used to screen for hybridomas that show positive reactivity with VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 immunogens. Hybridomas that bind to VSIG1 with high avidity are subcloned and further characterized. One clone from each hybridoma, which retains the parental cell reactivity (by ELISA), is selected for 5-10 vial cell banking and antibody purification stored at -140 ° C. be able to.

  To purify anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibodies, the selected hybridomas can be cultured in 2 liter spinner flasks for monoclonal antibody purification. The supernatant can be filtered and concentrated prior to affinity chromatography with protein A-Sepharose (Pharmacia, Piscataway, NJ). The eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to confirm purity. The buffer can be exchanged into PBS and the enrichment can be measured by OD280 using an extinction coefficient of 1.43. Monoclonal antibodies can be aliquoted and stored at -80 ° C.

  To determine if C1ORF32 or anti-FXYD3 monoclonal antibody binds to a unique epitope, the antibody can be biotinylated using commercially available reagents (Pierce, Rockford, Ill.), Respectively. Competition studies using unlabeled and biotinylated monoclonal antibodies can be performed using ELISA plates coated with VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 as described above. Biotinylated mAb binding can be detected with a strept-avidin-alkaline phosphatase probe.

  To determine the isotype of the purified antibody, an isotype ELISA can be performed using reagents specific for the antibody of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, the wells of a microplate are treated with anti-human immunoglobulin l. mu. Can be coated overnight at g / ml. After blocking with 1% BSA, the plates are reacted with 1 μg / ml or less of the tested monoclonal antibody or purified isotype control at ambient temperature for 1-2 hours. An alkaline phosphatase-conjugated probe specific for either human IgG1 or human IgM can then be reacted. Plates are developed and analyzed as described above.

  Anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 human IgG can be further tested for reactivity against VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen by Western blot, respectively. . Briefly, VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigen is transferred to a nitrocellulose membrane, blocked with 10% fetal calf serum, and probed with the monoclonal antibody to be tested. Human IgG binding can be detected using anti-human IgG alkaline phosphatase and developed using BCIP / NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).

Conjugates or immunoconjugates The present invention relates to conjugates for use in immunotherapy comprising a VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen and soluble portions thereof including extracellular regions or portions, or variants thereof. including. For example, the invention includes conjugates in which an ECD of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen is added to an immunoglobulin or fragment thereof. The present invention is used to promote or inhibit VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen activity, such as immune co-stimulation, and for the treatment of transplantation, autoimmunity and cancer indications described herein. Consider that.

In another aspect, the invention includes an anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibody or fragment thereof, such as a cytotoxin, drug (eg, immunosuppressant) or radioactive toxin Features immunoconjugates conjugated to a site for therapeutic treatment. Such conjugates are referred to herein as “immunoconjugates”. Immunoconjugates that contain one or more cytotoxins are called “immunotoxins”. A cytotoxin or cytotoxic agent includes any agent that is detrimental to (eg, kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mitramycin, Examples include mycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof.
Examples of therapeutic agents include antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildacarbazine), alkylating agents (eg, mechloretamine, thioepa, chlorambutyl, melphalan, karma Stin (BSNU) and Lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (eg, daunorubicin (formerly daunomycin) ) And doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mitramycin and anthramycin (AMC)), Antimitotic agents (e.g., vincristine and vinblastine).

  Other preferred examples of therapeutic cytotoxins that can be conjugated to the antibodies of the present invention include duocarmycins, calicheamicins, maytansines and auristatins. ), And derivatives thereof. An example of a calicheamicin antibody conjugate is commercially available. (Mylotarg.TM ™, Wyeth)

  Cytotoxins can be conjugated to the antibodies of the present invention using linker technology available in the art. Examples of linker types used to conjugate cytotoxins to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide-containing linkers. Linkers that are weak to resolution by low pH within the lysosomal compartment, or weak to resolution by proteases such as proteases that are differentially expressed in tumor tissues such as cathepsins (eg, cathepsins B, C, D) may be selected, for example Can be done.

  Further discussion of cytotoxin types, linkers and methods for conjugating therapeutic agents to antibodies can also be found in Saito, G. et al. et al. (2003) Adv. Drug Deliv. Rev. 55: 199-215; Trail, P.M. A. et al. (2003) Cancer Immunol. Immunother. 52: 328-337; Payne, G .; (2003) Cancer Cell 3: 207-212; Allen, T .; M.M. (2002) Nat. Rev. Cancer 2: 750-763; Pastan, I .; and Kreitman, R.A. J. et al. (2002) Curr. Opin. Investig. Drugs 3: 1089-1091; D. And Springer, C.I. J. et al. (2001) Adv. Drug Deliv. Rev. 53: 247-264.

  The antibodies of the present invention can also be conjugated to a radioisotope, producing a cytotoxic radiopharmaceutical called a radioimmunoconjugate. Radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine 131, indium 111, yttrium 90, and lutetium 177. Methods for preparing radioimmunoconjugates are established in the art. Zevalin. TM ™ (IDEC Pharmaceuticals) and Bexxar. Examples of radioimmunoconjugates containing TM ™ are commercially available. Similar methods can also be used to prepare radioimmunoconjugates using the antibodies of the present invention.

  The antibody conjugates of the invention can be used to modify a given biological response and the drug moiety should not be construed as limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide having the desired biological activity. Such proteins include, for example, enzymatically active toxins such as abrin, ricin A, Pseudomonas exotoxin or diphtheria toxin or active fragments thereof, proteins such as tumor necrosis factor or interferon gamma, or, for example, lymphokines , Interleukin 1 (“IL-1”), interleukin 2 (“IL-2”), interleukin 6 (“IL-6”), granulocyte-macrophage colony stimulating factor (“GM-CSF”), granulocytes Biological response modifiers such as colony stimulating factor ("G-CSF") or other growth factors may be mentioned.

  Techniques for conjugating such therapeutic sites to antibodies are well known, see, eg, Arnon et al. , "Monoclonal Antibodies For Immunization Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Theory. (Eds.), Pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al. "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (Eds.), Pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers of Cytotoxic Agents in Cancer Therapies: A Review”, in Monoclonal Aid. (Eds.), Pp. 475-506 (1985); “Analysis, Results, And Future Prospective of The Therapeutic Use of Radiolabeled Anti-Candal Therapeutic”. (Eds.), Pp. 303-16 (Academic Press 1985), and Thorpe et al. "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62: 119-58 (1982).

Bispecific Molecules In another aspect, the invention features a bispecific molecule comprising an anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibody of the invention, or a fragment thereof. (Features). In that regard, an antibody of the invention or antigen-binding portion thereof can be derivatized, or another functional molecule such as, for example, another peptide or protein (eg, another antibody or ligand for a receptor). It produces a bispecific molecule that binds to at least two different binding sites or target molecules. The antibodies of the invention can in fact be derivatized or operably linked to two or more functionalities to generate multispecific molecules that bind to three or more different binding sites and / or target molecules. obtain. Such multispecific molecules are also intended to be encompassed herein by the term “bispecific molecule”. To create a bispecific molecule of the invention, an antibody of the invention can be conjugated to one or more other binding molecules such as another antibody, antibody fragment, peptide or binding mimetic (eg, a chemical cup). Rings, gene fusions, non-covalent linkages, or the like) can be operably linked to result in bispecific molecules.

  Accordingly, the present invention includes bispecific molecules comprising at least a first binding specificity for VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 and a second binding specificity for a second target epitope. In a particular embodiment of the invention is a second target epitope Fc receptor such as, for example, human Fc gamma RI (CD64) or human Fc alpha receptor (CD89). Thus, the present invention relates to effector cells (eg monocytes, macrophages or polymorphonuclear cells (PMN)) that both express Fc gamma R, Fc alpha R or Fc epsilon R, and VSIG1, ILDR1, LOC253012, Bispecific molecules that can bind to both target cells expressing AI216611, C1ORF32 or FXYD3 are included. These bispecific molecules are cell effector cells that express VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 and phagocytosis of cells expressing VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3, antibody dependent cells Induces Fc receptor-mediated effector cell activity such as mediated cytotoxicity (ADCC), cytokine release or superoxide anion generation.

  In embodiments of the invention in which the bispecific molecule is multispecific, the molecule can further comprise a third binding specificity in addition to the anti-Fc binding specificity and the anti-6f binding specificity. . In one embodiment, the third binding specificity is, for example, an anti-enhancement factor such as a molecule that binds to a surface protein involved in cytotoxic activity, thereby increasing the immune response against the target cell. ) Part.

  An “anti-enhancement factor moiety” binds to a given molecule, eg, an antigen or receptor, thereby enhancing the effect of a binding determinant on an Fc receptor or target cell antigen. It can be an antibody, a functional antibody fragment or a ligand. An “anti-enhancement factor moiety” can bind to an Fc receptor or a target cell antigen. Alternatively, the anti-enhancement factor moiety can bind to an entity that is different from the entity to which the first and second binding specificities bind. The anti-enhancement factor moiety is responsible for cytotoxic T cells (eg, CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1, or other that results in an increased immune response to target cells. Can bind through immune cells).

  In one embodiment, as described in US Pat. No. 4,946,778 (Ladner et al.), The contents of which are expressly incorporated by reference, both specific molecules of the invention have binding specificity. For example, Fab, Fab ′, F (ab ′). sub. At least one antibody, or antibody fragment, such as 2, Fv, or single chain Fv. Antibodies can also be Fv or single chain constructs, light chains or

In one embodiment, the binding specificity for the Fey receptor is provided by a monoclonal antibody and the binding is not hindered by human immunoglobulin G (IgG). As used herein, the term “IgG receptor” refers to any of the eight gamma chain genes present in chromosome 1. These genes encode a total of 12 transmembrane or soluble receptor isoforms, which are grouped into three Fc gamma receptor classes: Fc gamma Rl (CD64), Fc gamma RII (CD32) , And Fc gamma RIII (CD16).
In one preferred embodiment, the Fc gamma receptor human high affinity Fc gamma RI. Human Fc gamma RI is a 72 kDa molecule that exhibits high affinity for monomeric IgG (10 ⁸ -10 ^-9 M ^-1 ).

  The production and characterization of certain preferred anti-Fc gamma monoclonal antibodies is described by Fanger et al. PCT Publication WO 88/00052 and US Pat. No. 4,954,617, the teachings of which are incorporated herein by reference in their entirety. These antibodies bind to an epitope of Fc gamma Rl, FcyRII, or FcyRIII at a site that is different from the Fc gamma binding site of the receptor. The binding is not substantially hindered by physiological levels of IgG. Specific anti-Fc gamma RI antibodies useful in the present invention are mAb22, mAb32, mAb44, mAb62 and mAb197. Hybridomas producing mAb32 are available from the American Type Culture Collection (ATCC Accession No. HB9469). In other embodiments, the anti-Fey receptor antibody is a humanized form of monoclonal antibody 22 (H22). Production and characterization of the H22 antibody is described in Graziano, R .; F. et al. (1995) J. MoI. Immunol. 155 (10): 4996-5002 and PCT Publication WO 94/10332. A cell line that acidifies the H22 antibody has been deposited with the American Type Culture Collection under HAO22CLI and is accession number CRL11177.

  In yet another preferred embodiment, the binding specificity for the Fc receptor is provided by an antibody that binds to a human IgA receptor. For example, Fc-alpha receptor (Fc alpha RI (CD89)), its binding is preferably not prevented by human immunoglobulin (IgA). The term “IgA receptor” is intended to include the gene product of one alpha-gene on chromosome 19 (Fc alpha RI). This gene is known to encode several alternatively spliced transmembrane isoforms of 55-10 kDa.

Fc alpha RI (CD89) is constitutively expressed on monocytes / macrophages, eosin and neutrophil granulocytes, but not on non-effector cell populations. Fc alpha RI has media affinity (approximately 5 × 10 ⁻⁷ M ⁻¹ ) for both IgA1 and IgA2, which is increased upon contact with cytokines such as G-CSF or GM-CSF (Morton, H C. et al. (1996) Critical Reviews in Immunology 16: 423-440). Four FcARI specific monoclonal antibodies have been described that bind to FcalphaRI outside the IgA ligand binding region, identified as A3, A59, A62 and A77 (Monteiro, RC et al. 1992) J. Immunol. 148: 1764).

  Fc alpha RI and Fc gamma RI are preferred trigger receptors for use in the bispecific molecules of the invention. Because (1) expressed primarily in immune effector cells (eg monocytes, PMN, macrophages, dendritic cells), (2) expressed at high levels (eg 5,000-100,000 per cell) (3) is a mediator of cytotoxic activity (eg ADCC, phagocytosis) and (4) mediates enhanced antigen presentation of targeted antigens such as self-antigens.

  While human monoclonal antibodies are preferred, other antibodies that can be employed in the bispecific molecules of the invention are mouse, chimeric and humanized monoclonal antibodies.

The bispecific molecules of the invention can be synthesized using methods known in the art, such as anti-FcR and anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 binding specificities. It can be prepared by conjugating the constitutive binding specifications. For example, each binding specificity of a bispecific molecule can be generated individually and then conjugated to each other. If the binding specificity is a protein or peptide, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), o-phenylene dimaleimide (oPDM),
N-succinimidyl-3- (2-pyridyldithio) propionate (N-succinimidyl-3- (2-pyridyl-thio) propionate) (SPDP), and succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate ( sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate) (sulfo-SMCC). (See, eg, Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82: 8648) and others. As a method, Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229: 81-83) and Glennie et al. (1987) J. Am. Immunol. 139: 2367-2375). Preferred conjugating agents are SATA and sulfo-SMCC, both of which are Pierce Chemical Co. (Rockford, Ill.).

  If the binding specificity is an antibody, they can be conjugated through a sulfhydryl bond in the C-terminal hinge region of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to include an odd number of sulfhydryl residues, preferably one, before conjugation.

Alternatively, both binding specificities can be encoded and expressed in the same vector and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAbXmAb, mAbXFab, FabXF (ab ′) 2 or ligand XFab fusion protein. The bispecific molecule of the present invention is a single chain molecule containing one single chain antibody and a binding determinant, or a single chain bispecific molecule containing two binding determinants Can do.
Bispecific molecules can include at least two single chain molecules. Methods for preparing bispecific molecules are described, for example, in US Pat. No. 5,260,203, US Pat. No. 5,455,030, US Pat. No. 4,881,175, US Pat. 405, US Pat. No. 5,091,513, US Pat. No. 5,476,786, US Pat. No. 5,013,653, US Pat. No. 5,258,498, and US Pat. No. 5,482. 858.

  Binding of bispecific molecules to specific targets can be achieved, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (eg, growth inhibition), or Western blot assay. Can be confirmed. Each of these assays generally detects the presence of a protein-antibody complex of interest, in particular, using a labeled reagent (eg, an antibody) specific for the complex of interest. For example, FcR-antibody complexes can be detected using, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to the antibody-FcR complex. Alternatively, the complex can be detected using any of a variety of other immunoassays. For example, antibodies can be radiolabeled and used in radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventeen Training Coin on Radiotechnology and Assay Assays, USA). Incorporated herein))) Radioisotopes can be detected by means of using a gamma counter or a scintillation counter, or by autoradiography.

Pharmaceutical Compositions In another aspect, the invention provides a composition (eg, pharmaceutical) comprising one or a combination of monoclonal antibodies of the invention or antigen-binding portion thereof formulated with a pharmaceutically acceptable carrier. A composition). Such compositions can include one or (eg, two or more different) antibodies or immunoconjugates of the invention or a combination of both specific molecules. For example, a pharmaceutical composition of the invention can comprise a combination of antibodies (or immunoconjugates or bispecific molecules) that bind to or have complementary activities to different epitopes on the target antigen.

  As previously discussed, VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 of the present invention specifically binds to the activity elicited by VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen and / or, respectively. Or identifying other molecules such as small organic molecules, peptides, ribozymes, carbohydrates, glycoproteins, siRNA, antisense RNA that modulate (enhance or inhibit). These molecules can be identified by known screening methods such as binding assays. In order to identify putative drug candidates that bind and / or modulate VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3-related activity, these assays are typically high-throughput, synthetic or natural compounds. Would be screened for large libraries.

  Specifically, the present invention includes the development of drugs comprising the corresponding nucleic acid sequence encoding the extracellular region of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen, or fragments or variants thereof. These conjugates can include targets such as immunoglobulin regions or other sites. These conjugates are expressed in known vector systems or vectors containing nucleic acid sequences corresponding to cells or nucleic acid sequences and are used in cancer therapy and immunotherapy, such as in the treatment of autoimmunity, transplantation, GVHD, cancer and immunodeficiency diseases or conditions. Can be used for

  Accordingly, the present invention features a pharmaceutical composition comprising a therapeutically effective amount of the therapeutic agent described in the present invention. According to the present invention, the therapeutic agent may be any one of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 extracellular region, or a fragment or variant thereof, or the corresponding nucleic acid sequence encoding. I will.

  The pharmaceutical composition according to the present invention is more preferably, but not limited to, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin Includes lymphoma, breast, prostate, lung, ovary, colon, spleen, kidney, bladder, head and neck, uterus, testicles, stomach, neck, liver, bone, skin, pancreas, brain cancer, including non-solid and solid tumors It is preferably further used for the treatment of cancers, including sarcomas, hematological malignancies, which can be non-metastatic, invasive or metastatic cancers.

  The pharmaceutical composition according to the invention is further used for the treatment of autoimmunity and is associated with multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, transplant rejection Immunodeficiency, benign lymphocyte vasculitis, lupus erythematosus, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin-dependent diabetes mellitus, Goodpasture's syndrome, severe muscle Asthenia, pemphigus, sympathetic ophthalmitis, autoimmune uveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic hepatitis, ulcerative colitis, Sjogren's syndrome , Rheumatic disease, polymyositis, scleroderma, mixed connective tissue disease, inflammatory rheumatism, degenerative rheumatism, non-articular rheumatism, collagen disease, chronic polymorphism Inflammation, arthritic psoriasis, ankylosing spondylitis, juvenile rheumatoid arthritis, shoulder arthritis, nodular polyarteritis, progressive systemic sclerosis, urate arthritis, dermatomyositis, rheumatoid muscle, myositis, muscle stiffness It is used for the treatment of autoimmune diseases selected from diseases and cartilage mineralization.

  The pharmaceutical composition according to the present invention is preferably used for the treatment of any host transplant disease that may develop after any transplant rejection and / or bone marrow transplant.

  “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder must be prevented. Thus, as used herein, a mammal being treated may have been diagnosed as having or prone to a disorder or susceptible to a disorder. “Mammals” for therapeutic purposes include, for example, humans, domestic animals and farm animals, and mammals, including zoo animals, sport animals, pet animals, such as dogs, horses, cats, cows, etc. Refers to any animal classified as an animal. Preferably the mammal is a human.

  The term “therapeutically effective amount” refers to an amount of an agent according to the present invention effective to treat a disease or disorder in a mammal.

  The therapeutic agents of the invention can be given to a subject alone or as part of a pharmaceutical composition mixed with a pharmacologically acceptable carrier.

  The pharmaceutical compositions of the invention can also be administered in combination therapy, ie in combination with other drugs. For example, the combination treatment is an anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibody, or VSIG1, ILDR1 according to the present invention, in combination with at least one other treatment or immunomodulator. , LOC253012, AI216611, C1ORF32 or the extracellular region of FXYD3 antigen or soluble polypeptides conjugates including small molecules such as peptides, ribozymes, siRNA or other drugs that bind to VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 , VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 modulators. Examples of therapeutic agents that can be used in combination therapy are described in more detail in the section below on the use of antibodies of the invention.

As used herein, “pharmacologically acceptable carrier” includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and agents that delay absorption, and the like. . Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion). Depending on the route of administration, the active compound, i.e. antibody, immunoconjugate or bispecific molecule, can be coated with a material from the natural state that can inactivate the action of acids and compounds. Protect. The pharmaceutical compounds of the present invention may contain one or more pharmacologically acceptable salts. “Pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesirable toxic effects. (See, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66: 1-19.) Examples of such salts include acid addition salts and base addition salts. It is done. Acid addition salts include non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy Examples include those derived from non-toxic organic acids such as alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids.
Base addition salts include alkaline earth metals such as sodium, potassium, magnesium and calcium, and non-toxic such as N, N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine and procaine And those derived from organic amines.

  The pharmaceutical composition of the present invention may also contain a pharmaceutically acceptable antioxidant. Pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfide, sodium metabisulfite, sodium sulfite, and (2) ascorbyl palmitate, butylhydroxy Oil-soluble antioxidants such as anisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, alpha tocopherol, and (3) citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc. A metal chelating agent is mentioned.

  The pharmaceutical composition of the present invention may also contain a pharmaceutically acceptable antioxidant. Pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfide, sodium metabisulfite, sodium sulfite, and (2) ascorbyl palmitate, butylhydroxy Oil-soluble antioxidants such as anisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, alpha tocopherol, and (3) citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc. A metal chelating agent is mentioned. Examples of suitable aqueous and non-aqueous carriers that may be employed in the pharmaceutical composition of the present invention include water, ethanol, polyhydric alcohols (such as glycerol, propylene glycol, polyethylene glycol) and suitable mixtures thereof, olive oil Vegetable oils such as, and injectable organic esters such as ethyl oleate. For example, suitable fluidity can be maintained by the use of a coating agent such as lecithin, in the case of dispersion, by maintaining the required particle size, and by the use of a surfactant.

  These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured both by pre-sterilization treatment and by inclusion of various antibacterial and antifungal substances (eg, parabens, chlorobutanol, phenol sorbic acid, etc.). It may also be desirable to include isotonic agents such as sugars, sodium chloride in the composition. In addition, prolonged absorption of the injectable dosage form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

  Pharmacologically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmacologically active substances is known in the art. Except insofar as any conventional medium or agent is not compatible with the active compound, its use in the pharmaceutical composition of the present invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

  The therapeutic composition typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyhydric alcohol (eg, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by dispersion, by the maintenance of the required particle size and by the use of surfactants. In many cases, it will be preferable to include isotonic agents such as sugar, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in one or a combination of the ingredients listed above and the required amount in a suitable solvent, followed by sterile microfilament, if necessary. Do a torsion. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is by vacuum drying and lyophilization to yield any additional desired ingredients from the powder of the active ingredient and the previously sterile filtered solution. is there.

  Sterile injectable solutions can be prepared by incorporating the active compound in one or a combination of the ingredients listed above and the required amount in a suitable solvent, followed by sterile microfilament, if necessary. Do a torsion. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is by vacuum drying and lyophilization to yield any additional desired ingredients from the powder of the active ingredient and the previously sterile filtered solution. is there.

  The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect. Generally, in 100 percent, this amount is about 0.01 percent to about 99 percent, preferably about 0.1 percent to about 70 percent, most preferably about 100 percent of the active ingredient in combination with a pharmaceutically acceptable carrier. It ranges from about 1 percent to about 30 percent.

Dosage regimens are adjusted to provide the optimum desired response (eg, a therapeutic effect). For example, a single bolus can be administered, several divided doses can be administered over time, or the dosage can be proportionally, as indicated in the requirements of the treatment situation. ) Can be reduced or increased. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to a physically discrete unit suitable as a single dosage for the patient being treated. Each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmacological carrier. The specification of the dosage unit form of the present invention is for (a) the specific characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the treatment of sensitivity in the individual. , Determined by the limitations inherent in the field of combining such active compounds and depends directly.

  For antibody administration, the dosage is about 0.0001 to 100 mg / kg (host body weight) and more usually 0.01 to 5 mg / kg. For example, the dosage can be in the range of 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight or 10 mg / kg body weight or 1-10 mg / kg. A typical treatment regimen is once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, and once every three to six months. With multiple doses. Preferred dosage regimes for the anti-VSIG1 antibodies of the invention include 1 mg / kg body weight or 3 mg / kg body weight via intravenous administration, and the antibody is given using one of the following dosing schedules: (i ) 6 times every 4 weeks, then every 3 months, (ii) every 3 weeks, (iii) 1 mg / kg body weight once, followed by 1 mg / kg body weight every 3 weeks.

  In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each antibody administered is within the indicated range. Antibodies are usually administered multiple times. The interval between each administration is, for example, every week, month, every three months or year. Intervals can also be irregular if indicated by measuring blood levels of antibodies against the target antigen in the patient. In some methods, dosage is adjusted to a plasma antibody concentration of about 1-1000 mug / ml, and in some methods, about 25-300 mu. g / ml.

  Alternatively, the antibody can be administered as a sustained release agent, in which case less frequent administration is required. Dosage and frequency will vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half life, followed by human antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive lifelong treatment. In therapeutic applications, relatively high doses at relatively short intervals are sometimes used until disease progression is reduced or stopped, preferably until the patient shows partial or complete improvement in disease symptoms. is necessary. Thereafter, the patient can be administered a prophylactic regime.

  The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention is to achieve the desired therapeutic response for a particular patient, composition and mode of administration without being toxic to the patient. Can be varied to obtain an amount of active ingredient that is effective. The selected dose depends on the activity of the particular composition of the invention employed or its ester, salt or amide, route of administration, time of administration, rate of elimination of the particular compound employed, duration of treatment, Other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, status, health status and previous medical history of the patient being treated, and well known in the medical field Will depend on a variety of pharmacokinetic factors, including

A “therapeutically effective amount” of an anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibody of the present invention is preferably a reduction in the severity of disease symptoms, frequency and duration of periods without disease symptoms Result in increased life span, increased life span, disease remission, or prevention of disability or disability due to disease. For example, for the treatment of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 positive tumors, such as lung tumors, ovarian tumors and colon tumors, a “therapeutically effective amount” refers to cell proliferation or Tumor growth is preferably inhibited by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, even more preferably at least about 80%. The ability of the composition to inhibit tumor growth can be evaluated in animal model systems that are predictive of efficacy in human tumors. Alternatively, the properties of the composition can be assessed by testing the ability of the composition to inhibit, in vitro inhibition by assays known to those skilled in the art. A therapeutically effective amount of the therapeutic compound can reduce the size of the tumor, otherwise it can ameliorate symptoms in the subject.
Those skilled in the art will be able to determine the amount based on factors such as the size of the subject's body, the severity of the subject's symptoms, and the particular composition or route of administration selected.

  The compositions of the present invention can be administered through one or more routes of administration using one or more various methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired result. Preferred routes of administration of the antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example, by injection or infusion. As used herein, the phrase “parenteral administration” includes other than enteral and topical administration. Usually means the mode of administration by injection, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transductal, subcutaneous, epidermal, joint Intrainjection, subcapsular, subcapsular, intraspinal, epidural, intrasternal injection and infusion.

  Alternatively, VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen-modulating antibodies or other VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 drugs or molecules and conjugates and combinations thereof according to the present invention For example, intranasally, orally, vaginally, rectally, sublingually or topically, such as topically or via a parenteral route such as the epidermal or mucosal route of administration Can be done.

  The active compound should be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Can do. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are patented or generally known to those skilled in the art. For example, Sustained and Controlled Release Drug Delivery Systems, J. Org. R. Robinson, ed. , Marcel Dekker, Inc. , New York, 1978. checking ...

  The therapeutic composition can be administered with medical devices known in the art. For example, in a preferred embodiment, the therapeutic composition of the present invention comprises US Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, It can be administered with a needle hypodermic injection device such as the devices disclosed in 4,941,880, 4,790,824, or 4,596,556. Examples of implants and modules useful in the present invention include US Pat. No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate, for administering drugs through the skin. U.S. Pat. No. 4,486,194 disclosing a therapeutic device of U.S. Pat. No. 4,447,233 disclosing a drug infusion pump for delivering a drug at a precise infusion rate, continuously with variable flow US Pat. No. 4,447,224 which discloses an implantable injector for drug delivery, US Pat. No. 4,439,196 which discloses an osmotic drug delivery system with a multi-chamber compartment, which is permeable No. 4,475,196, which discloses a drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

  In certain embodiments, the antibodies or other VSIG1-related agents of the invention can be formulated to ensure proper dispersion in vivo. For example, the blood brain barrier (BBB) excludes many highly lipophilic compounds. In order to ensure that the therapeutic compounds of the present invention exceed the BBB (if desired), the compounds can be formulated, for example, in liposomes. See, for example, US Pat. Nos. 4,522,811, 5,374,548, and 5,399,331 for methods of making liposomes. Liposomes can contain one or more sites that are selectively transported to specific cells or organs, thus enhancing targeted drug delivery. (See, for example, V. V. Ranade (1989) J. Clin. Pharmacol. 29: 685). Typical target sites include folic acid or biotin (see, eg, US Pat. No. 5,416,016 (Low et al)), mannoside (Umezawa et al, (1988) Biochem. Biophys. Res. Commun. 153: 1038), antibodies (PG Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180), surfactant protein A Receptor (Briscoe et al. (1995) Am. J Physiol. 1233: 134), pl20 (Schreier et al. (1994) J. Biol. Chem. 269: 9090) (see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346: 123; J. J. Killion; I. J. Fiddler (1994) Immunomethods 4: 273). .

Diagnostic use of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen and corresponding polynucleotides According to some embodiments, a sample taken from a subject (patient) for performing a diagnostic assay according to the present invention is: Blood, serum, urine, plasma, prostate fluid, seminal fluid, semen, skin, respiratory organs, intestinal and genitourinary exocrine secretions, tears, cerebrospinal fluid, sputum, saliva Secretions of breast milk, ascites, pleural fluid, cystic fluid, thoracic system (and / or its lavage), bronchoalveolar lavage, lavage of the genital system, and lavage of other parts of the body or system in the body, A sample of any organ containing isolated cells or tissues, where the cells or tissues include but are not limited to lung, colon, ovary and / Or it may be organ Karae selected from breast tissue possible), selected from the group consisting of bodily fluids or secretions containing bottle or tissue sample, or any combination thereof. In some embodiments, the term includes a sample of in vivo cell culture constituents. Prior to being subjected to a diagnostic assay, the sample can optionally be diluted with a suitable eluant.

  In some embodiments, in the context of the present invention, the phrase “marker” refers to the disease in a sample taken from a patient (subject) having one of the diseases or conditions described herein. Or refers to a nucleic acid fragment, peptide, or polypeptide that is differentially present compared to a comparative sample taken from a subject without one of the pathological conditions.

  In some embodiments, the term “polypeptide” should be understood to refer to a molecule comprising at least 2 to thousands or more amino acids. The term “polypeptide” includes is to be understood to include, in particular, native peptides (either degradation products, synthetic peptides or recombinant peptides), peptidomimetics or peptide analogs such as peptoids and semipeptoids, It should be understood that, for example, any desired modification may be included, including, among other things, making the peptide more stable in the body, penetrating by cells, or other modifications. is there. Such modifications include, but are not limited to, N-terminal modifications, C-terminal modifications, peptide bond modifications, backbone modifications, residue modifications or others. Inclusion of such peptides within the polypeptides of the invention can produce polypeptides that share identity with the polypeptides described herein, such as those provided in the sequence listing.

  In some embodiments, the phrase “differentially present” refers to the amount of marker present in a sample taken from a patient having one of the diseases or conditions described herein, or Refers to the difference in quality compared to a comparative sample taken from a subject who does not have one of the diseases or conditions described herein. For example, if the amount of nucleic acid fragments in one sample differs significantly from the amount of nucleic acid fragments in another sample, as measured, for example, in a hybridization and / or NAT based assay, Optionally, there may be a differential between the two samples. A polypeptide is differentially present between two samples when the amount of polypeptide in one sample is significantly different from the amount of polypeptide in the other sample. Notably, if a marker is detectable in one sample and not detectable in another sample, such a marker can be considered differentially present. Optionally, a relatively low amount of up-regulation can serve as a marker, as described herein. One skilled in the art will readily be able to determine such relative levels of markers. Further guidance is provided in each marker description below.

  In some embodiments, the phrase “diagnostic” means identifying the presence or nature of a disease state. Diagnostic methods differ in their sensitivity and specificity. The “sensitivity” of a diagnostic assay is the percentage of individuals with a disease determined to be positive (the percentage of “true positives”). Individuals with diseases that are not detected by the assay are “false negatives”. A subject who does not have a disease and is determined to be negative in the assay is called “true negative”. The “specificity” of a diagnostic assay is 1 minus the false positive rate. Here, the “false positive” rate is defined as the percentage of humans who are determined to be positive and have no disease. Although a particular diagnostic method may not provide a definitive diagnosis of the condition, it is sufficient if it provides a positive indication to assist in the diagnosis.

In some embodiments, the phrase “qualitative” includes the absence of expression when related to differences in the expression levels of the polynucleotides or polypeptides described herein, as will be appreciated by those skilled in the art. Or, in some embodiments, temporal control of expression, or in some embodiments, the timing of expression, in some embodiments, optional for the expressed molecule. This refers to post-translational modifications. In some embodiments, the phrase “quantitative” refers to a difference in the expression level of a polynucleotide or polypeptide described herein as measured by any means known in the art. , Refers to the absolute difference in the amount of expression. Or, in other embodiments, relative differences that may be substantially significant,
Or, in some embodiments, it shows a trend for differences in expression, such as when viewed over time or over time.

  In some embodiments, the term “diagnose” classifies the disease or condition, determines the severity of the disease, observes the progression of the disease, the outcome of the disease and / or the likelihood of recovery. Refers to expecting. The term “detect” may optionally include any of the above.

  In some embodiments, the diagnosis of a disease described in the present invention can be influenced in a biological sample obtained from a subject by measuring the level of a polynucleotide or polypeptide of the present invention, and the level Can be correlated to the predisposition of the disease or the presence or absence of the disease. It should be noted that, as described in more detail below, a “biological sample obtained from a subject” can also optionally include a sample that has not been physically removed from the subject.

  In some embodiments, the term “level” refers to the expression level of RNA and / or protein or the DNA copy number of a marker of the invention.

  Typically, the level of a marker in a biological sample obtained from a subject is different (ie, increased or decreased) from the level of the same marker in a similar sample obtained from a healthy individual. (Examples of biological samples are described herein.)

  In order to measure the level of DNA, RNA and / or polypeptide of a subject marker in a subject, a number of well known methods of collecting tissue or body fluids can be used to ingest a biological sample from the subject.

  Examples include, but are not limited to, fine needle biopsy, needle biopsy, core needle biopsy, and surgical biopsy (eg, brain biopsy) and lavage. Regardless of the procedure employed, once a biopsy / sample is obtained, the level of the marker can be measured and thus a diagnosis can be made.

  Measuring the level of the same marker in normal tissue of the same origin is preferably in parallel to detect elevated and / or amplified and / or decreased expression of the marker relative to normal tissue Executed.

  In some embodiments, the term “test amount” of a marker refers to the amount of marker in a subject sample that is consistent with the diagnosis of a particular disease or condition. The test amount can be either an absolute amount (eg, microgram / ml) or a relative amount (eg, the relative strength of the signal).

  In some embodiments, the term “control amount” of a marker can be any amount or range of amounts to be compared to a test amount of the marker. For example, a control amount of a marker can be the amount of marker in a patient with a particular disease or condition, or in a person who does not have such a disease or condition. A control amount can be either an absolute amount (eg, microgram / ml) or a relative amount (eg, the relative intensity of the signal).

  In some embodiments, the term “detection” refers to identifying the presence or absence or amount of an object to be detected.

  In some embodiments, the term “level” includes any moiety or item detectable by spectroscopic, photochemical, biochemical, immunochemical or chemical means. For example, useful labels include 32P, 35S, fluorescent dyes, electron-dense reagents, enzymes (eg, commonly used in ELISA) biotin-streptavidin, digoxigenin, hapten And nucleic acid molecules having a sequence complementary to the protein (if there is an antiserum or monoclonal antibody to the protein) or target. The label often produces a measurable signal, such as a radioactive, chromogenic or fluorescent signal, which can be used to quantify the amount of label bound to the sample. The label is incorporated or added to the primer or probe, either covalently or through ionic, van der Waals or hydrogen bonding, eg, incorporation of a biotinylated nucleotide recognized by a radioactive nucleotide or streptavidin. be able to. The label may be detectable directly or indirectly. Indirect detection involves the direct or indirect binding of the second label to the first label. For example, the label can be a ligand of a binding partner such as biotin, which is a binding partner of streptavidin, or a nucleotide sequence that is a binding partner of a complementary sequence and specifically hybridizes to it. Can be. The binding partner itself may be directly detectable. For example, the antibody itself can be labeled with a fluorescent molecule. The binding partner may also be indirectly detectable. For example, a nucleic acid having a complementary nucleotide sequence can be part of a branched DNA molecule that can be sequentially detected through hybridization with other labeled nucleic acid molecules. (See, for example, PD Fahrlander and A. Klausner, Bio / Technology 6: 1165 (1988).) Signal quantification is accomplished by scintillation counting, densitometry or flow cytometry.

  Typical detectable labels used with immunoassays are optionally or preferably, but not limited to, magnetic beads, fluorescent dyes, radioactive labels, enzymes (eg, horseradish peroxide, alkaline phosphatase and generally Others used in ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic beads. Alternatively, the marker in the sample may be an indirect assay in which, for example, a second, labeled antibody is used to detect an antibody specific for the marker bound, and / or, for example, a different epitope of the marker Monoclonal antibodies that bind to can be detected in competition or inhibition assays where the mixture is incubated simultaneously with the mixture.

  An “immunoassay” is an assay that uses an antibody that specifically binds to an antigen. An immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and / or quantify the antibody.

  The phrase “specifically (or selectively) binds” to an antibody, or “specifically (or selectively) immunoreacts” or “specifically interacts or binds” to a protein or peptide In referring to (or other epitopes), in some embodiments, it refers to a binding reaction that is determinative of the presence of a protein in a population of proteins and other biologics heterologous. Thus, under specified immunoassay conditions, a particular antibody binds to a particular protein at least twice as much as the background (non-specific signal) and is substantially significant to other proteins present in the sample. Do not combine by quantity. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, a polyclonal antibody raised against a semen basic protein from a particular species such as rat, mouse, or human can be selected and specifically immunoreacts with the semen basic protein. However, only polyclonal antibodies that do not specifically immunoreact with other proteins except polymorphic variants and sperm basic protein alleles are obtained. This selection may be achieved by removing antibodies that cross-react with semen elementary protein molecules from other species. A variety of immunoassay formats can be used to select antibodies that specifically immunoreact with a particular protein. For example, solid phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with proteins. (E.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay format and conditions that are used as used.) At least twice background signal or noise, more typically 10 to 100 times background.

  In another embodiment, the present invention provides a method for detecting a polypeptide of the present invention in a biological sample. The method includes contacting a biological sample with an antibody that specifically recognizes a polypeptide according to the present invention and detecting the interaction, wherein the presence of the interaction is determined by the polypeptide in the biological sample. Correlate with the presence of

  In some embodiments of the invention, the polypeptides described herein are non-limiting examples of markers for diagnosing disease and / or indicia conditions. Each marker of the present invention is diverse, including but not limited to prognosis, prediction, screening, early diagnosis, determination of progression, treatment selection and treatment observation of the disease and / or suggestive condition. It can be used alone or in combination for the application.

  For related purposes, the disease detected is not limited to acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma Non-solid and solid tumors, including breast, prostate, lung, ovary, colon, spleen, kidney, bladder, head and neck, uterus, testicles, stomach, neck, liver, bone, skin, pancreas, brain cancer, sarcoma, blood Cancers that can be non-metastatic, invasive or metastatic, such as malignant tumors, may be mentioned.

  For another related purpose, the detected diseases include multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, immunodeficiency associated with transplant rejection, benign lymphocyte blood vessels Inflammation, lupus erythematosus, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin-dependent diabetes, Goodpascher's syndrome, myasthenia gravis, pemphigus, sympathetic Ophthalmitis, autoimmune uveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic hepatitis, ulcerative colitis, Sjogren's syndrome, rheumatic disease, polymyositis , Scleroderma, mixed connective tissue disease, inflammatory rheumatism, degenerative rheumatism, non-articular rheumatism, collagen disease, chronic polyarthritis, arthritic psoriasis, ankylosing From the group consisting of spondylitis, juvenile rheumatoid arthritis, peri-shoulderitis, polyarteritis nodosa, progressive systemic sclerosis, urate arthritis, dermatomyositis, rheumatoid muscle, myositis, myopathy and cartilage calcification Selected autoimmune and neoplastic disorders may be mentioned.

  For another related purpose, the detected disease would include any living transplant rejection and / or graft host disease.

  Each polypeptide / polynucleotide of the present invention is not limited as described above, but includes prognosis, prediction, screening, early diagnosis, determination of progression, treatment selection and disease and / or indication conditions. ) Can be used alone or in combination for a variety of uses.

  Such combinations may optionally include combinations that feature any sub-combination of markers and / or at least one other marker such as, for example, a known marker. Further, such combinations are optionally and preferably as described above for quantitative or semi-quantitative measurement of any marker described herein and other markers and / or described herein. Used to measure the ratio between any other known marker and / or any other marker.

  According to further embodiments of the present invention, the markers of the present invention are optionally, alone or in lung cancer, including but not limited to TPA, CEA, CA15-3, beta-2-microglobulin, CA19-9. Can be used in combination with known markers and / or in combination with known proteins of the mutant markers described herein.

  According to a further embodiment of the present invention, the marker of the present invention is optionally, alone or without limitation, CEA, CA125 (mucin 16), CA72-4TAG, CA-50, CA54-61, CA. It can be used in combination with known markers of ovarian cancer, including -195 and CA19-9, and / or in combination with known proteins of the mutant markers described herein.

  According to further embodiments of the invention, the markers of the invention are optionally, alone or in combination with known markers of rectal cancer including, but not limited to, CEA, CA19-9, CA50, and / or Alternatively, the mutant markers described herein can be used in combination with known proteins.

  In some embodiments of the invention, methods, uses, devices and assays for the diagnosis of a disease or condition are provided. Optionally, multiple markers can be used with the present invention. The plurality of markers can optionally include the markers described herein and / or one or more known markers. The plurality of markers are preferably correlated with a disease or condition. For example, such a correlation may optionally include measuring the concentration of each of the plurality of markers and individually comparing the concentration of each marker to a threshold level. Optionally, if the concentration of the marker is above or below the threshold level (depending on the marker and / or the diagnostic test being performed), the concentration of the marker correlates with the disease or condition. Optionally and preferably, multiple marker concentrations correlate with the disease or condition.

  Alternatively, such correlation optionally includes measuring the concentration of each of the plurality of markers, calculating a single index value based on the concentration of each of the plurality of markers, and It may include comparing the index value to a threshold level.

  Alternatively, such correlation may optionally include measuring temporal changes used in the correlation stage at at least one marker.

  Alternatively, such correlation optionally determines whether at least “X” of the plurality of markers is at a concentration outside of a predetermined range and / or above or below (as described above). Can be included. The value of “X” can optionally be one marker, multiple markers, or all markers. Alternatively or additionally, rather than including any marker in the “X” count, one or more specific markers of the plurality of markers optionally correlate with a disease or condition (according to range and / or threshold). May be required.

  Alternatively, such correlation also optionally includes determining whether the ratio of marker concentration to two markers is out of range and / or above or below a threshold. Optionally, if the ratio is above or below the threshold level and / or out of range, the ratio correlates with the disease or condition.

  Optionally, a combination of two or more of these correlations can be used with a correlation between a single panel and / or multiple panels.

  Optionally, the method identifies diseases and conditions with a sensitivity of at least 70% at a specificity of at least 85% compared to normal subjects. As used herein, sensitivity refers to the number of positive (disease) samples detected out of the total number of positive samples present, and specificity refers to the detected negative (non-disease) out of the total number of negative samples present. B) relating to the number of samples. Preferably, the method identifies diseases and conditions with a sensitivity of at least 80% at a specificity of at least 90% compared to normal subjects. More preferably, the method identifies diseases and conditions with a sensitivity of at least 90% at a specificity of at least 90% compared to normal subjects. Even more preferably, the disease and condition are identified with a sensitivity of at least 70% at a specificity of at least 85% compared to a subject exhibiting symptoms mimicking the disease or condition.

  The marker panel can be analyzed in a number of ways well known to those skilled in the art. For example, each member of the panel can be compared to a “normal” value or a value indicative of a particular result. The specific diagnosis / prognosis may depend on the comparison of each marker against this value. Alternatively, if only a subset of markers is outside the normal range, this subset may be indicative of a particular diagnosis / prognosis. One skilled in the art will also appreciate that diagnostic markers, differential diagnostic markers, prognostic markers, time of onset markers, disease or status identification markers, and the like can be combined in a single assay or device. Markers are also typically used for a variety of purposes, such as by applying different weighting factors for different thresholds or markers for different purposes.

  In one embodiment, the panel includes markers for the following purposes: Diagnosis of the disease, if the disease is in the acute phase, and / or if an acute attack of the disease has occurred, diagnosis of the disease and condition, the disease is non-acute If a phase and / or non-acute seizure of the disease has occurred, diagnosis of the disease and pathology, indications of whether an acute and non-acute phase or combination of seizures is occurring, diagnosis of the disease and subsequent adverse consequences Prognosis, disease diagnosis and subsequent acute or non-acute phase or stroke, disease progression (eg, for cancer, such progression may include, for example, the onset or recurrence of metastases).

  The diagnosis may also optionally include a differential diagnosis of the disease to distinguish it from other diseases, including diseases that may be characterized by one or more similar or identical symptoms.

  In certain embodiments, one or more diagnostic or prognostic indicators correlate with a condition or disease only by the presence or absence of the indicator. In other embodiments, a threshold level of a diagnostic or prognostic indicator can be established and the level of the indicator in a patient sample can be compared to the threshold level. The sensitivity and specificity of a diagnostic and / or prognostic test depends more than just the “quality” of the analysis of the test, and they also depend on the definition of what constitutes an unusual result. In practice, by plotting the value of the variable against the relative frequency in the “normal” and “disease” populations and / or by comparing the results from subjects before, during and / or after treatment. A Receiver Operating Characteristic curve, or “ROC” curve, is typically calculated.

  According to embodiments of the present invention, VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 protein, polynucleotide or fragment thereof, as described above, as a biomarker for detecting disease and / or suggesting pathology, Can be characterized.

  According to yet another embodiment, the present invention provides an amino acid sequence, optionally encoded by a nucleic acid sequence corresponding to VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3, which is optionally and preferably described herein. Including fragments. Any oligopeptide or peptide relating to such an amino acid sequence or fragment thereof can also optionally be used as a biomarker (in addition or alternatively).

  According to yet other embodiments, the present invention provides a method of detecting a polynucleotide of the present invention in a biological sample using a NAT-based assay, which comprises an isolated nucleic acid molecule, or at least approximately Hybridizing the shortest length of the oligonucleotide fragment to the nucleic acid material in the biological sample and detecting the hybridized complex. Here, the presence of the hybridized complex correlates with the presence of the polynucleotide in the biological sample. Non-limiting examples of methods or assays are described below.

  The invention also relates to kits based on such diagnostic methods or assays.

Nucleic Acid Technology (NAT) Based Assays Detection of a nucleic acid of interest in a biological sample is also optionally performed by a NAT based assay, including, for example, PCR (or its variation nucleic acid amplification techniques such as real-time PCR). As used herein, a “primer” can anneal (hybridize) to a target sequence, thereby serving as a starting point for DNA synthesis under appropriate conditions. Define an oligonucleotide that creates a double stranded region Selection or amplification of a target nucleic acid sequence can be performed by a number of suitable methods known in the art, including, but not limited to, polymerase chaining Reaction (PCR), ligase chain reaction (LCR), strand displacement a (SDW), transcription based amplification, q3 replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi et al., 1988ol, BioTech). Malek et al., 1994, Methods MoI. Biol, 28: 253-260; and Sambrook et al., 1989, supra.) Non-limiting assays based on nucleic acid technology (NAT). Examples of PCR include real-time PCR, LCR, self-supporting synthesis reaction, Q-beta replicase, cycling probe (cycling probe reaction, branched DNA, RFLP analysis, DGG / TGGE, single strand conformation polymorphism, dideoxy fingerprinting, microarray, fluorescence in situ hybridization and relative genomic hybridization. As used herein, “amplification pair” "(Or" primer pair ") is used in conjunction with one of many types of amplification processes, preferably in amplifying selected nucleic acid sequences by polymerase chain reaction. A pair of oligonucleotides (oligos) of the invention, selected as follows: As is generally known in the art, oligos are designed to bind to complementary sequences under selected conditions. The In one particular embodiment, amplification of the nucleic acid sample from the patient is amplified under conditions that favor the amplification of the most abundantly differentially expressed nucleic acid. In one preferred embodiment, RT-PCR is performed on mRNA samples from patients under conditions that favor amplification of the most abundant mRNA. In other preferred embodiments, amplification of differentially expressed nucleic acids can occur simultaneously. One skilled in the art will recognize that such methods could be applied for the detection of differentially expressed proteins instead of differentially expressed nucleic acid sequences. Will. Nucleic acids (ie DNA or RNA) for practicing the invention can be obtained according to well-known methods.

The oligonucleotide primers of the invention can be of any suitable length depending on the particular assay format and the particular requirements and target genome used.
Optionally, the oligonucleotide primers are at least 12 nucleotides in length, preferably 15 to 24 molecules, and can be adapted to be particularly suitable for the selected nucleic acid amplification system. As is generally known in the art, an oligonucleotide primer can be designed taking into account its melting point for hybridization with its target sequence. (Sambrook et al., 1989, Molecular Cloning-A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1989, in Current Protocols in Moles.

Immunoassay In another embodiment of the invention, an immunoassay can be used to qualitatively or quantitatively detect and analyze a marker in a sample. The method includes providing an antibody that specifically binds to the marker, contacting the sample with the antibody, and detecting the presence of a complex of antibodies bound to the marker in the sample.

  To prepare an antibody that specifically binds to the marker, a purified protein marker can be used. Antibodies specific for protein markers can be prepared using any suitable method known in the art.

  After the antibody is prepared, the marker can be detected and / or quantified using any of a number of well-recognized immunobinding assays. Useful assays include, for example, enzyme immunosorbent assays (ELISA), radioimmunoassays (RIA), Western blot assays, or enzyme immunoassays (EIA) such as slot blot assays. See, for example, U.S. Pat. Nos. 4,366,241, 4,376,110, 4,517,288, and 4,837,168. In general, a sample obtained from a subject can be contacted with an antibody that specifically binds to a marker.

  Optionally, the antibody can be immobilized on a solid support to facilitate washing and subsequent isolation of the complex prior to contacting the antibody with the sample. Examples of solid supports include, but are not limited to, for example, glass or plastic in the form of microplates, sticks, beads or microbeads. The antibody can also be attached to a solid support.

  After incubating the sample with the antibody, the mixture can be washed and the antibody-marker complex can be detected. This is accomplished by incubating the washed mixture with a detection reagent. Alternatively, the marker in the sample is used, for example, to detect a second, labeled antibody to detect the bound marker-specific antibody, using an indirect assay, and / or, for example, a different marker Monoclonal antibodies that bind to the epitope can be detected in competition or inhibition assays that are incubated simultaneously with the mixture.

  Throughout the assay, incubation and / or wash steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend on the assay format, marker, volume of solution, concentration, etc. The assay can be performed over a range of temperatures, such as 10 ° C. to 40 ° C., but will usually be performed at ambient temperature.

  An immunoassay can be used to measure a test amount of a marker in a sample from a subject. Initially, a test amount of a marker in a sample can be detected using the immunoassay methods described above. If a marker is present in the sample, it will form an antibody-marker complex with an antibody that specifically binds to the marker under the appropriate incubation conditions described above. The amount of antibody-marker complex is optionally measured by comparison with a standard. As noted above, the test amount of marker need not be measured in absolute amounts as long as the unit of measurement is compared to the control amount and / or signal.

  Radioimmunoassay (RIA): In one type, this method involves the precipitation of the desired substrate and is in the method described in detail later herein. The methods are described in detail later herein with a radiolabeled protein-binding antibody (eg, Protein A labeled with I125) immobilized on a precipitable carrier such as a specific antibody and agarose beads. The number of counts in the precipitated precipitate is proportional to the amount of substrate.

  Another type of RIA employs labeled nature and unlabeled protein-bound antibodies. Samples containing unknown amounts of substrate are added in various amounts. The decrease in counts precipitated from the labeled substrate is proportional to the amount of substrate in the added sample.

  Enzyme-linked immunosorbent assay (ELISA): This method involves the immobilization of a sample (eg, an immobilized cell or protein-like solution) that contains a protein substrate on a surface, such as a well of a microplate. A substrate-specific antibody linked to the enzyme is added to and bound to the substrate. The presence of the antibody is then detected and quantified by a color reaction using an enzyme linked to the antibody. Enzymes commonly used in this method include horseradish peroxidase and alkaline phosphatase. If well standardized and within the linear range of reaction, the amount of substrate present in the sample is proportional to the amount of color developed. Substrate standards are commonly used to improve quantitative accuracy.

  Western blot: This method involves separation of the substrate from other proteins by means of transfer of the substrate to an acrylamide gel followed by a membrane (eg nylon or PVDF). The presence of the substrate is then detected by an antibody specific for the substrate, which is then detected by an antibody binding reagent. The antibody binding reagent can be, for example, protein A, or other antibody. The antibody binding reagent can be radiolabeled or enzyme linked as described above. Detection can be by autoradiography, a colorimetric reaction or chemiluminescence. This method allows both quantification of the amount of substrate and determination of its identity by relative position on the membrane indicating the distance traveled in the acrylamide gel during electrophoresis.

Immunohistochemistry: This method involves the detection of a substrate in situ in cells fixed by a substrate specific antibody. The substrate specific antibody can be linked to an enzyme or to a fluorophore. Detection is by microscopy and subjective evaluation. If an enzyme-linked antibody is used, a colorimetric reaction may be required.

Fluorescence activated cell sorting (FACS): This method involves the detection of in situ substrates in cells by substrate specific antibodies. The substrate specific antibody is linked to a fluorophore. Detection is by a cell sorter that reads the wavelength of light emitted from each cell as it passes through the beam. This method can use two or more antibodies simultaneously.

Radioactive Imaging Methods These include, but are not limited to, positron emission tomography (PET) single photon emission computed tomography (SPECT). Both of these techniques are non-invasive and can be used to detect and / or measure a wide variety of tissue events and / or functions such as, for example, detection of cancerous cells. Unlike PET, SPECT can optionally use two labels simultaneously. SPECT also has several other advantages, for example with respect to cost and the type of label that can be used. For example, US Pat. No. 6,696,686 describes the use of SPECT for the detection of breast cancer, which is incorporated herein by reference in its entirety.

Theranostics The term theranostics refers to the use of a diagnostic test to diagnose a disease, select the correct treatment plan according to the results of the diagnostic test, and / or observe the patient's response to treatment according to the results of the diagnostic test . Theranostics tests can be used to select patients for treatments that are particularly beneficial and are unlikely to have side effects. It also provides an early and objective indication of treatment effect in each patient so that treatment (if necessary) can be changed with minimal delay. For example, DAKO and Genentech have jointly created HercepTest and Herceptin (trastuzumab) for the treatment of breast cancer, which is the first theranostic study that was approved simultaneously with a new therapeutic agent. In addition to HercepTest (which is an immunohistochemistry test), other theranostic tests using conventional clinical chemistry immunoassays, cell-based techniques and nucleic acid tests are under development. PPGx's recently marketed TPMT (thiopurine S-methyltransferase) test allows physicians to identify patients at risk for potential fatal side effects of 6-mercaptopurine, a drug used to treat leukemia. it can. Nova Molecular also pioneered SNP genotyping of the apolipoprotein E gene to predict Alzheimer's disease patients' response to cholinergic treatment, and is currently widely used in clinical trials of this drug for this indicator. Accordingly, the field of theranostics is the intersection of treatment test information that predicts a patient's response to treatment with the selection of an appropriate treatment for a particular patient.

Surrogate markers Surrogate markers are markers used in clinical trials as an alternative to clinically meaningful endpoints that can be detected by laboratory and / or patient physical signs or symptoms. Surrogate markers are a direct measure of how a patient feels, functions, and survives and are expected to predict the effectiveness of the treatment. The need for surrogate markers is measured earlier, more easily, or more frequently than such endpoints, which are referred to as clinical endpoints, with respect to the target endpoint for the effect of treatment on the patient. This happens mainly when it can be done. Ideally, surrogate markers are biologically relevant and predict disease progression (including but not limited to conventional clinical chemistry immunoassays, immunoassays, cell-based techniques and nucleic acid testing and imaging Should be measurable by standardized assays (such as modalities).

  Surrogate endpoints were first used primarily in the cardiovascular field. For example, antihypertensive drugs have been approved based on their effectiveness in lowering blood pressure. Similarly, in the past, cholesterol-lowering drugs have been approved based on their ability to reduce serum cholesterol, rather than direct evidence to reduce death from atherosclerotic heart disease. In addition, two surrogate markers that are currently commonly used in HIV studies are CD4 + T cell counts and quantitative plasma HIV RNA (viral load). In some embodiments of the invention, the polypeptide / polynucleotide expression pattern may serve as a surrogate marker for a particular disease, as will be appreciated by those skilled in the art.

Uses and Methods of the Invention VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 Agents, in particular antibodies, in particular human antibodies, antibody compositions and cells of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigens according to the invention Soluble conjugates containing outer regions or fragments or variants thereof, or corresponding nucleic acid sequences or vectors or cells expressing the same, as well as methods of the invention are related to VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 antigen For example, disease and / or B7-related immune costimulation including VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen The modulation of immune co-stimulation, including, for example, has numerous in vitro and in vivo diagnostic and therapeutic utilities, including diagnosis and treatment of therapeutically desirable disorders. As noted above, these conditions include, among others, cancers that differentially express VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigens, including those that are invasive and metastatic, such as lung cancer, ovary Autoimmune pathologies where modulation of costimulation is therapeutically desirable, such as cancer, colon cancer, and / or those associated with B7. The subject anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibody prevents B7-mediated negative stimulation of T cell activity against cancer cells and / or prevents positive stimulation of T cell activity Can do. Such antibodies include but are not limited to acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, breast, prostate, lung, Non-metastatic, including non-solid and solid tumors, including ovarian, colon, spleen, kidney, bladder, head and neck, uterus, testis, stomach, cervix, liver, bone, skin, pancreas, brain cancer, sarcoma, hematologic malignancy Cancers that can be sex, invasive, metastatic, and non-malignant disorders such as but not limited to transplant rejection and immunodeficiency diseases including graft host disease, and pathologies including autoimmune deficiency diseases such as those mentioned above Can be used in the treatment of

  For example, these molecules can be administered into a human subject, eg, in vivo, to treat, prevent, or diagnose cells in culture in vitro or in vitro, or various disorders. Preferred subjects include cells expressing VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen and human patients with disorders mediated by cells with VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 activity. In particular, the method comprises treating human patients with abnormal VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen expression with AI581519_P3 (SEQ ID NO: 11), AI581519_P4 (SEQ ID NO: 12), AI581519_P5 (SEQ ID NO: 13). , AI581519_P7 (SEQ ID NO: 14), AI581519_P9 (SEQ ID NO: 15), AI581519_P10 (SEQ ID NO: 16), AA424839_P3 (SEQ ID NO: 22), AA424839_P5 (SEQ ID NO: 21), AA424839_P7 (SEQ ID NO: 23) or AA424839_1_P11 (SEQ ID NO: 23) H68654_1_P2 (SEQ ID NO: 35), H68654_1_P5 (SEQ ID NO: 36), H68654_1_P7 SEQ ID NO: 37), H68654_1_P12 (SEQ ID NO: 38), H68654_1_P13 (SEQ ID NO: 39), H68654_1_P14 (SEQ ID NO: 40), AI216611_P0 (SEQ ID NO: 43), AI216611_P1 (SEQ ID NO: 44), H19011_1_P8 (SEQ ID NO: 48_), H19011_ No. 50), R31375_P0 (SEQ ID NO: 70), R31375_P14 (SEQ ID NO: 72), R31375_P31 (SEQ ID NO: 73) or R31375_P33 (SEQ ID NO: 74), suitable for treating human patients with antibodies ing

  The VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 agent described in the present invention can be administered with other agents and the two agents can be administered sequentially or simultaneously.

  Given the specific binding of the antibodies of the present invention to VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3, the antibodies of the present invention are specific for the expression of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 on the cell surface. Can be used to detect the target, and further can be used to purify VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen via immunoaffinity purification.

  Furthermore, given the expression of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 in a variety of tumor cells, human antibodies, antibody compositions and methods of the present invention are suitable for tumor formation such as, for example, lung cancer and ovarian cancer as described above. It can be used to treat a subject with a disorder characterized by the presence of a tumor cell that expresses the disorder, VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen.

  In one embodiment, the antibodies of the invention (eg, human antibodies, multispecific and bispecific molecules and compositions) are VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 levels, or VSIG1, ILDR1, The levels of cells containing LOC253012, AI216611, C1ORF32 or FXYD3 on the cell surface can be used to detect, respectively, and these levels can be linked to certain disease symptoms. Alternatively, antibodies can be used to inhibit or block VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 function and can lead to prevention or amelioration of certain disease symptoms. Thereby, VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 are suggested as disease mediators, respectively. This is because the samples and control samples were combined with anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibody and the corresponding antibody and VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3, respectively. It can be achieved by contacting under conditions that allow formation. Any complexes formed between the antibody and VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 are detected and compared in the sample and control.

  In another embodiment, antibodies of the invention (eg, human antibodies, multispecific and bispecific molecules and compositions) are first tested for binding activity associated with therapeutic or diagnostic applications in vitro. Can do. For example, the compositions of the invention can be tested using a low color development assay.

  The antibodies (eg, human antibodies, multispecific and bispecific molecules and compositions) of the invention have additional uses in the treatment and diagnosis of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3-related diseases. For example, human antibodies, multispecific and bispecific molecules and immunoconjugates can be used to elicit one or more of the following biological activities in vivo or in vitro: VSIG1, ILDR1, Inhibiting the growth and / or killing of cells expressing LOC253012, AI216611, C1ORF32 or FXYD3, phagocytosis of cells expressing VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 in the presence of human effector cells Or mediate ADCC, or VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 ligand may be VSIG1, ILDR1, LOC253012, AI21661, respectively. , Preventing binding to C1ORF32 or FXYD3.

  In certain embodiments, antibodies (eg, human antibodies, multispecific and bispecific molecules and compositions) treat, prevent, or diagnose a variety of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3-related diseases. To be used in vivo. VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3-related diseases include, among others, for example, but not limited to, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, breast, prostate, spleen, kidney, bladder, head and neck, uterus, testes, stomach, neck, liver, bone, skin, pancreas, lung cancer including brain cancer, ovarian cancer, colon cancer, Cancers such as non-solid and solid tumor sarcomas, hematological malignancies, which may be non-metastatic, invasive or metastatic, and further as VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3-related diseases, among others Autoimmune disease, transplant rejection and It includes non-malignant disorders such as immunodeficiency diseases, including explants member host disease. Such disorders include multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, immunodeficiency associated with transplant rejection, benign lymphocyte vasculitis, lupus erythematosus, Hashimoto's thyroid Inflammation, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin-dependent diabetes, Goodpasture syndrome, myasthenia gravis, pemphigus, sympathetic ophthalmitis, autoimmune uveitis , Autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic hepatitis, ulceritis colitis, Sjogren's syndrome, rheumatic disease, polymyositis, scleroderma, mixed connective tissue Disease, inflammatory rheumatism, degenerative rheumatism, non-articular rheumatism, collagen disease, chronic polyarthritis, arthritic psoriasis, ankylosing spondylitis, juvenile rheumatoid arthritis Examples include autoimmune diseases selected from peri-shoulder arthritis, polyarteritis nodosa, progressive systemic sclerosis, urate arthritis, dermatomyositis, rheumatoid arthritis, myositis, myopathy and cartilage calcification .

  Suitable routes for in vivo and in vitro administration of antibody compositions of the invention (eg, human monoclonal antibodies, multispecific and bispecific molecules and immunoconjugates) are well known in the art and are Can be selected. For example, the antibody composition can be administered by infusion (eg, intravenous or subcutaneous). The appropriate dosage of the molecule used will depend on the age and weight of the subject and the concentration and / or dosage form of the antibody composition.

  As described above, the human anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibody of the present invention may be one or more other therapeutic agents, eg, cytotoxic agents, radiotoxic agents. Or it can be co-administered with an immunosuppressant or the like. The antibody can be linked to the agent (as an immune complex) or can be administered separately from the agent. In the latter case (separate administration), the antibody can be administered before, after, or at the same time as the drug, or can be co-administered with other known therapies such as anti-cancer therapy, radiation therapy, for example. it can. As such therapeutic agents, doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambutyl, cyclophosphamide hydroxyurea, among others, are effective only at levels that are toxic or semi-toxic to the patient Antitumor drugs are included. Cisplatin is administered intravenously at 100 mg / dose once every 4 weeks, and adriamycin is administered intravenously at 60-75 mg / ml once every 21 days. Co-administration of the human anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibody or antigen-binding fragment thereof of the present invention with a chemotherapeutic agent produces different mechanisms that produce cytotoxic effects on human tumor cells Provides two anticancer drugs that work through. Such co-administration can also solve problems due to the development of resistance to drugs, or changes in the antigenicity of tumor cells that render the tumor cells non-responsive to antibodies.

Alternatively, target-specific effector cells, such as effector cells linked to inventive compositions (eg, human antibodies, multispecific and bispecific molecules) can be used as therapeutic agents. Target effector cells can be human leukocytes such as macrophages, neutrophils or monocytes. Other cells include eosinophils, natural killer cells and cells with IgG- or IgA-receptors. If desired, effector cells can also be obtained from a subject undergoing treatment. Target effector cells can be administered as a cell suspension in a physiologically acceptable solution. The number of cells administered can be on the order of 10 ⁻⁸ to 10 ⁻⁹ , but will vary depending on the therapeutic purpose. In general, the amount is sufficient to localize the target cells, eg, tumor cells expressing VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3, and to achieve cell death, eg, by phagocytosis Will. The route of administration will also vary.

  Treatment with target effector cells can be performed in conjunction with other techniques for removal of target cells. For example, anti-tumor therapies using the compositions of the invention (eg, human antibodies, multispecific and bispecific molecules) and / or effected cells armed with these compositions include chemotherapy and Can be used together. Furthermore, combination immunotherapy can be used to direct two different cytotoxic effector populations to tumor cell rejection. For example, anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibodies linked to anti-Fc gamma RI or anti-CD3 can be used in conjunction with IgG- or IgA-receptor specific binding agents. .

  The bispecific and multispecific molecules of the present invention also allow Fc on effector cells to regulate the Roma or Fc gamma R levels on the cell surface by covering or removing receptors. Can be used. Mixtures of anti-Fc receptors can also be used for this purpose.

  Also, compositions of the invention (eg, human antibodies, multispecific and bispecific molecules and those with complement binding sites such as sites from IgG1, -2, or -3 or IgM that bind complement) The immunoconjugate) can be used in the presence of complement. In one embodiment, the in vitro treatment of a population of cells comprising a target cell with a binding agent of the invention and a suitable effector cell can be supplemented by the addition of complement or complement-containing serum. Phagocytosis of target cells coated with the binding agents of the present invention can be improved by binding of complement proteins. In another embodiment, target cells coated with the compositions of the invention (eg, human antibodies, multispecific and bispecific molecules) can also be lysed by complement. In yet another embodiment, the compositions of the invention do not activate complement.

  The compositions of the invention (eg, human antibodies, multispecific and bispecific molecules and immunoconjugates) can also be administered with complement. Thus, compositions comprising human antibodies, multispecific and bispecific molecules and serum or complement are within the scope of the invention. These compositions are advantageous in that the complement is located in close proximity to human antibodies, multispecific and bispecific molecules. Alternatively, the human antibodies, multispecific or bispecific molecules of the invention and complement or serum can be administered separately.

  VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen or VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 conjugate or antibody composition of the invention (eg, human antibody, bispecific or multispecific molecule or immunity Conjugates) and kits containing instructions for use are also within the scope of the invention. The kit may further comprise one or more additional reagents such as immunosuppressive, cytotoxic or radiotoxic agents, or one or more additional human antibodies of the invention (eg, VSIG1, ILDR1, LOC253012, AI216611, A human antibody different from the first human antibody with complementary activity that binds to an epitope in the C1ORF32 or FXYD3 antigen.

  Accordingly, a patient treated with an antibody composition of the present invention may be further treated with another therapeutic agent, such as a cytotoxic or radiotoxic drug, that enhances or increases the therapeutic effect of the human antibody (administration of the human antibody of the present invention Can be administered prior to, simultaneously with, subsequent to administration).

  In another embodiment, the subject is additionally treated with an agent that modulates (eg, enhances or inhibits) expression or activity of Fey or Fey receptors, such as by treating the subject with a cytokine. obtain. Preferred cytokines administered during treatment with multispecific molecules include granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-gamma (IFN-gamma). ), Tumor necrosis factor (TNF).

  Also, compositions of the invention (eg, human antibodies, multispecific and bispecific molecules), eg, expressing Fc gamma R or VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 by labeling cells Can be used to target cells. In such applications, the binding agent can be linked to a molecule that can be detected. Accordingly, the present invention provides a method of finding in vitro or in vivo localization of cells expressing Fc receptors such as Fc gamma R, or VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen. The detectable label can be, for example, a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.

  In certain embodiments, the invention measures the amount of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen in a sample, or measures the amount of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen. Provide each method. It consists of a human monoclonal antibody or antigen-binding portion thereof that specifically binds a sample and a control sample to VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3, respectively, an antibody or portion thereof and VSIG1, ILDR1, LOC253012, AI216611, Contacting under conditions that allow formation of a complex between C1ORF32 or FXYD3. Complex formation is then detected and the difference in sample complex formation compared to the reference sample suggests the presence of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen in the sample. As noted above, in particular, the present invention provides immunoassays, radioimmunoassays, radioassays, radioimaging assays, ELISAs, Western blots, FACS, slot blots, immunohistochemical assays, and / or others well known to those skilled in the art. Assays that detect VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen in vitro and in vivo.

  In another embodiment, the present invention is provided in a subject, for example, but not limited to, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, Hodgkin lymphoma, non- Hodgkin lymphoma, breast, prostate, lung, ovary, colon, spleen, kidney, bladder, head and neck, uterus, testes, stomach, neck, liver, bone, skin, pancreas, brain cancer, including solid tumor sarcoma, Cancers such as hematological malignancies, which can be non-metastatic, invasive or metastatic, and non-malignant disorders including but not limited to transplant rejection and graft host disease or the above Treat disorders mediated by VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3, such as immunodeficiency diseases such as autoimmune diseases selected from Any VSIG1, anti-ILDR1, anti-LOC216011, anti-C1ORF32 or anti-FXYD3 antibody or any pathologic condition where modulation of immune co-stimulation according to the method and VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 is therapeutically desirable. With soluble VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen conjugates or VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or other agents that target (modulate or inhibit) FXYD3 biological activity, Provide a method of treatment.

  Anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibody, soluble VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen conjugate, or VSIG1, ILDR1, LOC356311, X21RF11, C21RF11 By administering the portion to a subject, the ability of the VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen to elicit such activity is inhibited or promoted, thus treating the associated disorder. A soluble VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen, or antigen conjugate, or anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32 or anti-FXYD3 antibody, or a fragment containing the composition, or VSIG1 Other agents that target and modulate ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 can also be administered alone to treat or prevent VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen-mediated diseases, It can also be used in combination with other therapeutic agents such as cytotoxic or radiotoxic drugs acting in synergy with or synergistically with the antibody composition. That.

  In yet another embodiment, the immunoconjugates of the invention can be used to target compounds to cells with VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 cell surface receptors (eg, therapeutics) by linking the compound to an antibody. Agents, labels, cytotoxins, radiotoxins, immunosuppressants, etc.). Therefore, the present invention relates to in vitro or in vivo localization of cells expressing VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 (for example, radioisotopes, fluorescent compounds, enzymes, enzyme coenzymes, etc. Provides a method of finding (using a detectable label). Alternatively, the immunoconjugate targets cells with VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 cell surface receptors and cytotoxins or radiotoxins to VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigen. Can be used to destroy.

  The present invention is now directed to DNA transcripts encoding VSIG1, ILDR1, LOC253012, AI216611, C1ORF32 or FXYD3 antigens, their regions and the sequence characterization of expression data in normal and cancerous tissues, and production of fully human antibodies This will be further described with reference to a desk-top example. This information and examples are illustrative and should not be construed as limiting. All figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

Example 1: Method used to analyze the expression of RNA encoding the protein of the present invention The target of the present invention is directed to its expression in various cancerous and non-cancerous tissue samples and / or various immunity The cells and their expression in a panel of extensive human samples including malignant blood diseases and blood cell lines and several samples of several normal tissues were examined. A list of blood-specific RNA samples used for qRT-PCR analysis is shown in Table 1 below. Table 2 describes the samples used for the normal tissue panel. A description of the samples used in the lung cancer test panel is shown in Table 3 below. A description of the samples used in the ovarian cancer test panel is shown in Table 4 below. A description of the samples used in the rectal cancer test panel is shown in Table 5 below. The keys of Tables 3, 4, and 5 are shown in Tables 3_1, 4_1, and 5_1. The test was performed as described in the “Materials and Experimental Procedures” section below.

Table 1: Samples in a blood-specific panel

Table 2: Tissue samples in normal panels

Table 3: Lung cancer test panel

Table 3_1

Table 4: Tissue samples in the ovarian panel

Table 4_1

Table 5: Rectal cancer test panel

Table 5_1

Materials Used to Obtain Expression Data and Experimental Procedures RNA Preparation RNA was obtained from ABS (Wilmington, DE 19001, USA, http://www.absbioagents.com), BioChain Inst. Inc. (Hayward, CA 94545 USA www.biochain.com), GOG (ovary sample -Pediatic Cooperative Human Tissue Network, Gynecologic Oncology Group Tissue Bank, Children Hospital of Columbus (Columbus OH 43205 USA)), Clontech (Franklin Lakes, NJ USA 07417 , Www.clontech.com), Ambion (Austin, TX 78744 USA, http://www.amion.com), Asternad (Detroit, MI 48202-3420, USA, www.asterand.com), and Gen. mics Collaborative Inc. a Division of Seracare (Cambridge, MA 02139, USA, www.genomicsinc.com). RNA was generated from blood cells, cell lines, and tissue samples using TRI-Reagent (Molecular Research Center) according to the manufacturer's instructions. Tissue and RNA samples were obtained from patients or postmortem specimens. Most of the RNA samples were treated with DNase I (Ambion).

  RT-PCR—Purified RNA (2-10 μg) was mixed with 300-1500 ng of random hexamer primer (Invitrogen), 500 μM dNTPs, for a total volume of 31.2-156 μl, and the mixture was mixed at 65 ° C. for 5 Incubated for minutes and quenched on ice. Then 10-50 μl of 5X Superscript II first strand buffer (Invitrogen), 4.8-24 μl of 0.1 MDTT, 80-400 units of RNasin (Promega) were added. The mixture was incubated at 25 ° C. for 10 minutes and further incubated at 42 ° C. for 2 minutes. Thereafter, 2-10 μl (400-2000 units) Superscript II (Invitrogen) was added and the reaction (final volume 50-250 μl) was incubated at 42 ° C. for 50 minutes and then at 70 ° C. for 15 minutes. The obtained cDNA was diluted 1:20 using TE buffer (10 mM Tris pH = 8, 1 mM EDTA pH = 8).

  Real-time RT-PCR analysis was performed as follows. Using the SYBR Green I assay (PE Applied Biosystem), cDNA prepared as described above (5 μl) in a real-time PCR reaction (specific volume 20 μl) containing specific primers and UNG Enzyme (Eurogentech or ABI or Roche), Used as a mold. Amplification was accomplished as follows: 50 ° C. for 2 minutes, 95 ° C. for 10 minutes, followed by 40 cycles of 95 ° C. for 15 seconds, followed by a dissociation step at 60 ° C. for 1 minute. Detection was performed using PE Applied Biosystem SDS 7000. The cycle when the reaction reached the threshold level of fluorescence (Ct = Threshold Cycle, details are given below) was recorded and used to calculate the relative transcription level during the RT reaction. The relative amount was calculated using the formula, Q = efficiency ^ -Ct. The efficiency of the PCR reaction was calculated from a standard curve generated using different dilutions of several reverse transcription (RT) reactions. In order to minimize inherent differences during the RT reaction, the relative amounts obtained were normalized using a normalization factor calculated as follows.

  The expression of several housekeeping (HSKP) genes was examined in each panel. Divide the relative amount of each housekeeping gene (relative quantity (Q)) in each sample, calculated as above, by the intermediate amount of that gene in all panel samples and calculate “relative Q rel to MED”. Obtained. Next, for each sample, an intermediate value of “relative Q rel to MED” of the selected housekeeping gene was calculated and used as a normalization factor for further calculation of this sample. A schematic summary of quantitative real-time PCR analysis is shown in FIG. As shown, the X axis indicates the number of cycles. CT = Threshold Cycle Point, the cycle at which the amplification curve intersects the fluorescence threshold set in the experiment. This is the number of calculated cycles where the PCR product signal exceeds the background level (passive dye ROX) and is still in the geometric / exponential phase. (As shown in the figure, once the level of fluorescence crosses the measurement threshold, it is the geometrical increase phase where the measurement is most accurately made, followed by a linear phase and a horizontal phase. The latter two phases do not give an accurate measurement.) The Y-axis shows normalized reporter fluorescence. Note that this type of analysis provides relative quantification.

  For individual RT samples, specific amplicon expression was normalized to normalization factors calculated from the expression of various housekeeping genes as described in the section above.

  These housekeeping genes are different in each panel. In the rectal panel, HPRT1 (GenBank accession number NM — 000194 (SEQ ID NO: 118), amplicon-HPRT1-amplicon (SEQ ID NO: 181)), PBGD (GenBank accession number BC019323 (SEQ ID NO: 117)), amplicon-PBGD-amplicon. (SEQ ID NO: 178)), and G6PD (GenBank accession number NM_000402 (SEQ ID NO: 119), G6PD amplicon (SEQ ID NO: 184)). In the lung panel, HPRT1 (GenBank accession number NM — 000194 (SEQ ID NO: 118), amplicon-HPRT1-amplicon (SEQ ID NO: 181)), PBGD (GenBank accession number BC0193323 (SEQ ID NO: 117)), amplicon-PBGD-amplicon. (SEQ ID NO: 178)), SDHA (GenBank accession number NMJ) 004168 (SEQ ID NO: 116); amplicon-SDHA-amplicon (SEQ ID NO: 175)) and ubiquitin (GenBank accession number BC000449 (SEQ ID NO: 115)), amplicon- Ubiquitin-amplicon (SEQ ID NO: 172)). In the ovarian panel, SDHA (GenBank accession number NM_004168 (SEQ ID NO: 116), amplicon-SDHA-amplicon (SEQ ID NO: 175)), HPRT1 (GenBank accession number NM_000194 (SEQ ID NO: 118)), amplicon-HPRT1-amplicon. (SEQ ID NO: 181)) and G6PD (GenBank accession number NM_000402 (SEQ ID NO: 119), G6PD amplicon (SEQ ID NO: 184)). In the normal panel, SDHA (GenBank accession number NM_004168 (SEQ ID NO: 116), amplicon-SDHA-amplicon (SEQ ID NO: 175)), ubiquitin (GenBank accession number BC000449 (SEQ ID NO: 115)), amplicon-ubiquitin-amplicon. (SEQ ID NO: 172)) and TATA box (GenBank accession number NM_003194 (SEQ ID NO: 114), TATA amplicon (SEQ ID NO: 169)). In the blood panel, HSB1L_HUMAN (Accession No. Q9Y450) (SEQ ID NO: 109), DHSA_HUMAN (SEQ ID NO: 110) (Accession No. P31040), SFRS4_HUMAN (SEQ ID NO: 111) (Accession No. Q08170) and SLC25A3 (Accession No. Q7Z7N7) (SEQ ID NO. 112).

  The sequence of housekeeping genes measured in all blood panel examples was as follows:

  HSB1L_HUMAN (SEQ ID NO: 109) (Accession number Q9Y450)

  T05337_seg30-34F1-forward primer (SEQ ID NO: 152): GCTCCCAGGCCATAGAGACTTC

  T05337_seg30-34R1 (SEQ ID NO: 153) -reverse primer: CAGCTTCAAACTCTCCCCCTGC

  Amplicon (SEQ ID NO: 154): GCTCCAGGCCATAAGGACTTCATTCCAAATATGATTACAGGAGCAGCCCGAGCGGATGTAGCTGTTTAGTCGCAGGGGAGAGTTTGAAGCTG

  DHSA_HUMAN (SEQ ID NO: 110) (Accession number P31040)

  M78124_seg45-48F1 (SEQ ID NO: 155) -forward primer: TTCCTTGCCAGGACCTAGAG

  M78124_seg45-48R1-reverse primer (SEQ ID NO: 156): CATAAAACCTTTCGCCTTGAC

  Amplicon (SEQ ID NO: 157): TTCCTTGCCAGGACCTAGAGTTTGTTCAGGTCCCACCCCAGGGCATATATGGGTCTGGTGTCTCATTACCGGAAGGATGTCTGTGAGAGGGAGGGATTCTCATTAAGAGTCAAGGTGAAGG

  SFRS4_HUMAN (SEQ ID NO: 111) (Accession number Q08170)

  HUMSRP75Aseg30-33F1 (SEQ ID NO: 158) -forward primer: AATTTGTCAAGTCGGTGCAGC

  HUMSRP75Aseg30-33R1 (SEQ ID NO: 159)-reverse primer: TCACCCCTTCATTTTTGCCG

  Amplicon (SEQ ID NO: 160): AATTTGTCAAGTCGGTGCAGCTGGCAAGACCTAAAGGATTATATGCGTCAGGCAGGAGAAGTGACTTATGCAGATGTCCACAAGGACGCAAAAAATGAAGGGGTGA

  SLC25A3 (Accession number Q7Z7N7) (SEQ ID NO: 112)

  SSMPCPseg24-25-29F1-forward primer (SEQ ID NO: 161): CCCAAAATGTATAAGGAAGAAGGC

  SSMPCPseg24-25-29R1-reverse primer (SEQ ID NO: 162): TTCAAAGCAGCGCACTACTA

  Amplicon (SEQ ID NO: 163): CAGCCAGGTTATGCCACAACTTTGAGGGATGCAGCTCCCCAAAATGTATAAGGAAGAAGGCCTAAAAGCATTCTACAAGGGGGTTGCTCCTCCTGGATGAGACAGATACCATAGATCTCGCTGAT

  The sequence of housekeeping genes measured in all examples on a normal tissue sample panel was as follows:

TATA box (GenBank accession number NM_003194 (SEQ ID NO: 114))

  TATA box forward primer (SEQ ID NO: 167): CGGTTTGCTGCGGTAATCAT

  TATA box reverse primer (SEQ ID NO: 168): TTTCTTGCTGCCAGTCTGGAC

  TATA box-amplicon (SEQ ID NO: 169): CGGTTTGCTGCCGTAATCATGAGGATATAGAGAGCCACGAACCACGGCACTGATTTTCAGTTCTGGAAAATGGGTGCCAAGAGGAGCCAAGAGTGGAAGAACAGCTCCAGA

  Ubiquitin (GenBank accession number BC000449 (SEQ ID NO: 115))

  Ubiquitin forward primer (SEQ ID NO: 170): ATTTGGGTCCGCGTTCTTG

  Ubiquitin reverse primer (SEQ ID NO: 171): TGCCTTGACATTCTCGATGGT

  Ubiquitin-amplicon (SEQ ID NO: 172)

  ATTTGGGTCCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGACAATGCAGATTCTTCGTGAAGACTCTGAACTGGTAAGACCCATCACCCTCGAGGTTGAGCCCCAGGACATCACAGCCA

  SDHA (GenBank accession number NM_004168 (SEQ ID NO: 116))

  SDHA forward primer (SEQ ID NO: 173): TGGGAACAAGAGGGCATCTTG

  SDHA reverse primer (SEQ ID NO: 174): CCACCACTGCCATCAAATTCATG

  SDHA-Amplicon (SEQ ID NO: 175): TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATCTCATTTCTGCTCAGTATCCAGTAGTGGATCATGAATTTGATGCAGTGGTGG

  The housekeeping gene primers and amplicon sequences measured in all cancerous examples are listed below. HPRT1, PBGD and G6PD were used for the rectal panel. PBGD, HPRT1, ubiquitin and SDHA were used for the lung panel. HPRT1, SDHA and G6PD were used for the ovarian panel.

  SDHA (GenBank accession number NM_004168 (SEQ ID NO: 116))

  SDHA forward primer (SEQ ID NO: 173): TGGGAACAAGAGGGCATCTTG

  SDHA reverse primer (SEQ ID NO: 174): CCACCACTGCCATCAAATTCATG

  SDHA-Amplicon (SEQ ID NO: 175): TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATCTCATTTCTGCTCAGTATCCAGTAGTGGATCATGAATTTGATGCAGTGGTGG

  PBGD (GenBank accession number BC019323 (SEQ ID NO: 117))

  PBGD forward primer (SEQ ID NO: 176): TGAGAGTGATTCGCGTGGG

  PBGD reverse primer (SEQ ID NO: 177): CCAGGGTACGAGGCTTTCAAT

  PBGD-Amplicon (SEQ ID NO: 178): TGAGAGTGATTCGCGGTGGTACCCCGCAAGAGCCAGCTTGCTCGCATACAGACGACAGGTGTGGTGCAACAATTGAAAGCCTCGTACCCCTGG

  HPRT1 (GenBank accession number NM_000194 (SEQ ID NO: 118))

  HPRT1 forward primer (SEQ ID NO: 179): TGACACTGGCAAAAACATGCA

  HPRT1 reverse primer (SEQ ID NO: 180): GGTCCTTTTCACCAGCAAGCT

  HPRT1-Amplicon (SEQ ID NO: 181): TGACACTGGGCAAAAAATGCAGAACTTTGCTTTCCTTGGTCAGGCAGTATAATCCAAAGATGTCAAGGTCGCAAGCTTGCTGGTGAAAAGGACC

  G6PD (GenBank accession number NM_000402 (SEQ ID NO: 119))

  G6PD forward primer (SEQ ID NO: 182): gagccgtcaccagaagacat

  G6PD reverse primer (SEQ ID NO: 183): ggabagccgggtagagctc

  G6PD-Amplicon (SEQ ID NO: 184): gagccgtcaccagaagaacattcacgagtcctgcatgagccagagatagctggaaccgcatcatcgtggagaagcccttcgggggggaccgtgccatgacctcc

  Ubiquitin (GenBank accession number BC000449 (SEQ ID NO: 115))

  Ubiquitin forward primer (SEQ ID NO: 170): ATTTGGGTCCGCGTTCTTG

  Ubiquitin reverse primer (SEQ ID NO: 171): TGCCTTGACATTCTCGATGGT

  Ubiquitin amplicon (SEQ ID NO: 172): ATTTGGGTCCGCGGTTCTGTTTGTGGATCGCTGTGATCGTCACTGACAATGCAGATCTTCGTGAAGACTCTGATCGGATAGACCCATGCATCATGCAGTGGACC

  Another method used to predict the expression pattern of the protein of the present invention was the MEDD discovery engine.

  MED is a publicly available collection of gene expression data, normalization, annotation, and performance of various queries. Download expression data from the most commonly used Affymetrix microarray from Gene Expression Omnibus (GEO-www.ncbi.nlm.nih.gov/GEO). The data was 95% percentile with respect to the steady-state value, multiplicatively normalized (normalized expression = 1200), and noise was removed from low 30% to 0. The experiment is automatically annotated initially, then manually, the tissues and conditions are identified, the chips are grouped by this annotation, and the overall expression pattern of the genes on each chip is This grouping was cross-validated by comparing with the average expression pattern of genes. Each probe set in each group was assigned an expression value that was an intermediate value of the expression of the probe sets in all the chips included in the group. The vector of expression for all probe sets within a group is the virtual chip of that group, and a collection of all such virtual chips is a virtual panel. is there. A panel (or subpanel) can be queried to identify probe sets with the required properties (eg, specific expression in a subset of tissues, or differential expression between diseased and healthy tissues). For subsequent analysis, these probe sets are linked to LEADS contigs and RefSeqs (http://www.ncbi.nlm.nih.gov/RefSeq/) by probe level mapping.

  The downloaded Affymetrix platform is the HG-U95A and HG-U133 family (A, B, A2.0 and PLUS2.0). Three virtual panels were then created: U95 and U133 Plus 2.0 based on the corresponding platform, and a set of common probe sets for HG-U133A, HG-U133A2.0 and HG-U133 PLUS 2.0+ U133 using

  The results of the MED discovery engine are shown in the scatter diagram. A scatter plot is a compact representation of a given panel (collection of groups). The Y axis describes (normalized) expression and the X axis describes the group of panels. For each group, intermediate expression is indicated by a solid clogging, and expression values for different chips within the group are represented by small dashes ("-"). Groups should be ordered and marked as follows: “other” groups (eg, benign, non-cancerous disease, etc.) triangles, treated cells square, normal circles, fit Is a cross, cancer is a rhombus. In addition, the number of chips in each group is noted beside the group name.

Example 2: Description of Cluster AI581519 The present invention relates to VSIG1 polypeptides, novel splice variants and diagnostic and therapeutic agents based thereon.

  According to the present invention, cluster AI581519 (internal ID 72756422) featured 10 subject transcripts and 2 subject sections, and their names are shown in Tables 6 and 7, respectively. Selected protein variants are shown in Table 8.

Table 6: Target transcripts

Table 7: Target sections

Table 8: Target proteins

  These sequences are variants of known proteins, V-set and immunoglobulin region-containing 1 (RefSeq accession number NP — 87413, aka: RP5-889N15.1, 1700062D20Rik, GPA34, MGC44287, dJ889N15.1). Yes, referred to herein as a known protein.

  VSIG1 is a V-set and immunoglobulin region-containing 1 protein, also known as glycoprotein A34 (GPA34). This gene was originally identified as a transcript that encodes a protein with similarity to the rectal cancer antigen glycoprotein A33 (GPA33) (Scanlan et al. Cancer Immunotherapy 6: 2 2006), with 32% of GPA33 There is homology. The authors have shown that A34 mRNA and protein expression is very tissue-limited, mainly expressed in the stomach and testis. A34 mRNA and protein expression was also detected in gastric cancer, esophageal cancer and ovarian cancer. In their studies, expression was not detected in lung, breast or rectal cancer (Scanlan et al. 2006, Cancer Immunity 6: 2).

  Known wild-type VSIG1 nucleic acid sequences have been reported in various patent and non-patent references. For example, the sequence of AI581519_P3 (SEQ ID NO: 11) was disclosed in WO2004037999, where it was called glycoprotein A34 (GPA34). This PCT application includes the sequence of AI581519_P3 (SEQ ID NO: 11) encoding the A34 antigen disclosed herein. The corresponding antigen A34 has been shown to be expressed in the test gastric cancer (29%), esophageal (63%), and to a much lower extent (9%) in the test ovarian cancer. The authors suggest that this antigen can be used for therapy and may be a suitable target for antibody cancer therapy.

  WO 9926972 describes that this antigenic protein controls (up or down) the growth and proliferation of, for example, T and / or B lymphocytes and is also of NK cells and other cell populations. It is disclosed that it can affect cytotoxic activity and exhibit immunostimulatory or immunosuppressive activity, such as for the treatment of various immunodeficiencies and disorders (including severe combined immunodeficiency (SCID)).

  In addition, the same protein sequence shown in AI581519_P3 (SEQ ID NO: 11) herein is described in WO9960020 and US2002193567, where it is identified as human secreted protein # 62 Has been.

  A sequence homologous to the VSIG1 mutant shown in AI581519_P4 (SEQ ID NO: 12) of the present specification (including two mismatches corresponding to known SNPs) is disclosed in WO2003027228. An extensive list of various differentially expressed sequences is disclosed.

  In addition, a sequence closely related to AI581519_P5 (SEQ ID NO: 13), including two mismatches corresponding to known SNPs, is disclosed in PCT application WO2004100774, which also includes many other It teaches sequences that are said to be differentially expressed.

  In addition, a sequence closely related to AI581519_P7 (SEQ ID NO: 14), including one mismatch corresponding to a known SNP, is disclosed in PCT application WO 2004048550, which is similarly It contains an extensive list of sequences that are said to be expressed constitutively.

  According to the present invention, VSIG1 is expected to be a novel B7 member based on the presence of IgV and IgC2 regions. The majority of proteins with one region of each Ig subtype are costimulatory molecules. Like other known B7 members, VSIG1 is also a type I membrane protein. In the present invention, several alternative splice variants of VSIG1 have been identified that contain unique regions within the extracellular region, as described below. It was shown in the present invention that a new variant of VSIG1 is overexpressed in lung adenocarcinoma and ovarian cancer.

  The MED discovery engine described in Example 1 herein was used to assess the expression of the VSIG1 transcript. Expression data for the Affymetrix probe set 234370 representing the VSIG1 gene data is shown in FIG. As is apparent from the scatter diagram of FIG. 2, the expression of the VSIG1 transcript detectable with the probe set was higher in lung cancer than in normal lung samples.

  As stated above, cluster AI581519 features the 10 transcripts listed in Table 6 above. These transcripts encode a protein that is a variant of protein V-set and immunoglobulin region containing 1. Each mutant protein of the present invention will be described.

  The mutant protein AI581519_P3 (SEQ ID NO: 11) of the present invention comprises transcripts AI581519_T0 (SEQ ID NO: 1), AI581519_T1 (SEQ ID NO: 2), AI581519_T2 (SEQ ID NO: 3), AI581519_T3 (SEQ ID NO: 4) and AI581519_T4 (SEQ ID NO: 5). It has an amino acid sequence encoded by

  Mutant protein localization was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, mutant proteins are thought to be localized next: membranes.

  The mutant protein AI581519_P3 (SEQ ID NO: 11) has the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 9. (The alternative amino acids are listed in the light of their position on the amino acid sequence.)

Table 9: Amino acid mutations

  The mutant protein has the following regions as determined using InterPro. The region is shown in Table 10.

Table 10: InterPro area

  Mutant protein AI581519_P3 (SEQ ID NO: 11) is transferred to the following transcripts AI581519_T0 (SEQ ID NO: 1), AI581519_T1 (SEQ ID NO: 2), AI581519_T2 (SEQ ID NO: 3), AI581519_T3 (SEQ ID NO: 4) and AI581519_T4 (SEQ ID NO: 5). Coded.

  The coding part of the transcript AI581519_T0 (SEQ ID NO: 1) starts at position 171 and ends at position 1331. The transcript also has the following SNPs listed in Table 11. (The alternative nucleic acids were listed in the light of their position on the nucleotide sequence (SEQ ID NO: 11).)

Table 11: Nucleic acid SNPs

  The coding part of the transcript AI581519_T1 (SEQ ID NO: 2) starts at position 171 and ends at position 1331. The transcript also has the following SNPs listed in Table 12. (The alternative nucleic acids were listed in light of their position on the nucleic acid sequence.)

Table 12: Nucleic acid SNPs

  The coding part of the transcript AI581519_T2 (SEQ ID NO: 3) starts at position 171 and ends at position 1331. The transcript also has the following SNPs listed in Table 13. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 13: Nucleic acid SNPs

  The coding part of the transcript AI581519_T3 (SEQ ID NO: 4) starts at position 171 and ends at position 1331. The transcript also has the following SNPs listed in Table 14. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 14: Nucleic acid SNPs

  The coding part of the transcript AI581519_T4 (SEQ ID NO: 5) starts at position 171 and ends at position 1331. The transcript also has the following SNPs listed in Table 15. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 15: Nucleic acid SNPs

  The mutant protein AI581519_P4 (SEQ ID NO: 12) of the present invention has the amino acid sequence encoded by the transcript AI581519_T5 (SEQ ID NO: 6). An alignment to published protein sequences is shown in FIG. 3A. A brief description of the relationship of the mutant protein of the present invention to each of the aligned proteins is as follows.

  2. AI581519_P4 (SEQ ID NO: 12) and a comparison report between known proteins NP_87413 (SEQ ID NO: 11) and Q86XK7_HUMAN (FIG. 3A)

  A. An isolated chimeric polypeptide encoding AI581519_P4 (SEQ ID NO: 12), corresponding to amino acids 1 to 71 of the known proteins NP_87413 and Q86XK7_HUMAN (SEQ ID NO: 11), and amino acids of AI581519_P4 (SEQ ID NO: 12) MVFAFWKVFLILSCLAQQVSVVQVTIPDGFVNVTVGSNVTLICICYTTTVASREQ LSIQWSFFHKKEMEPIS, which is at least 90% homologous to the polymorphic amino acid sequence RV GST % A second amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90% and even more preferably at least 95, 96, 97, 98 or 99% homologous to the selection and the known proteins NP_87413 and Q86XK7_HUMAN (sequence corresponds to amino acids 72 to 387 of No. 11), also corresponds to amino acids 108-423 of AI581519_P4 (SEQ ID NO: 12), AiwaiefuesukyujijikyueibuieiaijikyuefukeidiaruaitijiesuenudiPijienueiesuaitiaiesueichiemukyuPieidiesujiaiwaiaishidibuienuenuPiPidiefuerujikyuenukyujiaieruenubuiesubuierubuikeiPiesukeiPierushiesubuikyujiaruPiitijieichitiaiesueruesushieruesueierujitiPiesupibuiwaiwaidaburyueichikeieruijiarudiaibuiPibuikeiienuefuEnupitiTGILVIGNLTNFEQGYYQCTAINRL Comprises a third amino acid sequence that is at least 90% homologous to EnuesuesushiiaidierutiesuesueichiPiibuijiaiaibuijieieruaijiesuerubuijieieiaiaiaiesubuibuishiefueiaruenukeieikeieikeieikeiiaruenuesukeitiaieiieruiPiemutikeiaienuPiarujiiesuieiemuPiaruidieitikyueruibuitieruPiesuesuaieichiitiJipiditiaikyuiPidiwaiiPikeiPitikyuiPiEipiiPiEipijiesuiPiemueibuiPidierudiaiieruieruiPiitikyuesuieruiPiiPiEPEPESEPGVVVEPLSEDEKGVVKA, the first amino acid sequence, the second amino acid sequence and the third amino acid sequence, the polypeptide is a continuous, in this marked order.

  C. At least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and even more preferably at least about 95, to the sequence HSSCLSTEGMMEKAVGQCLKMTHVRDARGRCSWTSE (SEQ ID NO: 284) of AI581519_P4 (SEQ ID NO: 12); An isolated polypeptide encoding the edge portion of AI581519_P4 (SEQ ID NO: 12) comprising an amino acid sequence that is 96, 97, 98 or 99% homologous

  Mutant protein localization was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, mutant proteins are thought to be localized to: membranes.

  In addition, mutant protein AI581519_P4 (SEQ ID NO: 12) contains the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 16 (in terms of amino acid sequence positions, alternative amino acids). (SEQ ID NO: 12)).

Table 16: Amino acid mutations

  The mutant protein has the following regions as determined using InterPro. Table 17 shows the area.

Table 17: InterPro area

  The mutant protein AI581519_P4 (SEQ ID NO: 12) is encoded by AI581519_T5 (SEQ ID NO: 6), the coding part starting at position 171 and ending at position 1439. The transcript also has the following SNPs listed in Table 18. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 18: Nucleic acid SNPs

  The mutant protein AI581519_P5 (SEQ ID NO: 13) of the present invention has the amino acid sequence encoded by the transcript AI581519_T6 (SEQ ID NO: 7). An alignment alignment for published protein sequences is shown in FIG. 3B. A brief description of the relationship of the mutant protein of the present invention to each of the aligned proteins is as follows.

  2. A comparison report between AI581519_P5 (SEQ ID NO: 13) and the known proteins NP_87413 and Q86XK7_HUMAN (SEQ ID NO: 11) (FIG. 3B)

  A. An isolated chimeric polypeptide encoding AI581519_P5 (SEQ ID NO: 13), corresponding to amino acids 1 to 71 of the known proteins NP_87413 and Q86XK7_HUMAN (SEQ ID NO: 11), and amino acids of AI581519_P5 (SEQ ID NO: 13) MVFAFWKVFLLSCLAGQVSVVQVTIPDGFVNVTVGSNVTLICIYTTTV QLDQLDVGSKVGSK At least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and even more preferably at least 95, 96, 97, 98 or 99% homologous to the polypeptide with column number 285) there second amino acid sequence, and corresponds to a known protein NP_872413 and amino acids 72-387 of Q86XK7_HUMAN (SEQ ID NO: 11), also corresponds to amino acids 134-449 of AI581519_P5 (SEQ ID NO: 13), AiwaiefuesukyujijikyueibuieiaijikyuefukeidiaruaitijiesuenudiPijienueiesuaitiaiesueichiemukyuPieidiesujiaiwaiaishidibuienuenuPiPidiefuerujikyuenukyujiaieruenubuiesubuierubuikeiPiesukeiPierushiesubuikyujiaruPiitijieichitiaiesueruesushieruesuALGTPSPVYYWHKLEGRDIVPVKEN Comprises a third amino acid sequence that is at least 90% homologous to EnupititijiaierubuiaijienuerutienuefuikyujiwaiwaikyushitieiaienuaruerujienuesuesushiiaidierutiesuesueichiPiibuijiaiaibuijieieruaijiesuerubuijieieiaiaiaiesubuibuishiefueiaruenukeieikeieikeieikeiiaruenuesukeitiaieiieruiPiemutikeiaienuPiarujiiesuieiemuPiaruidieitikyueruibuitieruPiesuesuaieichiitiJipiditiaikyuiPidiwaiiPikeiPitikyuiPiEipiiPiEipijiesuiPiemueibuiPidierudiaiieruieruiPiitikyuesuieruiPiiPiEPEPESEPGVVVEPLSEDEKGVVKA, the first amino acid sequence, the second amino acid sequence and the third amino acid sequence is a continuous, marked here sequentially polypeptide .

  C. AI581519_P5 (SEQ ID NO: 13) sequence HSSCLSTEGMEEKAVGQCLKMTHVRDARGRCSWTSESSPWEEGKWPDVEAVKGTLDGQQAELQ (SEQ ID NO: 285) at least 70%, optionally at least about 80%, preferably at least about 85%, most preferably at least about 90% An isolated polypeptide encoding the edge portion of AI581519_P5 (SEQ ID NO: 13) comprising an amino acid sequence that is 96, 97, 98 or 99% homologous

  Mutant protein localization was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, mutant proteins are thought to be localized next: membranes.

  In addition, mutant protein AI581519_P5 (SEQ ID NO: 13) contains the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 19 (in terms of amino acid sequence positions, alternative amino acids). (SEQ ID NO: 13)).

Table 19: Amino acid mutations

  The mutant protein has the following regions as determined using InterPro. These areas are shown in Table 20.

Table 20: InterPro area

  The mutant protein AI581519_P5 (SEQ ID NO: 13) is encoded by the next transcript AI581519_T6 (SEQ ID NO: 7), the coding part starting at position 171 and ending at position 1517. What is the transcript? It also has the following SNPs listed in Table 21. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 21: Nucleic acid SNPs

  The mutant protein AI581519_P7 (SEQ ID NO: 14) of the present invention is encoded by the transcript AI581519_T8 (SEQ ID NO: 8). An alignment to one or more published protein sequences is shown in FIG. 3C. A brief description of the relationship of the mutant protein of the present invention to each of its aligned proteins is as follows.

  2. Comparison report between AI581519_P7 (SEQ ID NO: 14) and the known proteins NP_87413 and Q86XK7_HUMAN (SEQ ID NO: 11) (FIG. 3C) (FIG. 3C)

  A. An isolated chimeric polypeptide encoding AI581519_P7 (SEQ ID NO: 14), corresponding to amino acids 1 to 96 of the known proteins NP_87413 and Q86XK7_HUMAN (SEQ ID NO: 11), and amino acids of AI581519_P7 (SEQ ID NO: 14) MFAFAFWKVFLILSCLAQQVSVVQVTIPDGFVNVTVGSNVTLICICYTTTVASREQLSIQWSFFHHKKEMEPISIYFSQGGQAVAIGQFKDRITGSNDP Corresponding to amino acids 97-446 of issue 14), wherein the second amino acid sequence that is at least 90% homologous to BuikeiPiesukeiPierushiesubuikyujiaruPiitijieichitiaiesueruesushieruesueierujitiPiesupibuiwaiwaidaburyueichikeieruijiarudiaibuiPibuikeiienuefuEnupititijiaierubuiaijienuerutienuefuikyujiwaiwaikyushitieiaienuaruerujienuesuesushiiaidierutiesuesueichiPiibuijiaiaibuijieieruaijiesuerubuijieieiaiaiaiesubuibuishiefueiaruenukeieikeieikeieikeiiaruenuesukeitiaieiieruiPiemutikeiaienuPiarujiiesuieiemuPiaruidieitikyueruibuitieruPiesuesuaieichiitiJipiditiaikyuiPidiwaiiPikeiPitikyuiPiEipiiPiEipijiesuiPiemueibuiPidierudiaiieruieruiPiitikyuesuieruiPiiPiEPEPESEPGVVVEPLSEDEKGVVKA, the first amino acid sequence and a second amino acid sequence is a continuous, noted here Polypeptides that are in order.

  C. An isolated chimeric polypeptide encoding an edge portion of AI581519_P7 (SEQ ID NO: 14), comprising a polypeptide having a length “n”, wherein n is at least about 10 amino acids in length, any Optionally comprises at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length, more preferably at least about 50 amino acids in length, and at least two amino acids comprise PV; It has the following structure: sequence starting with any amino acid number 96-x to 96 and ending with any amino acid number 97 + ((n-2) -x), where x varies from 0 to n-2 .

  Mutant protein localization was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, mutant proteins are thought to be localized next: membranes.

  In addition, mutant protein AI581519_P7 (SEQ ID NO: 14) lists the following non-silent SNPs (single nucleotide polymorphisms) (single nucleotide polymorphisms) listed in Table 22 in the light of amino acid sequence positions. (SEQ ID NO: 14)).

Table 22: Amino acid mutations

  The mutant protein has the following regions as determined using InterPro. The area is shown in Table 23.

Table 23: InterPro area

  The mutant protein AI581519_P7 (SEQ ID NO: 14) is encoded by AI581519_T8 (SEQ ID NO: 8), the coding part starting at position 171 and ending at position 1208. The transcript also has the following SNPs listed in Table 24. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 24: Nucleic acid SNPs

  The mutant protein AI581519_P9 (SEQ ID NO: 15) of the present invention has the amino acid sequence encoded by the transcript AI581519_T10 (SEQ ID NO: 9). An alignment to one or more previously published protein sequences is shown in FIG. 3D. A brief description of the relationship of the mutant protein of the present invention to each of the aligned proteins is as follows.

  2. Comparison report between AI581519_P9 (SEQ ID NO: 15) and the known proteins NP_87413 and Q86XK7_HUMAN (SEQ ID NO: 11) (FIG. 3D) (FIG. 3D)

  A. An isolated chimeric polypeptide encoding AI581519_P9 (SEQ ID NO: 15), corresponding to amino acids 1-189 of the known proteins NP_87413 and Q86XK7_HUMAN (SEQ ID NO: 11), and amino acids of AI581519_P9 (SEQ ID NO: 15) It corresponds to 1-189, at least 9 to MVFAFWKVFLILSCLAGQVSVVQVTIPDGFVNVTVGSNVTLICIYTTTVASREQLSIQWSFFHKKEMEPISIYFSQGGQAVAIGQFKDRITGSNDPGNASITISHMQPADSGIYICDVNNPPDFLGQNQGILNVSVLVKPSKPLCSVQGRPETGHTISLSCLSALGTPSPVYYWHKLEGRDIVPVKENF A polypeptide having the first amino acid sequence that is% homologous and the sequence TNHRDFGHWKSDKF (SEQ ID NO: 286) corresponding to amino acids 190-203 of AI581519_P9 (SEQ ID NO: 15), preferably at least 80%, preferably at least 80% Comprises a second amino acid sequence that is at least 85%, more preferably at least 90% and most preferably at least 95, 96, 97, 98 or 99% homologous, wherein said first amino acid sequence and said second amino acid sequence are A polypeptide that is sequential and in the order noted herein.

  C. At least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and even more preferably at least about 95, to the sequence TNHRDFGHWKSDKF (SEQ ID NO: 286) of AI581519_P9 (SEQ ID NO: 15); An isolated polypeptide encoding the edge portion of AI581519_P9 (SEQ ID NO: 15) comprising an amino acid sequence that is 96, 97, 98 or 99% homologous

  Mutant protein localization was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, mutant proteins are thought to be localized next: membranes.

  In addition, mutant protein AI581519_P9 (SEQ ID NO: 15) contains the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 25 (in terms of amino acid sequence positions, alternative amino acids). (SEQ ID NO: 15)).

Table 25: Amino acid mutations

  The mutant protein has the following regions as determined using InterPro. These regions are shown in Table 26.

Table 26: InterPro area

  The mutant protein AI581519_P9 (SEQ ID NO: 15) is encoded by the transcript AI581519_T10 (SEQ ID NO: 9), the coding part starting at position 171 and ending at position 779. What is the transcript? It also has the following SNPs listed in Table 27. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 27: Nucleic acid SNPs

  The mutant protein AI581519_P10 (SEQ ID NO: 16) of the present invention has the amino acid sequence encoded by the transcript AI581519_T11 (SEQ ID NO: 10). An alignment to published protein sequences is shown in FIG. 3E. A brief description of the relationship of the mutant protein of the present invention to each of the aligned proteins is as follows.

  2. Comparison report between AI581519_P10 (SEQ ID NO: 16) and the known proteins NP_87413 and Q86XK7_HUMAN (SEQ ID NO: 11) (FIG. 3E) (FIG. 3E)

  A. An isolated chimeric polypeptide encoding AI581519_P10 (SEQ ID NO: 16), corresponding to amino acids 1 to 229 of the known proteins NP_87413 and Q86XK7_HUMAN (SEQ ID NO: 11), and amino acids of AI581519_P10 (SEQ ID NO: 16) equivalent to 1~229, MVFAFWKVFLILSCLAGQVSVVQVTIPDGFVNVTVGSNVTLICIYTTTVASREQLSIQWSFFHKKEMEPISIYFSQGGQAVAIGQFKDRITGSNDPGNASITISHMQPADSGIYICDVNNPPDFLGQNQGILNVSVLVKPSKPLCSVQGRPETGHTISLSCLSALGTPSPVYYWHKLEGRDIVPVKENFNPTTG A first amino acid sequence that is at least 90% homologous to LVIGNLTNFEQGYYQCTAINRLGNSSSCEILTSS, and a second amino acid sequence RQ (SEQ ID NO: 287) corresponding to amino acids 230-231 of AI581519_P10 (SEQ ID NO: 16), said first amino acid sequence, The second amino acid sequence and the third amino acid sequence are consecutive and are in the order noted here.

  Mutant protein localization was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, mutant proteins are thought to be localized next: membranes.

  In addition, mutant protein AI581519_P10 (SEQ ID NO: 16) contains the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 28 (in terms of amino acid sequence positions, alternative amino acids). (SEQ ID NO: 16)).

Table 28: Amino acid mutations

  The mutant protein has the following regions as determined using InterPro. These regions are shown in Table 29.

Table 29: InterPro area

  The mutant protein AI581519_P10 (SEQ ID NO: 16) is encoded by the transcript AI581519_T11 (SEQ ID NO: 10), the coding part starting at position 171 and ending at position 863. The transcript also has the following SNPs listed in Table 30. (The alternative nucleic acids were listed in light of their position on the nucleic acid sequence.)

Table 30: Nucleic acid SNPs

  According to an optional embodiment of the present invention, a short section associated with the upper cluster is also provided. These sections are up to about 120 bp in length and are included in a separate description.

  The section cluster AI581519_N7 (SEQ ID NO: 120) of the present invention is supported by six libraries. The number of libraries was determined as described above. This section is found in the following transcripts: AI581519_T5 (SEQ ID NO: 6) and AI581519_T6 (SEQ ID NO: 7). Table 31 below lists the start and end positions of this section on each transcript.

Table 31: Section position on transcript

  The section cluster AI581519_N9 (SEQ ID NO: 121) of the present invention is supported by one library. The number of libraries was determined as described above. This section is found in the following transcript: AI581519_T6 (SEQ ID NO: 7). Table 32 below lists the start and end positions of this section on each transcript.

Table 32: Location of sections on transcripts

  V-set and immunoglobulin region-containing 1 (VSIG1) AI581519 transcripts detectable by the amplicon shown in SEQ ID NO: AI581519_seg7 (SEQ ID NO: 190), normal and cancerous ovarian tissue, normal and cancerous lung tissue and different Expression in normal tissues

  seg7-AI581519_seg7 (SEQ ID NO: 190) amplicon and primers AI581519_seg7F1 (SEQ ID NO: 188) and AI581519_seg7R1 (SEQ ID NO: 189) or thus detectable V-set and immunoglobulin region containing 1 (VSIG1) transcripts can be expressed in the ovary Measured by real-time PCR on panels, lung panels and normal panels. The samples used are detailed in Table 4, Table 3 and Table 2, respectively.

  Ovarian panel—Non-detected samples (Sample Nos. 80, 83, 100 and 109, Table 4) were calculated as Ct value 41. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1. The normalized amount of each RT sample is then divided by the median value of the normal sample (sample numbers 52-78, Table 4 above), and each sample is increased by a factor of up to the median value of the normal sample (up -Regulation) value was obtained.

  FIG. 4 is a histogram showing overexpression in cancerous ovary samples versus normal samples of the V-set and immunoglobulin region containing 1 (VSIG1) transcripts shown above.

  As is apparent from FIG. 4, the expression of V-set and immunoglobulin region-containing 1 (VSIG1) transcripts detectable by the above amplicons in mucinous and endometrioid cancer samples is non-cancerous. It was significantly higher than in the samples (sample numbers 52-78, Table 4 above). In particular, at least a 40-fold overexpression was seen in 4 of 12 mucinous cancer samples and 3 of 10 endometrioid cancer samples.

  The significance of these results was verified using statistical analysis as described below. As checked by Fisher's exact test, a threshold of 40-fold overexpression was observed between mucinous and endometrioid cancers and normal samples with P values of 6.02e-003 and 1.54e, respectively. At 002, it was found to make a difference. The upper value indicates that the result is statistically significant.

  Lung Panel—For each RT sample, the expression of the above amplicon was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1. The normalized amount of each RT sample is then divided by the median value of normal samples (sample numbers 51-64 and 69-70, Table 3 above) to determine what each sample is relative to the normal value of the normal sample. The value of up-regulation was obtained.

  FIG. 5 is a histogram showing overexpression in cancerous lung samples versus normal samples of the V-set and immunoglobulin region containing 1 (VSIG1) transcripts shown above.

  As is apparent from FIG. 5, the expression of V-set and immunoglobulin region-containing 1 (VSIG1) transcripts detectable by the above amplicons in adenocarcinoma samples is shown in non-cancerous samples (sample numbers 51-64 and 69). ~ 70, significantly higher than in Table 3) above. In particular, at least a 6-fold overexpression was seen in 11 of the 23 adenocarcinoma samples.

  The significance of these results was verified using statistical analysis as described below. The T value of the difference P-value in the expression level of V-set and immunoglobulin region containing 1 (VSIG1) transcripts detectable by the amplicon in lung adenocarcinoma samples relative to normal tissue samples as 9.36e-003 I decided on. When checked with Fisher's exact test, the 6-fold overexpression threshold was found to make a difference between adenocarcinoma and normal samples with a P value of 8.07e-004.

  The upper value indicates that the result is statistically significant.

  Normal panel—For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1. The normalized amount of each RT sample is then divided by the median value of the ovarian samples (sample numbers 31, 32, 33 and 34, Table 2 above) and the relative expression of each sample relative to the median value of the ovary sample Was obtained. This is shown in FIG. 6A. Also, the normalized amount of each RT sample is divided by the intermediate value of the amount of ovarian samples (sample numbers 31-34, table 2 above) and lung samples (sample numbers 26, 28, 29 and 30, table 2 above). The relative expression value of each sample relative to the intermediate value of) was obtained. This is shown in FIG. 6B.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as just one example to serve as a non-limiting illustration of a suitable primer pair: AI581519_seg7F1 (SEQ ID NO: 188) forward primer and AI581519_seg7R1 (SEQ ID NO: 189) reverse primer

  The present invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to serve as a non-limiting illustration of a suitable amplicon: AI581519_seg7 (SEQ ID NO: 190)

  Forward primer> AI581519_seg7F1 (SEQ ID NO: 188): CACAGCTCTGGCTCCAGTACTTG

  Reverse primer> AI581519_seg7R1 (SEQ ID NO: 189): AGCTACACTCTTCCCGAGCG

  Amplicon> AI581519_seg7 (SEQ ID NO: 190)

  CACAGCTCGTGCCTCAGTACTGAGGGTATGGAGGAAAAGGCAGTCGGTCAGTGTCTAAAAATGACGCACGTAAGAGACGCTCGGGGGAAGATGTAGCT

  V-set and immunoglobulin region-containing 1 (VSIG1) AI581519 transcript detectable by the amplicon shown in SEQ ID NO: AI581519_seg7-9 (SEQ ID NO: 187) in normal and cancerous ovarian tissues and normal and cancerous lung tissues Expression

  seg7-9-AI581519_seg7-9 (SEQ ID NO: 187) amplicon and primers AI581519_seg7-9F1 (SEQ ID NO: 185) and AI581519_seg7-9R1 (SEQ ID NO: 186) or thus detectable V-set and immunoglobulin region containing 1 (VSIG1) ) Transcript expression was measured by real-time PCR in ovary and lung panels. The samples used are detailed in Table 4 and Table 3 above, respectively.

  The ovarian panel-non-detected sample (Sample No. 40, Table 4) was calculated as a Ct value of 41. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1. The normalized amount of each RT sample is then divided by the median value of the normal sample (sample numbers 52-78, Table 4 above), and each sample is increased by a factor of up (up− The value of whether or not (regulation) was obtained.

  FIG. 7 is a histogram showing overexpression in cancerous ovary samples relative to normal samples of the V-set and immunoglobulin region containing 1 (VSIG1) transcripts shown above.

  As is apparent from FIG. 7, the expression of V-set and immunoglobulin region-containing 1 (VSIG1) transcripts detectable by the above amplicons in mucinous cancer samples is shown in non-cancerous samples (sample numbers 52-78, It was significantly higher than in Table 4) above, with a small number of adenocarcinoma samples being higher than in non-cancerous samples. In particular, at least 6-fold overexpression was seen in 6 of 9 mucinous cancer samples and 9 of 37 endometrioid cancer samples.

  The significance of these results was verified using statistical analysis as described below. When checked with Fisher's exact test, the 6-fold overexpression threshold was found to produce a difference between mucinous cancer and normal samples with a P value of 2.75e-004. When checked with Fisher's exact test, the 6-fold overexpression threshold was found to produce a difference between adenocarcinoma and normal samples with a P value of 2.44e-002.

  The upper value indicates that the result is statistically significant.

  Lung Panel—For each RT sample, the expression of the above amplicon was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1. The normalized amount of each RT sample is then divided by the median value of normal samples (sample numbers 51-64 and 69-70, Table 3 above) to determine what each sample is relative to the normal value of the normal sample. The value of up-regulation was obtained.

  FIG. 8 is a histogram showing overexpression in cancerous lung samples versus normal samples of V-set and immunoglobulin region containing 1 (VSIG1) transcripts shown above.

  As is apparent from FIG. 8, expression of V-set and immunoglobulin region-containing 1 (VSIG1) transcripts detectable by the above amplicons in adenocarcinoma samples is shown in non-cancerous samples (sample numbers 51-64 and 69). ~ 70, significantly higher than in Table 3) above, with a small number of non-small cell cancer samples being higher than in non-cancerous samples. In particular, at least 17-fold overexpression was seen in 8 of 18 adenocarcinoma samples.

  The significance of these results was verified using statistical analysis as described below. The P value of the difference in the expression level of V-set and immunoglobulin region-containing 1 (VSIG1) transcripts detectable by the amplicon of lung adenocarcinoma relative to normal tissue samples was determined by T test as 1.17e-002. It was.

  When checked with Fisher's exact test, it was found that the 17-fold overexpression threshold produced a difference between adenocarcinoma and normal samples with a P value of 2.41e-003.

  The upper value indicates that the result is statistically significant.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as an example to serve as a non-limiting illustration of a suitable primer pair: AI581519_seg7-9F1 (SEQ ID NO: 185) forward primer and AI581519_seg7-9R1 (SEQ ID NO: 186) ) Reverse primer

  The present invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to serve as a non-limiting illustration of a suitable amplicon: AI581519_seg7-9 (SEQ ID NO: 187)

  Forward primer> AI581519_seg7-9F1 (SEQ ID NO: 185): AATGACGCACGTAAGAGACGC

  Reverse primer> AI581519_seg7-9R1 (SEQ ID NO: 186): GAGTGCCCTTCACAGCCCTCA

Amplicon> AI581519_seg7-9 (SEQ ID NO: 187)
AATGACGCACGTAAGAGAGGCCTCGGGGAAGATTGTAGCTGGACCTCTGAGTCTCCTGTGGAGGGAGGGAAGTGGCCAGAGTTGAGGCTGTGGAGGGCACTC

  Expression in a blood-specific panel of a V-set and immunoglobulin region containing 1 (VSIG1) AI581519 transcript detectable by the amplicon shown in SEQ ID NO: AI581519seg7-9 (SEQ ID NO: 196)

  Seg7-9-AI581519 seg7-9F3R3 (SEQ ID NO: 196) amplicon and primers AI581519 seg7-9F3 (SEQ ID NO: 194) and AI581519 seg7-9R3 (SEQ ID NO: 195) or thus detectable VSIG1 transcript expression in the blood panel in real time Measured by PCR. The samples used are detailed in Table 1 above. Non-detected samples (sample numbers 28, 33, 83, 85, 90 and 63, Table 1) were calculated according to the Ct value 41. The samples used are detailed in Table 1 above. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1. The normalized amount of each RT sample is then divided by the median value of the normal sample (sample numbers 64, 69-72 and 74-76, Table 1 above) and the relative value of each sample relative to the normal value of the normal sample An expression value was obtained.

  The result of this analysis is shown in the histogram of FIG. The expression of the VSIG1 transcript was high in CD8, CD4 untreated and CD4 + CD25− samples, and higher in normal small intestine and normal stomach samples.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as just one example to serve as a non-limiting illustration of a suitable primer pair: seg7-9F3 forward primer (SEQ ID NO: 194) and seg7-9R3 reverse primer ( SEQ ID NO: 195)

  The present invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as an example to serve as a non-limiting illustration of a suitable amplicon pair: seg7-9F3R3 (SEQ ID NO: 196)

  Forward primer> AI581519seg7-9F3 (SEQ ID NO: 194)

  ATGACGCACGTAAGAGAGGCCTCG

  Reverse primer> AI581519seg7-9R3 (SEQ ID NO: 195)

  GGAGTTCAGCCCTGCTGTCCATCAAG

  Amplicon> AI581519seg7-9F3R3 (SEQ ID NO: 196)

  ATGACGCACGTAAGAGACGCCTCGGGGAAGATGTTAGCTGACCTCTGAGTCTCCTTGGGAGGAGGGGAAGTGGCCAGAGTGTGAGGCTGTGAAGGGCACTCTTGATGGACAGCAGGCGGAACTCC

  Normal and cancerous lung tissue, normal and cancerous ovarian tissue of a V-set and immunoglobulin region-containing 1 (VSIG1) AI581519 transcript detectable by the amplicon represented by the sequence name AI581519_junc7-11F2R2 (SEQ ID NO: 193), Expression in other normal tissues and blood-specific panels

  Junc7-11F2R2-AI581519_junc7-11F2R2 (SEQ ID NO: 193) amplicon and primers AI581519 Junc7-11F2 (SEQ ID NO: 191) and AI581519_junc7-11R2 (SEQ ID NO: 192) or hence detectable VSIG1 transcript expression in the lung panel, Measured by real-time PCR on panels, normal panels and blood panels. The samples used are detailed in Table 3, Table 4, Table 2 and Table 1, respectively. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1.

  Lung Panel—Non-detected sample (Sample No. 49, Table 3) was calculated with a Ct value of 41, then the normalized amount of each RT sample was calculated as normal samples (Sample Nos. 51-64, 69 and 70, divided by the intermediate value of the amount in Table 3) above, to obtain a value indicating how many times each sample was up-regulated with respect to the intermediate value of the normal sample.

  FIG. 10 is a histogram showing overexpression in cancerous lung samples versus normal samples of VSIG1 transcripts shown above.

  As is apparent from FIG. 10, the expression of the VSIG1 transcript detectable by the above amplicon in the adenocarcinoma sample is more prominent than in the non-cancerous samples (sample numbers 51-64, 69 and 70, Table 3 above). it was high. In particular, at least 16-fold overexpression was seen in 10 of the 23 adenocarcinoma samples.

  The significance of these results was verified using statistical analysis as described below. The P value of the difference in the expression level of the VSIG1 transcript detectable by the amplicon of lung adenocarcinoma relative to a normal tissue sample was determined by T test as 5.13e-003.

  When checked with Fisher's exact test, the 16-fold overexpression threshold was found to produce a difference between adenocarcinoma and normal samples with a P value of 1.80e-003.

  The upper value indicates that the result is statistically significant.

  Ovarian panel—Non-detected samples (sample numbers 16, 23, 57, 60-62, 67, 68, 71-74, 77 and 78, Table 4) were set to a Ct value of 41 and calculated accordingly. The normalized amount of each RT sample is then divided by the median value of normal samples (sample numbers 52, 53, 55 and 57-67, Table 4 above), and each sample relative to the median value of normal samples The value of how many times up-regulation was obtained.

  FIG. 11 is a histogram showing overexpression in cancerous ovary samples versus normal samples of VSIG1 transcripts shown above.

  As is clear from FIG. 11, the expression of the VSIG1 transcript detectable by the above amplicon in the mucinous cancer sample is in non-cancerous samples (sample numbers 52, 53, 55 and 57-67, Table 4 above). It was significantly higher, with a small number of adenocarcinoma samples being higher than in non-cancerous samples. In particular, at least 25-fold overexpression was found in 6 of 9 mucinous cancer samples and 10 of 37 adenocarcinoma samples.

  The significance of these results was verified using statistical analysis as described below. When checked by Fisher's exact test, the 25-fold overexpression threshold was different between the mucinous and adenocarcinoma and normal samples with P values of 3.95e-004 and 1.88e-002, respectively. It was found to produce. The above value indicates that the result is statistically significant

  Normal panels—non-detected samples (sample numbers 11-20, 28, 30, 32-34, 36, 38-40, 49 and 56, Table 2) were set to a Ct value of 41 and calculated accordingly. The normalized amount of each RT sample is then divided by the median value of the lung sample (sample numbers 26 and 28-30, Table 2 above) to determine the relative expression of each sample relative to the median value of the lung sample. Got the value. This is shown in FIG. 12A. The normalized amount of each RT sample was divided by the median value of the ovarian sample (sample numbers 31-34, Table 2 above) to give a value for the relative expression of each sample relative to the median value of the ovary sample. This is shown in FIG. 12B.

  Blood panel—non-detected samples (sample numbers 6, 15-19, 22-24, 27, 29, 40, 41, 46-50, 52-58, 60-64, 71 and 76, Table 1), Ct values 41 was calculated accordingly. The normalized amount of each RT sample is then divided by the median value of the normal kidney sample (sample numbers 65-67, Table 1 above) and the relative expression value of each sample relative to the normal value of the normal sample. Obtained.

  The result of this analysis is shown in the histogram of FIG. The above VSIG1 transcript expression is very high in CD8, CD4 naïve and CD4 + CD25− samples, high in some lymphomas, and very high in normal small intestine and normal gastric samples.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as an example to serve as a non-limiting illustration of a suitable primer pair: AI581519_junc7-11F2 (SEQ ID NO: 191) forward primer and AI581519_junc7-11R2 (SEQ ID NO: 192) ) Reverse primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as an example only to serve as a non-limiting description of a suitable amplicon: AI581519_junc7-11F2R2 (SEQ ID NO: 193)

  Forward primer> AI581519_junc7-11F2 (SEQ ID NO: 191)

  GAAGATTGTAGCTGGACCTCTGGAATTTA

  Reverse primer> AI581519_junc7-11R2 (SEQ ID NO: 192)

  GTTGGACCCTGTAATTCGATCTTT

  Amplicon> AI581519_junc7-11F2R2 (SEQ ID NO: 193)

  GAAGATGTAGCTGACCTCTGAGATTTACTTTTCTCAAGGGTGGACAAGCTGTTAGCCATCGGGCAATTTAAAGATCGAATTACAGGGTCCAAC

Example 3 Description of Cluster AA424839 The present invention relates to ILDR1 polypeptides, novel splice variants, and diagnostic and therapeutic agents based thereon.

  According to the present invention, cluster AA424839 (internal ID 71418261) features four target transcripts and one target section, and their names are shown in Tables 33 and 34, respectively. Selected protein variants are shown in Table 35.

Table 33: Target transcripts

Table 34: Target sections

Table 35: Target proteins

  These sequences are variants of a known protein, the immunoglobulin-like region containing receptor 1 (also referred to as RefSeq accession number NP_787120 (SEQ ID NO: 21), ILDR1 alpha, ILDR1 beta, MGC50831), Called a known protein.

  ILDR1, which is the shown immunoglobulin-like region-containing receptor 1 (SEQ ID NO: 21), is described in Hauge et al. (2004) BBRC 323: 970-978, where differential expression of transcripts encoding this protein was shown in painless follicular lymphoma (FL) and diffusely transformed Consistent with large B-cell lymphoma (DLBCL). Gene identification was performed using the cDNA subtraction method on biopsy tissues matched with FL and DLBCL patients. This protein has been shown to have a signal peptide and transmembrane region, as well as an Ig region in the extracellular part, and has 31% identity to the lipolysis-stimulated remnant receptor (LSR). It was found to be a membrane-bound protein.

  According to the present invention, ILDR1 protein and ILDR1 splice variants were expected to be novel B7 / CD28 members. According to the present invention, ILDR1 and ILDR1 splice variants have been shown to be overexpressed in ovarian cancer.

  Expression of the ILDR1 transcript was assessed using the MED Discovery engine described in Example 1 herein. Expression data for Affymetrix probe set 235583_at representing ILDR1 gene data is shown in FIGS. As is clear from the scatter diagram shown in FIG. 14, the expression of ILDR1 transcripts detectable with the above probe set was higher in ovarian cancer than in normal ovary samples. As is apparent from the scatter diagram shown in FIG. 15, the expression of ILDR1 transcripts detectable with the above probe set was higher in colon cancer compared to normal colon samples.

  As indicated above, cluster AA424839 features the four transcripts listed in Table 33 above. These transcripts encode proteins that are variants of the protein immunoglobulin-like region containing receptor 1 (SEQ ID NO: 21). Here, each protein of the present invention will be described.

  The mutant protein AA424839_P3 (SEQ ID NO: 22) of the present invention has the amino acid sequence encoded by the transcript AA424839_T0 (SEQ ID NO: 17). An alignment to published protein sequences is shown in FIG. 16A. A brief description of the relationship between the mutant proteins of the invention and each of their aligned proteins is as follows:

  1. Comparison report between AA424839_P3 (SEQ ID NO: 22) and the known proteins Q86SU0_HUMAN (SEQ ID NO: 21) and NP_787120 (SEQ ID NO: 21) (FIG. 16A):

  A. An isolated chimeric polypeptide encoding AA424839_P3 (SEQ ID NO: 22), corresponding to amino acids 1-215 of the known proteins Q86SU0_HUMAN (SEQ ID NO: 21) and NP_787120 (SEQ ID NO: 21), and AA424839_P3 (SEQ ID NO: 22) MAWPKLPAPWLLLCTWLPAGCLSLLVTVQHTERYVTLFASIILKCDYTTSAQLQDVVVTWRFKSFCKDPIFDYYSASYQAALSLGQDPSNDCNDNQREVRIVAQRRGQNEPVLGVDYRQRKITIQNRADLVINEVMWWDHGVYYCTIEAPGDTSGDPDKEVKLIVLHWLTVIFIILGALLLLLLIGVCWCQ which also corresponds to amino acids 1 to 215 of) A first amino acid sequence that is at least 90% homologous to CPQYCCCYIRCPCCPAHCCCPEEE and a polypeptide having the sequence ALARHRYMKQAQALGPQMMGKPLYWGADRSSQVSSYPMHPPLLLQR (SEQ ID NO: 288) corresponding to amino acids 216-259 of AA424839_P3 (SEQ ID NO: 22) A second amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95, 96, 97, 98, or 99% homologous to the known protein Q86SU0_HUMAN ( SEQ ID NO: 21) and NP_787120 (SEQ ID NO: 21) corresponding to amino acids 216 to 502, AA424839_P (SEQ ID NO: 22) third in amino acids 260-546 at least 90% homologous to the corresponding DLSLPSSLPQMPMTQTTNQPPIANGVLEYLEKELRNLNLAQPLPPDLKGRFGHPCSMLSSLGSEVVERRIIHLPPLIRDLSSSRRTSDSLHQQWLTPIPSRPWDLREGRSHHHYPDFHQELQDRGPKSWALERRELDPSWSGRHRSSRLNGSPIHWSDRDSLSDVPSSSEARWRPSHPPFRSRCQERPRRPSPRESTQRHGRRRRHRSYSPPLPSGLSSWSSEEDKERQPQSWRAHRRGSHSPHWPEEKPPSYRSLDITPGKNSRKKGSVERRSEKDSSHSGRSVVI of An amino acid sequence, wherein the first amino acid sequence, the second amino acid sequence, and the third amino acid sequence are consecutive and in the stated order.

  B. An isolated polypeptide encoding the end of AA424839_P3 (SEQ ID NO: 22), wherein the sequence is AALHRRYMKQAQALGPQMMMGPLKYWGADRSSQVSSYPMHPLLQR (SEQ ID NO: 288) of AA424839_P3 (SEQ ID NO: 22), optionally at least about 80% A polypeptide comprising an amino acid sequence that is preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95, 96, 97, 98, or 99% homologous.

  The localization of this mutant protein was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, this mutant protein is thought to be localized next: membrane.

  Mutant protein AA424839_P3 (SEQ ID NO: 22) has the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 36. (Substituted amino acids were listed in the light of their position on the amino acid sequence (SEQ ID NO: 22).)

Table 36: Amino acid mutations

  The mutant protein has the following regions as determined using InterPro. The region is shown in Table 10.

Table 37: InterPro area

  Mutant protein AA424839_P3 (SEQ ID NO: 22) is encoded by transcript AA424839_T0 (SEQ ID NO: 17), the coding part starting at position 204 and ending at position 1841. This transcript also has the following SNPs listed in Table 38. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 38: Nucleic acid SNPs

  The protein AA424839_P5 (SEQ ID NO: 21) of the present invention has the amino acid sequence encoded by the transcript AA424839_T2 (SEQ ID NO: 18).

  The localization of this protein was determined according to the results of a number of different software programs and analyses, including analysis by SignalP and other special programs. For cells, this protein is thought to be localized next: membrane.

  Protein AA424839_P5 (SEQ ID NO: 21) has the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 39. (Substituted amino acids were listed in the light of their position on the amino acid sequence (SEQ ID NO: 21).)

Table 39: Amino acid mutations

  This protein has the following regions as determined using InterPro. The region is shown in Table 40:

Table 40: InterPro area

  The protein AA424839_P5 (SEQ ID NO: 21) is encoded by the transcript AA424839_T2 (SEQ ID NO: 18), the coding part starting at position 204 and ending at position 1709. This transcript also has the following SNPs listed in Table 41. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 41: Nucleic acid SNPs

  As shown in Table 42, the genomic structure of protein AA424839_P5 (SEQ ID NO: 21) (number of exons associated with the extracellular region of the protein, the length of these exons, the frame of the codon into which the intron was inserted, and the gene structure) The properties of the proteins and the location of the regions are characteristic of the B7 / costimulatory protein family of ligands.

Table 42: Genomic structure and protein properties

  The mutant protein AA424839_P7 (SEQ ID NO: 23) of the present invention has the amino acid sequence encoded by the transcript AA424839_T4 (SEQ ID NO: 19). An alignment to one or more published protein sequences is shown in FIG. 16B. A brief description of the relationship between the mutant proteins of the invention and each of the aligned proteins is as follows:

  1. Comparative report between AA424839_P7 (SEQ ID NO: 23) and known proteins Q86SU0_HUMAN and NP_787120 (SEQ ID NO: 21) (FIG. 16B):

  A. An isolated chimeric polypeptide encoding AA424839_P7 (SEQ ID NO: 23), which corresponds to amino acids 1-126 of the known proteins Q86SU0_HUMAN and NP_787120 (SEQ ID NO: 21), and amino acids 1 to 1 of AA424839_P7 (SEQ ID NO: 23) MAWPKLPAPWLLLCTWLPAGCLSLLVTVQH126 which also corresponds to 126 is an amino acid corresponding to the amino acid sequence of the seventh amino acid, which is identical to the amino acid sequence of the seventh amino acid, which is identical to the amino acid sequence of the seventh amino acid, which is identical to the amino acid sequence of the seventh amino acid, which is equivalent to the amino acid At least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95, 96, 97, 98, or 99% homologous to a polypeptide having KQAQALGPQMMGKPLYWGADRSSQVSSYPMHPLLQR A second amino acid sequence and DLSLPSLLPSLDLLSLGLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPLSLPNL And a third amino acid sequence that is at least 90% homologous, against EsuerueichikyukyudaburyuerutiPiaiPiesuaruPidaburyudieruaruijiaruesueichieichieichiwaiPidiefueichikyuierukyudiaruJipikeiesudaburyueieruiaruaruierudiPiesudaburyuesujiarueichiaruesuesuarueruenujiesuPiaieichidaburyuesudiarudiesueruesudibuiPiesuesuesuieiarudaburyuaruPiesueichipipiefuaruesuarushikyuiaruPiaruaruPiesupiaruiesutikyuarueichijiaruaruaruarueichiaruesuwaiesupipierupiesujieruesuesudaburyuesuesuiidikeiiarukyuPikyuesudaburyuarueieichiaruarujiesueichiesuPieichidaburyuPiiikeiPiPiesuwaiaruesuerudiaitiPijikeiNSRKKGSVERRSEKDSSHSGRSVVI, wherein said first amino acid sequence, the second amino acid sequence, and a third amino acid sequence is a continuous, this serial A chimeric polypeptide that is in the order in which it is produced.

  The localization of this mutant protein was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, this mutant protein is thought to be localized: secretion.

  Mutant protein AA424839_P7 (SEQ ID NO: 23) has the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 43. (The alternative amino acids are listed in the light of their position on the amino acid sequence.)

Table 43: Amino acid mutations

  Mutant protein AA424839_P7 (SEQ ID NO: 23) is encoded by transcript AA424839_T4 (SEQ ID NO: 19), the coding part of which starts at position 204 and ends at position 1574. This transcript also has the following SNPs listed in Table 44. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 44: Nucleic acid SNPs

  The mutant protein AA424839_1_P11 (SEQ ID NO: 24) of the present invention has an amino acid sequence encoded by the transcript AA424839_1_T7 (SEQ ID NO: 20). An alignment to one or more published protein sequences is shown in FIG. 16C.

  The localization of this mutant protein was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, this mutant protein is thought to be localized next: membrane.

  Mutant protein AA424839_1_P11 (SEQ ID NO: 24) has the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 45. (Substituted amino acids were listed in the light of their position on the amino acid sequence (SEQ ID NO: 24).)

Table 45: Amino acid mutations

  The mutant protein has the following regions as determined using InterPro. The region is shown in Table 46:

Table 46: InterPro area

  Mutant protein AA424839_1_P11 (SEQ ID NO: 24) is encoded by transcript AA424839_1_T7 (SEQ ID NO: 20), the coding part starting at position 204 and ending at position 1670. This transcript also has the following SNPs listed in Table 47. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 47: Nucleic acid SNPs

  The section cluster AA424839_N18 (SEQ ID NO: 122) of the present invention is supported by 10 libraries. The number of libraries was determined as described above. This section is found in the following transcripts: AA424839_T0 (SEQ ID NO: 17), AA424839_T2 (SEQ ID NO: 18), AA424839_T4 (SEQ ID NO: 19), and AA424839_1_T7 (SEQ ID NO: 20). Table 48 below lists the start and end positions of this section on each transcript.

Table 48: Section position on transcript

  Expression of immunoglobulin-like region-containing receptor 1 (ILDR1) AA424839 transcripts detectable in normal and cancerous ovarian tissues, as well as in different normal tissues, by the amplicon represented by the sequence name AA424839_seg18wt (SEQ ID NO: 199)

  seg18wt-AA424839_seg18wt (SEQ ID NO: 199) amplicon and of immunoglobulin-like region containing receptor 1 (ILDR1) transcripts detectable by or according to primers AA424839_seg18wtF1 (SEQ ID NO: 197) and AA424839_seg18wtR1 (SEQ ID NO: 198) Expression was measured by real-time PCR in ovarian and normal panels. Details of the used samples are shown in Tables 4 and 2 above. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1.

  Ovarian panel—Next, the normalized amount of each RT sample is divided by the median value of the normal sample (sample numbers 52-78, Table 4 above), and each sample is compared to the median value of the normal sample. A value of how many times it was enhanced was obtained.

  FIG. 17 is a histogram showing overexpression in a cancerous ovary sample versus a normal sample of the immunoglobulin-like region containing receptor 1 (ILDR1) transcript shown above.

  As is apparent from FIG. 17, expression of an immunoglobulin-like region-containing receptor 1 (ILDR1) transcript that is detectable by the amplicon described above in serous carcinoma, mucinous cancer, and adenocarcinoma samples Was significantly higher than the non-cancerous samples (sample numbers 52-78, Table 4 above). In particular, at least 25 times more than 36 samples in 37 adenocarcinoma samples, specifically 17 samples in 18 serous cancer samples, 9 samples in 9 mucinous cancer samples, and 10 samples in class 10 endometrial cancer samples. Overexpression was seen.

  The significance of these results was verified using statistical analysis as described below. Normal tissue samples of the level of expression of immunoglobulin-like region containing receptor 1 (ILDR1) transcripts detectable by the amplicons described above in ovarian adenocarcinoma samples, serous cancer samples, mucinous cancers, and endometrioid cancers P values for the difference to were determined by T-test as 3.85e-010, 6.21e-005, 1.10e-003, and 2.94e-004, respectively.

  When checked with Fisher's exact test, a 25-fold overexpression threshold was found between adenocarcinoma, serous cancer, mucinous cancer, endometrioid cancer, and normal samples with a P value of 3.31e-017, Differences were found to occur at 1.63e-011, 1.06e-008, and 2.87e-009.

  The above values indicate that this result is statistically significant.

  Normal Panel—Next, as shown in FIG. 18, the normalized amount of each RT sample is divided by the median value of the amount of ovarian samples (sample numbers 31, 32, 33, and 34, Table 2 above). The value of the relative expression of each sample relative to the median value of the ovary sample was obtained.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as an example to serve as a non-limiting illustration of a suitable primer pair: AA424839_seg18wtF1 (SEQ ID NO: 197) forward primer; and AA424839_seg18wtR1 (SEQ ID NO: 198) reverse direction Primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to serve as a non-limiting description of a suitable amplicon: AA424839_seg18wt (SEQ ID NO: 199).

  Forward primer> AA424839_seg18wtF1 (SEQ ID NO: 197)

  AGCACCACCACTCATCCCTGA

  Reverse primer> AA424839_seg18wtR1 (SEQ ID NO: 198)

  TGCCTTCCCACTCCACGATG

  Amplicon> AA424839_seg18wt (SEQ ID NO: 199)

  AGCACCACCATCATCCCTGATTTCCCACCAGGAGCTCCCAGGACCGGGGGCAAAGTCTTGGGGCATTGGAAAGAAGGGAGTTGGACCCATCGTGGATGTGGAAGGCA

  Expression of an immunoglobulin-like region-containing receptor 1 (ILDR1) AA424839 transcript detectable in normal and cancerous ovarian tissue, or in different normal tissues, by the amplicon shown in SEQ ID NO: AA424839_seg14-16 (SEQ ID NO: 202)

  The expression of ILDR1 transcripts detectable by or according to seg14-16-AA424839_seg14-16 (SEQ ID NO: 202) amplicon and primers AA424839_seg14-16F1 (SEQ ID NO: 200) and AA424839_seg14-16R1 (SEQ ID NO: 201) Measured by real-time PCR on ovarian panels or normal panels. Details of the used samples are shown in Tables 4 and 2 above. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1.

  Ovarian panel—The normalized amount of each RT sample is then expressed as the median of the amount of normal samples (sample numbers 52-55, 58, 59, 63-69, and 71-78, Table 4 above). The value of how many times each sample was enhanced relative to the intermediate value of the normal sample was obtained.

  FIG. 19 is a histogram showing overexpression of the ILDR1 transcript shown above in cancerous ovary samples compared to normal samples.

  As is apparent from FIG. 19, the expression of ILDR1 transcripts detectable by the above amplicons in serous, mucinous, and adenocarcinoma samples is shown in non-cancerous samples (sample numbers 52-55, 58 59, 63-69, and 71-78, significantly higher than Table 4) above. In particular, at least 14-fold overexpression was seen in 33 samples of 37 adenocarcinoma samples: 14 samples of 18 serous cancer samples, 9 samples of 9 mucinous cancer samples, and 10 samples of 10 adenocarcinoma samples.

  The significance of these results was verified using statistical analysis as described below.

  P value of the difference in expression levels of ILDR1 transcripts detectable by the amplicons described above from normal tissue samples in ovarian adenocarcinoma samples, ovarian serous cancer samples, ovarian mucinous cancer samples, and ovarian endometrial cancer samples Were determined by T test as 1.00e-008, 4.79e-004, 4.97e-004, and 6.93e-005, respectively.

  When checked by Fisher's exact test, a 14-fold overexpression threshold was found between adenocarcinoma, serous cancer, mucinous cancer, endometrioid cancer, and normal samples with a P value of 3.78e-012, 2.03e-007, 6.99e-008, and 2.25e-008 were found to make a difference.

  The above values indicate that this result is statistically significant.

  Normal panel—The normalized amount of each RT sample is then divided by the median value of the ovary sample (sample numbers 31-34, Table 2 above) and the relative value of each sample to the median value of the ovary sample Expression values were obtained.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as an example to serve as a non-limiting illustration of a suitable primer pair: AA424839_seg14-16F1 (SEQ ID NO: 200) forward primer; and AA424839_seg14-16R1 (SEQ ID NO: 201) Reverse primer

  Results showing the expression in different normal tissues of the ILDR1 AA424839 transcript, detectable by the amplicon shown in SEQ ID NO: AA424839_seg14-16 (SEQ ID NO: 202) are presented in FIG.

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained by way of example only to serve as a non-limiting illustration of a suitable amplicon: AA424839_seg14-16F1R1 (SEQ ID NO: 202).

  Forward primer> AA424839_seg14-16F1 (SEQ ID NO: 200)

  GCCACCGCTCATACATGAAGCA

  Reverse primer> AA424839_seg14-16R1 (SEQ ID NO: 201)

  CTGGACGGCAGGGGACAAAT

  Amplicon> AA424839_seg14-16F1R1 (SEQ ID NO: 202)

  GCCACCGCTACATGAAGCAGGCCCCAGCCCTAGGTCCTCCAGATGATGGGAAAACCCTGTACTGGGGGGCGCGACAGGAGCTCCCAGGTTTCATCTTATCCAATGCACCCGCTGCTGCAGCGGAATTTTGCTCCCTGCCGTCCAG

  Expression of an immunoglobulin-like region-containing receptor 1 (ILDR1) AA424839 transcript detectable in the blood-specific panel by the amplicon represented by the sequence name AA424839_seg11-14F3R3 (SEQ ID NO: 205)

  The expression of ILDR1 transcripts detectable by or according to seg11-14-AA424839seg11-14F3R3 (SEQ ID NO: 205) amplicon and primers AA424839seg11-14F3 (SEQ ID NO: 203) and AA424839seg11-14R3 (SEQ ID NO: 204) Measured by real-time PCR on a blood panel. Details of the used samples are shown in Table 1 above. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1. Next, the normalized amount of each RT sample is divided by the median of the amount of normal kidney samples (sample numbers 65-67, Table 1 above) and the relative expression of each sample relative to the median value of normal kidney samples. Was obtained.

  The result of this analysis is shown in the histogram of FIG. The expression of the ILDR1 transcript described above was seen in several lymphomas and cell lines, but this expression was as high as in normal kidney samples.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pairs were used as just one example to serve as a non-limiting illustration of suitable primer pairs: seg11-14F3 forward primer; and seg11-14R3 reverse primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as an example to serve as a non-limiting illustration of a suitable amplicon: seg11-14F3R3

  Forward primer> AA424839_seg11-14F3 (SEQ ID NO: 203)

  TCCTCCCTCCTCGCTGCTGATTG

  Reverse primer> AA424839_seg11-14R3 (SEQ ID NO: 204)

  TGGGCCTGTCTTCATGTAGCG

  Amplicon> AA424839_seg11-14F3R3 (SEQ ID NO: 205)

  TCCTCCTCCTGCTGCTGGATGGAGGTGGCTGTGCTCCAGTGCTGTCCCTAGTATTGCTGCTCTCATATCCGCTGTCCCCTCGCTCCTGCCCCACTGCTGCTGTCCCTGAGAGACCCTGGCCCCGCCACG

Example 4: Description of Cluster AI216611 The present invention relates in particular to a putative B7 / CD28 member, referred to as AI216611, and diagnostic and therapeutic agents based thereon. According to the present invention, cluster AI216611 (internal ID 70605934) features two target transcripts and three target sections, the names of which are shown in Tables 49 and 50, respectively. Selected proteins are shown in Table 51.

Table 49: Target transcripts

Table 50: Target sections

Table 51: Target proteins

  AI216611 is an unidentified gene whose full-length mRNA has not been deposited in Genbank. The protein corresponding to AI216611_P0 is found in the human genome annotation by Celera based on computer analysis and genome translation (DNA sequence accession number CH4701065) (Venter, J. C et al., 2001 Science 291, 1304-1351) . The protein corresponding to AI216611_P0 is also listed in other sequences disclosed in WO2003025148. However, this application does not characterize its function, and more particularly does not teach that it is a B7 / CD28 costimulatory protein.

  The protein corresponding to the AI216611_P1 sequence is a novel protein and is only partially similar to the polypeptide reported in WO205108415 assigned to Biogen-Idec (of 199 amino acids). 186), this international publication claims that this polypeptide is a transmembrane protein that can be targeted in the treatment of hyperproliferative disorders. WO205108415 does not report the function of this polypeptide. More specifically, it does not indicate that this is a B7 / CD28 costimulatory protein.

  According to the present invention, AI216611 is expected to be a novel B7 / CD28 family member based on the presence of the IgV region, a characteristic structural property of B7 / CD28 family members. Furthermore, AI216611 is similar to known CD28 family members with respect to the size of its exons and the location of IgV and transmembrane regions within these exons. Like all known B7 / CD28 members, AI216611 is a type I membrane protein. In accordance with the present invention, two transcripts of alternatively spliced AI216611 are provided, each containing a unique region within the intracellular region. It is demonstrated in the present invention that the expression of AI216611 and its variants is down-regulated in colon cancer, further supporting the role of immune costimulation.

  As mentioned above, contig AI216611 features the two transcripts listed in Table 49 above. Here, each protein of the present invention will be described.

  The protein AI216611_P0 (SEQ ID NO: 43) of the present invention has an amino acid sequence encoded by the transcription product AI216611_T0 (SEQ ID NO: 41).

  The localization of this protein was determined according to the results of a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, this mutant protein is thought to be localized next: membrane.

  Protein AI216611_P0 (SEQ ID NO: 43) has the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 52. (Substituted amino acids are listed in the light of their positions on the amino acid sequence (SEQ ID NO: 43).)

Table 52: Amino acid mutations

  This protein has the following regions as determined using InterPro. The region is shown in Table 53:

Table 53: InterPro area

  The protein AI216611_P0 (SEQ ID NO: 43) is encoded by the transcript AI216611_T0 (SEQ ID NO: 41), the coding part starting at position 1 and ending at position 600. This transcript also has the following SNPs listed in Table 54. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 54: Nucleic acid SNPs

  As shown in Table 55, the genomic structure of protein AI216611_P0 (SEQ ID NO: 43) (number of exons associated with the extracellular region of the protein, length of these exons, frame of codons inserted with introns, and within gene structure) The properties of the proteins and the location of the regions are characteristic of the B7 / costimulatory protein family of receptors.

Table 55: Genomic structure and protein properties

  The protein AI216611_P1 (SEQ ID NO: 44) of the present invention has the amino acid sequence encoded by the transcript AI216611_T1 (SEQ ID NO: 42).

  The localization of this protein was determined according to the results of a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, this mutant protein is thought to be localized next: membrane.

  Protein AI216611_P1 (SEQ ID NO: 44) has the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 56. (Substituted amino acids were listed in the light of their position on the amino acid sequence (SEQ ID NO: 44).)

Table 56: Amino acid mutations

  This protein has the following regions as determined using InterPro. The region is shown in Table 57:

Table 57: InterPro area

  The protein AI216611_P1 (SEQ ID NO: 44) is encoded by the transcript AI216611_T1 (SEQ ID NO: 42), the coding part starting at position 1 and ending at position 597. This transcript also has the following SNPs listed in Table 58. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 58: Nucleic acid SNPs

  As noted above, cluster AI216611 features the three intercepts listed in Table 50 above. These sections are part of the nucleic acid sequences described elsewhere herein because they are of particular interest. Here, each section of the present invention will be described.

  The slice cluster AI216611_N2 (SEQ ID NO: 126) of the present invention is supported by 4 libraries. The number of libraries was determined as described above. This section is found in the following transcripts: AI216611_T0 (SEQ ID NO: 41) and AI216611_T1 (SEQ ID NO: 42). Table 59 below lists the start and end positions of this section on each transcript.

Table 59: Section position on transcript

  The slice cluster AI216611_N4 (SEQ ID NO: 127) of the present invention is supported by 4 libraries. The number of libraries was determined as described above. This section is found in the following transcripts: AI216611_T0 (SEQ ID NO: 41) and AI216611_T1 (SEQ ID NO: 42). Table 60 below lists the start and end positions of this section on each transcript.

Table 60: Section position on transcript

  The section cluster AI216611_N6 (SEQ ID NO: 128) of the present invention is supported by two libraries. The number of libraries was determined as described above. This section is found in the following transcripts: AI216611_T0 (SEQ ID NO: 41) and AI216611_T1 (SEQ ID NO: 42). Table 61 below lists the start and end positions of this section on each transcript.

Table 61: Section position on transcript

  Expression of AI216611 transcripts detectable in normal and cancerous colon tissues, as well as in different normal tissues, by the amplicon shown as SEQ ID AI216611_junc4-6F2R2 (SEQ ID NO: 208)

  Expression of AI216611 transcripts detectable by or in accordance with junc4-6-AI216611_junc4-6F2R2 (SEQ ID NO: 208) amplicon and primers AI216611_junc4-6F2 (SEQ ID NO: 206) and AI216611 junc4-6R2 (SEQ ID NO: 207) Was measured by real-time PCR in colon and normal panels. Details of the samples used are shown in Tables 5 and 2 above. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1.

  Colon panel—The normalized amount of each RT sample was then divided by the median of the amount of normal samples (sample numbers 42-70, Table 5 above). Then, the reciprocal of this ratio was calculated to obtain a value indicating how many times each sample was controlled downward with respect to the intermediate value of the normal sample.

  FIG. 22 is a histogram showing downregulation of the AI216611 transcript shown above in cancerous colon samples compared to normal samples.

  As is apparent from FIG. 22, the expression of AI216611 transcripts detectable by the above amplicons in cancer samples was significantly lower than in non-cancerous samples (sample numbers 42-70, Table 5 above). In particular, 27 out of 55 adenocarcinoma samples showed at least a 5-fold down-regulation.

  The significance of these results was verified using statistical analysis as described below. The P value of the difference in the expression level of AI216611 transcripts detectable in the above amplicon relative to normal tissue samples in colon cancer samples was determined by T test as 2.39e-005.

  When checked with Fisher's exact test, it was found that a threshold of 5 times down-regulation produced a difference between cancer and normal samples with a P value of 5.09e-007.

  The above values indicate that this result is statistically significant.

  Normal panels—Non-detected samples (sample numbers 50, 52, 54, and 56, Table 2) were Ct values 41 and calculated accordingly. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1. Next, as shown in FIG. 23, the normalized amount of each RT sample is divided by the median value of the amount of the colon sample (sample numbers 3, 4, and 5, Table 2 above) to obtain the median of the colon sample. Relative expression values for each sample relative to values were obtained.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as an example to serve as a non-limiting illustration of a suitable primer pair: AI216611_junc4-6F2 (SEQ ID NO: 206) forward primer; and AI216611_junc4-6R2 (SEQ ID NO: 207) Reverse primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to serve as a non-limiting illustration of a suitable amplicon: AI216611_junc4-6F2R2 (SEQ ID NO: 208)

  Forward primer> AI216611_junc4-6F2 (SEQ ID NO: 206): CCGCCAGTATTAATCAGCCCTCATG

  Reverse primer> AI216611_junc4-6R2 (SEQ ID NO: 207): AATCTCCCTCAGTTGTCTTTCTTTG

  Amplicon> AI216611_junc4-6F2R2 (SEQ ID NO: 208)

  CCGCAGTATTATAATCAGCCTCATGTGGGTTTGATAATAAGTGTGCATATAAAATTTCAGAGGAAGAGAAGACACAAAACTCAAAGAAAAGCACAACTGAGGGAGATT

  Expression of AI216611 transcripts detectable in normal and cancerous colon tissues, as well as in different normal tissues, by the amplicon shown as SEQ ID AI216611_seg2WT (SEQ ID NO: 211)

  Expression of AI216611 transcripts detectable by or according to seg2WT-AI216611_seg2WT (SEQ ID NO: 211) amplicon and primers AI216611_seg2WTF1 (SEQ ID NO: 209) and AI216611_seg2WTR1 (SEQ ID NO: 210) is real-time in colon and normal panels Measured by PCR. Details of the samples used are shown in Tables 5 and 2 above.

  Colon panel—Non-detected sample (Sample No. 33, Table 5) was given a Ct value of 41 and calculated accordingly. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1. Next, the normalized amount of each RT sample was divided by the median value of the amount of normal samples (sample numbers 42-70, Table 5 above). Then, the reciprocal of this ratio was calculated to obtain a value indicating how many times each sample was controlled downward with respect to the intermediate value of the normal sample.

  FIG. 24 is a histogram showing downregulation of the AI216611 transcript shown above in cancerous colon samples compared to normal samples.

  As is clear from FIG. 24, expression of AI216611 transcripts detectable by the amplicons in cancer samples was significantly lower than in non-cancerous samples (sample numbers 42-70, Table 5 above). In particular, 25 out of 55 adenocarcinoma samples showed at least 5-fold down-regulation.

  The significance of these results was verified using statistical analysis as described below. When checked with Fisher's exact test, it was found that a threshold of 5 times down-regulation produced a difference between cancer and normal samples with a P value of 2.00e-006.

  The above values indicate that this result is statistically significant.

  Normal panel—For each RT sample, the expression of the above amplicon was normalized to the normalization factor calculated from the expression of these housekeeping genes described in Example 1 herein. Next, as shown in FIG. 25, the normalized amount of each RT sample is divided by the median value of the amount of colon samples (sample numbers 3, 4, and 5, Table 5 above) to obtain the median of colon samples. The value of the relative expression of each sample relative to the value was obtained.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as just one example to serve as a non-limiting illustration of a suitable primer pair: AI216611_seg2WTF1 (SEQ ID NO: 209) forward primer; and AI216611_seg2WTR1 (SEQ ID NO: 210) reverse direction Primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to serve as a non-limiting description of a suitable amplicon: AI216611_seg2WT (SEQ ID NO: 211)

  Forward primer> AI216611_seg2WTF1 (SEQ ID NO: 209)

  GAACCGCAGAAGATCGTGGAGT

  Reverse primer> AI216611_seg2WTR1 (SEQ ID NO: 210)

  CTGAAGAGCTGGATGGAGCC

  Amplicon> AI216611_seg2WT (SEQ ID NO: 211)

  GAACGCAGAAGAGTCGGATGTGGAAACCAGGGACTCAGGCCAACATCTCTCAAAGCCCAAGGACAGAGTTCGCACCTTTGACAACGGCTCCATCCAGCTCTTCAG

  Expression of AI216611 transcripts detectable by the amplicon shown in SEQ ID NO: AI216611_junc4-6 (SEQ ID NO: 214) in a blood specific panel

  The expression of AI216611 transcripts detectable by or according to the junc4-6-junc4-6F4R4 (SEQ ID NO: 214) amplicon and the primers junc4-6F4 (SEQ ID NO: 212) and junc4-6R4 (SEQ ID NO: 213) are Measured by real-time PCR on a blood panel. Details of the used samples are shown in Table 1 above. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1. Next, the normalized amount of each RT sample is divided by the median of the amount of normal kidney samples (sample numbers 65-67, Table 1 above) and the relative expression of each sample relative to the median value of normal kidney samples. Was obtained.

  The result of this analysis is shown in the histogram of FIG. The expression of the AI216611 transcript described above was significantly higher in the normal samples tested compared to the different blood specific samples in the panel.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pairs were used as just one example to serve as a non-limiting illustration of suitable primer pairs: junc4-6F4 (SEQ ID NO: 212) forward primer; and junc4-6R4 reverse primer (SEQ ID NO: 213)

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to serve as a non-limiting description of a suitable amplicon: junc4-6F4R4 (SEQ ID NO: 214)

  Forward primer AI216611junc4-6F4 (SEQ ID NO: 212): CTGCACTTTGTCGCTGTCATC

  Reverse primer AI216611junc4-6R4 (SEQ ID NO: 213): CAATCTCCCTCAGTTGTCTTTCTTTG

  Amplicon AI216611junc4-6F4R4 (SEQ ID NO: 214)

  CTGCACTTTGTGCGCTGTCATCTCTGCTCTTTCCTGCTGCTGTGGCCGCAGTATTATAATCAGCTCCATGTGGGTTTGTATAATAGTGTGCATATAAATTTTCAGAGAGAAGAAGACACAAACTCAAGAGAAAGCACATGAG

  Expression of AI216611 transcripts detectable in the blood-specific panel by the amplicon shown as SEQ ID AI216611_junc2-4seg5 (SEQ ID NO: 217)

  The expression of AI216611 transcripts detectable by or according to junc2-4seg5-junc2-4seg5F3R4 amplicon (SEQ ID NO: 217) and primers junc2-4seg5F3 (SEQ ID NO: 215) and junc2-4seg5R4 (SEQ ID NO: 216) Measured by real-time PCR on a blood panel. Details of the used samples are shown in Table 1 above. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1. Next, the normalized amount of each RT sample is divided by the median of the amount of normal kidney samples (sample numbers 65-67, Table 1 above) and the relative expression of each sample relative to the median value of normal kidney samples. Was obtained.

  The result of this analysis is shown in the histogram of FIG. The expression of the AI216611 transcript described above was significantly higher in the normal samples tested compared to the different blood specific samples in the panel.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as just one example to serve as a non-limiting illustration of a suitable primer pair: junc2-4seg5F3 (SEQ ID NO: 215) forward primer; and junc2-4seg5R4 (SEQ ID NO: 216) Reverse primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to serve as a non-limiting description of a suitable amplicon: junc2-4seg5F3R4 (SEQ ID NO: 217)

  Forward primer AI216611 junc2-4seg5F3 (SEQ ID NO: 215): TCGTCCACGTCTCTGAGATCC

  Reverse primer AI216611 junc2-4seg5R4 (SEQ ID NO: 216): CACCTCTGGCCCTAAAAACCACTC

  Amplicon> AI216611junc2-4seg5F3R4 (SEQ ID NO: 217)

  TGCTGCACGTCTCTGAGATCCTCTATGAAGACCTGCACTTTTGTCCGCTGTCATGGTCGGTGTGTCTGGTCGTGTG

  Expression of AI216611 transcripts detectable in normal and cancerous colon tissue by the amplicon shown as SEQ ID AI216611_junc2-4seg5F2R2 (SEQ ID NO: 220)

  Junc2-4seg5F2R2-AI216611_junc2-4seg5F2R2 (SEQ ID NO: 220) amplicon, and primers AI216611_junc2-4seg5F2 (SEQ ID NO: 218) and AI216611_junc2-4seg5R2 (SEQ ID NO: 219) can be detected according to these, or product A21 can be detected according to A11 Measured by real-time PCR on colon panels. Details of the used samples are shown in Table 5 above. Non-detected samples (sample numbers 28, 33, 83, 85, 90, and 63, Table 5) were calculated according to the Ct value 41. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1. Next, the normalized amount of each RT sample was divided by the median value of the amount of normal samples (sample numbers 42-62 and 64-70, Table 5 above). Then, the reciprocal of this ratio was calculated to obtain a value indicating how many times each sample was controlled downward with respect to the intermediate value of the normal sample.

  FIG. 28 is a histogram showing downregulation of the AI216611 transcript shown above in a cancerous colon sample compared to a normal sample.

  As is clear from FIG. 28, the expression of AI216611 transcripts that can be detected by the above amplicons in cancer samples is from non-cancerous samples (sample numbers 42-62 and 64-70, Table 5 above). Was also significantly lower. In particular, at least 5-fold down-regulation was observed in 31 out of 55 adenocarcinoma samples.

  The significance of these results was verified using statistical analysis as described below. The P value of the difference in the expression level of AI216611 transcripts detectable by the amplicon above in the colon cancer sample relative to the normal tissue sample was determined by T test as 5.29e-003.

  When checked with Fisher's exact test, it was found that a threshold of 5 times down-regulation produced a difference between cancer and normal samples with a P value of 4.18e-008.

  The above values indicate that this result is statistically significant.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as an example to serve as a non-limiting illustration of a suitable primer pair: AI216611_junc2-4seg5F2 (SEQ ID NO: 218) forward primer; and AI216611_junc2-4seg5R2 (SEQ ID NO: 219) Reverse primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to serve as a non-limiting illustration of a suitable amplicon: AI216611_junc2-4seg5F2R2 (SEQ ID NO: 220)

  Forward primer> AI216611_junc2-4seg5F2 (SEQ ID NO: 218)

  GCTGCACGTCTCTGGAGATCCT

  Reverse primer> AI216611_junc2-4seg5R2 (SEQ ID NO: 219)

  CACCTCTGGCCTCAAAAACCA

  Amplicon> AI216611_junc2-4seg5F2R2 (SEQ ID NO: 220)

  GCTGCATGGTGCTGCGTCGTGGTCGTGATGCATGG

Example 5: Description of Cluster H68654_1 The present invention relates to LOC25303012 polypeptides, novel splice variants, and diagnostic and therapeutic agents based on them.

  According to the present invention, cluster H68654_1 (internal ID 76432882) features 10 target transcripts and 3 target sections, the names of which are shown in Tables 62 and 63, respectively. Selected protein variants are shown in Table 64.

Table 62: Target transcripts

Table 63: Target sections

Table 64: Proteins of interest

  These sequences are variants of known proteins, hypothetical protein LOC253012 isoform 1 (RefSeq accession number NP_001034461 (SEQ ID NO: 35), NP_937794 (SEQ ID NO: 36)). Call.

  The known LOC253012 is based on a hypothesis discovered computationally in the secreted protein discovery strategy project (SPDI), a large-scale effort to identify novel human secreted and transmembrane proteins It is a protein (Clark et al 2003, Genome Res. 13: 2265-2270). The closest paralog confirmed experimentally is a hepatocyte adhesion molecule (Refseq accession number NP — 687935) (E value e-21).

  LOC253012 antigen is available from Genentech Inc. LOC253012 (PRO346) corresponding to H68654_1_P2 (SEQ ID NO: 35) is reported to be differentially expressed in lung tumor samples. The patent claims that the corresponding polypeptide can be used in the development of antibodies against the disclosed PRO antigens including PRO346. This patent application further suggests that antibodies against the class of polypeptides reported therein, including PRO346, can be used in the treatment and diagnosis of cancer, specifically in the diagnosis and treatment of lung cancer. However, this Genentech patent application does not teach that LOC253012 (PRO346) is differentially expressed in small cell lung cancer. In addition, Genentech Inc. There is no specific teaching regarding that anti-PRO346 antibodies can be used for the treatment of small cell lung cancer and / or the modulation of costimulation of APC / T cell activity. In addition, Genentech Inc. There is no teaching that PRO346 is an immune co-stimulatory protein, or more specifically a B7 family member. Genentech Inc. There is no teaching regarding the use of antibodies to the PRO346 antigen for immune co-stimulation, or in particular for modulation of the B7 costimulatory pathway.

  According to the present invention, LOC253012 was expected to be a novel immune costimulatory protein, in particular the B7 costimulatory protein. This expectation was based on the presence of both the IgV region and IgC2, which are characteristic structural features of B7 family members. Like other B7 members, LOC253012 is a type I membrane protein. LOC253012 and variants thereof have been demonstrated in the present invention to be overexpressed in lung cancer.

  Using the MED Discovery engine described in Example 1 herein, the expression of the LOC253012 transcript was evaluated. Expression data for the Affymetrix probe set 242601_at representative of LOC253012 gene data is shown in FIG. As is clear from the scatter diagram shown in FIG. 29, the expression of the LOC2530112 transcript detectable with the above probe set was higher in lung cancer than in normal lung samples.

  As indicated above, cluster H68654 features the 10 transcripts listed in Table 62 above. These transcripts encode a protein, a protein that is a variant of the hypothetical protein LOC25303012 isoform 1 (SEQ ID NO: 35). Here, each mutant protein of the present invention will be described.

  The mutant protein H68654_1_P2 (SEQ ID NO: 35) of the present invention has the amino acid sequence encoded by the transcripts H68654_1_T0 (SEQ ID NO: 25) and H68654_1_T5 (SEQ ID NO: 27).

  The localization of this mutant protein was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, this mutant protein is thought to be localized next: membrane.

  In addition, mutant protein H68654_1_P2 (SEQ ID NO: 35) has the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 65. (The alternative amino acids are listed in the light of their position on the amino acid sequence.)

Table 65: Amino acid mutations

  The mutant protein has the following regions as determined using InterPro. The region is shown in Table 66:

Table 66: InterPro area

  The coding part of the transcript H68654_1_T0 (SEQ ID NO: 25) starts at position 79 and ends at position 1464. This transcript also has the following SNPs listed in Table 67. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 67: Nucleic acid SNPs

  The coding part of the transcript H68654_1_T5 (SEQ ID NO: 27) starts at position 79 and ends at 1464. This transcript also has the following SNPs listed in Table 68. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 68: Nucleic acid SNPs

  As shown in Table 69, the genomic structure of protein H68654_1_P2 (SEQ ID NO: 35) (number of exons associated with the extracellular region of the protein, length of these exons, the frame of the codon into which the intron was inserted, and within the gene structure The properties of the proteins and the location of the regions are characteristic of the B7 / costimulatory protein family of ligands.

Table 69: Genomic structure and protein properties

  The mutant protein H68654_1_P5 (SEQ ID NO: 36) of the present invention has an amino acid sequence encoded by the transcript H68654_1_T4 (SEQ ID NO: 26).

  The localization of this mutant protein was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, this mutant protein is thought to be localized next: membrane.

  In addition, mutant protein H68654_1_P5 (SEQ ID NO: 36) has the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 70. (Substituted amino acids were listed in the light of their position on the amino acid sequence (SEQ ID NO: 36).)

Table 70: Amino acid mutations

  The mutant protein has the following regions as determined using InterPro. The region is shown in Table 71:

Table 71: InterPro area

  Mutant protein H68654_1_P5 (SEQ ID NO: 36) is encoded by transcript H68654_1_T4 (SEQ ID NO: 26), the coding portion of which begins at position 102 and ends at position 1451. This transcript also has the following SNPs listed in Table 72. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 72: Nucleic acid SNPs

  The mutant protein H68654_1_P7 (SEQ ID NO: 37) of the present invention has an amino acid sequence encoded by the transcript H68654_1_T8 (SEQ ID NO: 28). Alignments of H68654_1_P7 (SEQ ID NO: 37) to one or more previously published protein sequences are shown in FIGS. 30A and 30B. A brief description of the relationship between the mutant proteins of the invention and each of the aligned proteins is as follows:

  1. Comparative report between H68665_1_P7 (SEQ ID NO: 37) and the known proteins NP_937794 and Q6UXI0_HUMAN (SEQ ID NO: 36) (FIG. 30A):

  A. An isolated chimeric polypeptide encoding H68654_1_P7 (SEQ ID NO: 37), which corresponds to amino acids 1 to 367 of the known proteins NP_937794 and Q6UXIO_HUMAN (SEQ ID NO: 36), amino acids 1 to H68654_1_P7 (SEQ ID NO: 37) MWLKVFTTFLSFATGACSGLKVTVPSHTVHGVRGQALYLPVHYGFHTPASDIQIIWLFERPHTMPKYLLGSVNKSVVPDLEYQHKFTMMPPNASLLINPLQFPDEGNYIVKVNIQGNGTLSASQKIQVTVDDPVTKPVVQIHPPSGAVEYVGNMTLTCHVEGGTRLAYQWLKNGRPVHTSSTYSFSPQNNTLHIAPVTK also corresponds to the 367 A first amino acid sequence that is at least 90% homologous to DiaijienuwaiesushierubuiaruenuPibuiesuiemuiesudiaiaiemuPiaiaiwaiwaiJipiwaijierukyubuienuesudikeijierukeibuijiibuiefutibuidierujiieiaieruefudishiesueidiesueichiPiPienutiwaiesudaburyuaiaruarutidienutitiwaiaiaikeieichiJipiarueruibuieiesuikeibuieikyukeitiemudiwaibuishishieiwaienuenuaitijiarukyudiitieichiefutibuiaiaitiesubuijieruikeierueikyukeijikeiesueruesuPierueiesuITGISLFLIISMCLLFLWKKYQPYK, also H68654_1_P7 amino 368-455 corresponding to sequence GQKQNTGKLKHFQAMKMLWMTSEYMNLLLFQMFLVFPGSQAGLFQPLIVYRGKICTVQCMKLFSTSLPSSKTIQSELSWAKQYIRVKF of (SEQ ID NO: 37) (SEQ ID NO: 290) A second that is at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95, 96, 97, 98, or 99% homologous to the polypeptide. A chimeric polypeptide wherein the first amino acid sequence and the second amino acid sequence are consecutive and in the stated order.

  B. An isolated polypeptide encoding the end of H68654_1_P7 (SEQ ID NO: 37), the sequence of H68654_1_P7 (SEQ ID NO: 37) GQKQNTGKLHKHFQAMKMMLWMTSQMNFS% 80 A polypeptide comprising an amino acid sequence that is preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95, 96, 97, 98, or 99% homologous.

  2. Comparative report between H68654_1_P7 (SEQ ID NO: 37) and the known protein NP_001034461 (SEQ ID NO: 35) (FIG. 30B):

  A. An isolated chimeric polypeptide encoding H68654_1_P7 (SEQ ID NO: 37), at least 70 against a polypeptide having the sequence MWLKVFTTFLSFAT (SEQ ID NO: 289) corresponding to amino acids 1-14 of H68654_1_P7 (SEQ ID NO: 37) %, Optionally at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95, 96, 97, 98 or 99% homologous to the first amino acid sequence, known GACSGLKVTVPSHTVHGVGLGLYLPVHYGFHTPAS corresponding to amino acids 27 to 379 of protein NP_001034461 (SEQ ID NO: 35) and also corresponding to amino acids 15 to 367 of H68654_1_P7 (SEQ ID NO: 37) At least 90% homologous der against IQIIWLFERPHTMPKYLLGSVNKSVVPDLEYQHKFTMMPPNASLLINPLQFPDEGNYIVKVNIQGNGTLSASQKIQVTVDDPVTKPVVQIHPPSGAVEYVGNMTLTCHVEGGTRLAYQWLKNGRPVHTSSTYSFSPQNNTLHIAPVTKEDIGNYSCLVRNPVSEMESDIIMPIIYYGPYGLQVNSDKGLKVGEVFTVDLGEAILFDCSADSHPPNTYSWIRRTDNTTYIIKHGPRLEVASEKVAQKTMDYVCCAYNNITGRQDETHFTVIITSVGLEKLAQKGKSLSPLASITGISLFLIISMCLLFLWKKYQPYK A second amino acid sequence and the sequence GQKQNTGKLKHFQAMKMMLWMTSEYMNLLLFQMFLVFPGSQAGLFQPLIVYRGKICTVQCMKLFSLSPSKTIQ80 %, More preferably at least 90%, and most preferably at least 95, 96, 97, 98, or 99% homologous amino acid sequence, wherein said first amino acid sequence, second A chimeric polypeptide wherein the amino acid sequence of and of the third amino acid sequence are consecutive and in the order noted.

  B. An isolated polypeptide encoding the head of H68654_1_P7 (SEQ ID NO: 37), wherein the polypeptide is at least 70%, optionally at least about 80%, relative to the sequence MWLKVFTFLSFAT (SEQ ID NO: 289) of H68654_1_P7 (SEQ ID NO: 37); Preferably a polypeptide comprising a polypeptide that is at least about 85%, more preferably at least about 90%, and most preferably at least about 95, 96, 97, 98, or 99% homologous.

  C. An isolated polypeptide encoding the end of H68654_1_P7 (SEQ ID NO: 37), the sequence of H68654_1_P7 (SEQ ID NO: 37) GQKQNTGKLHKHFQAMKMMLWMTSQMNFS% 80 A polypeptide comprising an amino acid sequence that is preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95, 96, 97, 98, or 99% homologous.

  The localization of this mutant protein was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, this mutant protein is thought to be localized next: membrane.

  In addition, mutant protein H68654_1_P7 (SEQ ID NO: 37) has the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 73. (Substituted amino acids were listed in the light of their position on the amino acid sequence (SEQ ID NO: 37).)

Table 73: Amino acid mutations

  The mutant protein has the following regions as determined using InterPro. The region is shown in Table 74:

Table 74: InterPro area

  Mutant protein H68654_1_P7 (SEQ ID NO: 37) is encoded by transcript H68654_1_T8 (SEQ ID NO: 28), the coding portion of which begins at position 102 and ends at position 1466. This transcript also has the following SNPs listed in Table 75. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 75: Nucleic acid SNPs

  The mutant protein H68654_1_P12 (SEQ ID NO: 38) of the present invention has the amino acid sequence encoded by the transcripts H68654_1_T15 (SEQ ID NO: 29), H68654_1_T16 (SEQ ID NO: 30), and H68654_1_T18 (SEQ ID NO: 32). The alignment of H68665_1_P12 (SEQ ID NO: 38) to the published protein sequence is shown in FIGS. 30C and 30D. A brief description of the relationship between the mutant proteins of the invention and each of the aligned proteins is as follows:

  1. Comparative report between H68665_1_P12 (SEQ ID NO: 38) and the known proteins NP_937794 and Q6UXIO_HUMAN (SEQ ID NO: 36) (FIG. 30C):

  A. An isolated chimeric polypeptide encoding H68654_1_P12 (SEQ ID NO: 38), corresponding to amino acids 1 to 413 of the known proteins NP_937794 and Q6UXIO_HUMAN (SEQ ID NO: 36), and amino acids 1 to 1 of H68654_1_P12 (SEQ ID NO: 38) MWLKVFTTFLSFATGACSGLKVTVPSHTVHGVRGQALYLPVHYGFHTPASDIQIIWLFERPHTMPKYLLGSVNKSVVPDLEYQHKFTMMPPNASLLINPLQFPDEGNYIVKVNIQGNGTLSASQKIQVTVDDPVTKPVVQIHPPSGAVEYVGNMTLTCHVEGGTRLAYQWLKNGRPVHTSSTYSFSPQNNTLHIAPV also corresponds to the 413 A first amino acid sequence that is at least 90% homologous to KeiidiaijienuwaiesushierubuiaruenuPibuiesuiemuiesudiaiaiemuPiaiaiwaiwaiJipiwaijierukyubuienuesudikeijierukeibuijiibuiefutibuidierujiieiaieruefudishiesueidiesueichiPiPienutiwaiesudaburyuaiaruarutidienutitiwaiaiaikeieichiJipiarueruibuieiesuikeibuieikyukeitiemudiwaibuishishieiwaienuenuaitijiarukyudiitieichiefutibuiaiaitiesubuijieruikeierueikyukeijikeiesueruesuPierueiesuaitijiaiesueruefueruaiaiesuemushierueruefuerudaburyukeikeiwaikyuPiwaikeibuiaikeikyukeieruijiaruPiitiiwaiarukeieikyutiefuesuGHEDALDDFGIYEFVAFPDVSGVSR, at least 70% identity to the polypeptide having the H68654_1_P12 sequence corresponding to amino acids 414-419 of (SEQ ID NO: 38) VGFPSG (SEQ ID NO: 291), Optionally at least 80% A second amino acid sequence that is preferably at least 85%, more preferably at least 90%, and most preferably at least 95, 96, 97, 98, or 99%, wherein said first amino acid A chimeric polypeptide wherein the sequence and the second amino acid sequence are consecutive and in the order noted.

  B. An isolated polypeptide encoding an end of H68654_1_P12 (SEQ ID NO: 38), wherein the polypeptide is at least 70%, optionally at least about 80%, relative to the sequence VGFPSG (SEQ ID NO: 291) of H68654_1_P12 (SEQ ID NO: 38) A polypeptide comprising an amino acid sequence that is preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95, 96, 97, 98, or 99% homologous.

  2. Comparative report between H68654_1_P12 (SEQ ID NO: 38) and the known protein NP_001034461 (SEQ ID NO: 35) (FIG. 30D):

  A. An isolated chimeric polypeptide encoding H68654_1_P12 (SEQ ID NO: 38), at least 70 against a polypeptide having the sequence MWLKVFTTFLSAT (SEQ ID NO: 289) corresponding to amino acids 1-14 of H68654_1_P12 (SEQ ID NO: 38) %, Optionally at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95, 96, 97, 98 or 99% homologous to the first amino acid sequence, known GACSGLKVTVPSHTVHGVGLGLYLPVHYGFHT corresponding to amino acids 27 to 425 of protein NP_001034461 (SEQ ID NO: 35) and also corresponding to amino acids 15 to 413 of H68654_1_P12 (SEQ ID NO: 38) ASDIQIIWLFERPHTMPKYLLGSVNKSVVPDLEYQHKFTMMPPNASLLINPLQFPDEGNYIVKVNIQGNGTLSASQKIQVTVDDPVTKPVVQIHPPSGAVEYVGNMTLTCHVEGGTRLAYQWLKNGRPVHTSSTYSFSPQNNTLHIAPVTKEDIGNYSCLVRNPVSEMESDIIMPIIYYGPYGLQVNSDKGLKVGEVFTVDLGEAILFDCSADSHPPNTYSWIRRTDNTTYIIKHGPRLEVASEKVAQKTMDYVCCAYNNITGRQDETHFTVIITSVGLEKLAQKGKSLSPLASITGISLFLIISMCLLFLWKKYQPYKVIKQKLEGRPETE At least 70% for a polypeptide having a second amino acid sequence that is at least 90% homologous to RKAQTFSGHEDALDDFGIYEFVAFPDVSGVSR and a sequence VGFPSG (SEQ ID NO: 291) corresponding to amino acids 414-419 of H68654_1_P12 (SEQ ID NO: 38) A third amino acid sequence that is optionally at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95, 96, 97, 98, or 99% homologous, wherein The chimeric polypeptide wherein the first amino acid sequence, the second amino acid sequence, and the third amino acid sequence are consecutive and in the order described.

  B. An isolated polypeptide encoding the head of H68654_1_P12 (SEQ ID NO: 38), wherein the polypeptide is at least 70%, optionally at least about 80%, relative to the sequence MWLKVFFTFLSFAT (SEQ ID NO: 289) of H68654_1_P12 (SEQ ID NO: 38); Preferably a polypeptide comprising a polypeptide that is at least about 85%, more preferably at least about 90%, and most preferably at least about 95, 96, 97, 98, or 99% homologous.

  C. An isolated polypeptide encoding an end of H68654_1_P12 (SEQ ID NO: 38), wherein the polypeptide is at least 70%, optionally at least about 80%, relative to the sequence VGFPSG (SEQ ID NO: 291) of H68654_1_P12 (SEQ ID NO: 38) A polypeptide comprising an amino acid sequence that is preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95, 96, 97, 98, or 99% homologous.

  The localization of this mutant protein was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, this mutant protein is thought to be localized next: membrane.

  Moreover, mutant protein H68654_1_P12 (SEQ ID NO: 38) has the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 76. (Substituted amino acids were listed in the light of their position on the amino acid sequence (SEQ ID NO: 38).)

Table 76: Amino acid mutations

  The mutant protein has the following regions as determined using InterPro. The region is shown in Table 77:

Table 77: InterPro area

  The coding part of the transcript H68654_1_T15 (SEQ ID NO: 29) starts at position 102 and ends at position 1358. This transcript also has the following SNPs listed in Table 78. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 78: Nucleic acid SNPs

  The coding part of the transcript H68654_1_ begins at position 102 and ends at position 1358. This transcript also has the following SNPs listed in Table 79. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 79: Nucleic acid SNPs

  The coding part of the transcript H68654_1_T18 (SEQ ID NO: 32) starts at position 102 and ends at position 1358. This transcript also has the following SNPs listed in Table 80. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 80: Nucleic acid SNPs

  The mutant protein H68654_1_P13 (SEQ ID NO: 39) of the present invention has the amino acid sequence encoded by the transcripts H68654_1_T17 (SEQ ID NO: 31) and H68654_1_T19 (SEQ ID NO: 33). Alignments to one or more published protein sequences are shown in FIGS. 30E and 30F. A brief description of the relationship between the mutant proteins of the invention and each of the aligned proteins is as follows:

  1. Comparative report between H68665_1_P13 (SEQ ID NO: 39) and the known proteins NP_937794 and Q6UXI0_HUMAN (SEQ ID NO: 36) (Figure 30E):

  A. An isolated chimeric polypeptide encoding H68654_1_P13 (SEQ ID NO: 39), which corresponds to amino acids 1 to 376 of the known proteins NP_937794 and Q6UXIO_HUMAN (SEQ ID NO: 36), and amino acids 1 to 1 of H68654_1_P13 (SEQ ID NO: 39) MWLKVFTTFLSFATGACSGLKVTVPSHTVHGVRGQALYLPVHYGFHTPASDIQIIWLFERPHTMPKYLLGSVNKSVVPDLEYQHKFTMMPPNASLLINPLQFPDEGNYIVKVNIQGNGTLSASQKIQVTVDDPVTKPVVQIHPPSGAVEYVGNMTLTCHVEGGTRLAYQWLKNGRPVHTSSTYSFSPQNNTLHIAPV also corresponds to the 376 Comprises an amino acid sequences at least 90% homologous with respect KeiidiaijienuwaiesushierubuiaruenuPibuiesuiemuiesudiaiaiemuPiaiaiwaiwaiJipiwaijierukyubuienuesudikeijierukeibuijiibuiefutibuidierujiieiaieruefudishiesueidiesueichiPiPienutiwaiesudaburyuaiaruarutidienutitiwaiaiaikeieichiJipiarueruibuieiesuikeibuieikyukeitiemudiwaibuishishieiwaienuenuaitijiarukyudiitieichiefutibuiaiaitiesubuijieruikeierueikyukeijikeiesueruesuPierueiesuaitijiaiesueruefueruaiISMCLLFLWKKYQPYKVIKQKLEGR, where said and first amino acid sequence is a continuous, it is this marked the order, chimeric polypeptides.

  2. Comparative report between H68654_1_P13 (SEQ ID NO: 39) and the known protein NP_001034461 (SEQ ID NO: 35) (FIG. 30F):

  A. An isolated chimeric polypeptide encoding H68654_1_P13 (SEQ ID NO: 39), at least 70 against a polypeptide having the sequence MWLKVFTTFLSAT (SEQ ID NO: 289) corresponding to amino acids 1-14 of H68654_1_P13 (SEQ ID NO: 39) %, Optionally at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95, 96, 97, 98 or 99% homologous to the first amino acid sequence, known GACSGLKVTVPSHTVHGVRGQALYLPVHYGFHT which corresponds to amino acids 27 to 388 of protein NP_001034461 (SEQ ID NO: 35) and also corresponds to amino acids 15 to 376 of H68654_1_P13 (SEQ ID NO: 39) Against ASDIQIIWLFERPHTMPKYLLGSVNKSVVPDLEYQHKFTMMPPNASLLINPLQFPDEGNYIVKVNIQGNGTLSASQKIQVTVDDPVTKPVVQIHPPSGAVEYVGNMTLTCHVEGGTRLAYQWLKNGRPVHTSSTYSFSPQNNTLHIAPVTKEDIGNYSCLVRNPVSEMESDIIMPIIYYGPYGLQVNSDKGLKVGEVFTVDLGEAILFDCSADSHPPNTYSWIRRTDNTTYIIKHGPRLEVASEKVAQKTMDYVCCAYNNITGRQDETHFTVIITSVGLEKLAQKGKSLSPLASITGISLFLIISMCLLFLWKKYQPYKVIKQKLEGR It includes a second amino acid sequence is 90% homologous even without, and wherein said first amino acid sequence and a second amino acid sequence is a continuous, is this marked the order, chimeric polypeptides.

  B. An isolated polypeptide encoding the head of H68654_1_P13 (SEQ ID NO: 39), wherein the polypeptide is at least 70%, optionally at least about 80%, relative to the sequence MWLKVFFTFLSFAT (SEQ ID NO: 289) of H68654_1_P13 (SEQ ID NO: 39); Preferably a polypeptide comprising a polypeptide that is at least about 85%, more preferably at least about 90%, and most preferably at least about 95, 96, 97, 98, or 99% homologous.

  The localization of this mutant protein was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, this mutant protein is thought to be localized next: membrane.

  In addition, mutant protein H68654_1_P13 (SEQ ID NO: 39) has the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 81. (Substituted amino acids were listed in the light of their position on the amino acid sequence (SEQ ID NO: 39).)

Table 81: Amino acid mutations

  The mutant protein has the following regions as determined using InterPro. The region is shown in Table 82:

Table 82: InterPro area

  The coding part of the transcript H68654_1_T17 (SEQ ID NO: 31) starts at position 102 and ends at position 1229. This transcript also has the following SNPs listed in Table 83. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 83: Nucleic acid SNPs

  The coding part of the transcript H68654_1_T19 (SEQ ID NO: 33) starts at position 102 and ends at position 1229. This transcript also has the following SNPs listed in Table 84. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 84: Nucleic acid SNPs

  The mutant protein H68654_1_P14 (SEQ ID NO: 40) of the present invention has the amino acid sequence encoded by the transcript H68654_1_T20 (SEQ ID NO: 34). Alignments to published protein sequences are shown in FIGS. 30G and 30H. A brief description of the relationship between the mutant proteins of the invention and each of the aligned proteins is as follows:

  1. Comparative report between H68665_1_P14 (SEQ ID NO: 40) and the known proteins NP_937794 and Q6UXI0_HUMAN (SEQ ID NO: 36) (FIG. 30G):

  A. An isolated chimeric polypeptide encoding H68654_1_P14 (SEQ ID NO: 40), which corresponds to amino acids 1 to 389 of known proteins NP_937794 and Q6UXI0_HUMAN (SEQ ID NO: 36), amino acids 1 to H68654_1_P14 (SEQ ID NO: 40) MWLKVFTTFLSFATGACSGLKVTVPSHTVHGVRGQALYLPVHYGFHTPASDIQIIWLFERPHTMPKYLLGSVNKSVVPDLEYQHKFTMMPPNASLLINPLQFPDEGNYIVKVNIQGNGTLSASQKIQVTVDDPVTKPVVQIHPPSGAVEYVGNMTLTCHVEGGTRLAYQWLKNGRPVHTSSTYSFSPQNNTLHIAPV also corresponds to the 389 A first amino acid sequence that is at least 90% homologous to KeiidiaijienuwaiesushierubuiaruenuPibuiesuiemuiesudiaiaiemuPiaiaiwaiwaiJipiwaijierukyubuienuesudikeijierukeibuijiibuiefutibuidierujiieiaieruefudishiesueidiesueichiPiPienutiwaiesudaburyuaiaruarutidienutitiwaiaiaikeieichiJipiarueruibuieiesuikeibuieikyukeitiemudiwaibuishishieiwaienuenuaitijiarukyudiitieichiefutibuiaiaitiesubuijieruikeierueikyukeijikeiesueruesuPierueiesuaitijiaiesueruefueruaiaiesuemushierueruefuerudaburyukeikeiwaikyuPYKVIKQKLEGRPETEYRKAQTFSG, at least 70% identity to the polypeptide having the H68654_1_P14 sequence corresponding to amino acids 390-423 of (SEQ ID NO: 40) EfuemuerueieiPiesukyuaruEEEKKIWQGPGLLLCPHCNPHYHQY (SEQ ID NO: 292), At least optional A second amino acid sequence that is 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95, 96, 97, 98, or 99% homologous, wherein said A chimeric polypeptide wherein the amino acid sequence of 1 and the second amino acid sequence are consecutive and in the order noted.

  B. An isolated polypeptide encoding an end of H68665_1_P14 (SEQ ID NO: 40), wherein the polypeptide is at least 70%, optionally at least about 80% relative to the sequence FMLAAPSQREEEKKIWQGPLLLLPHCHNPPHYHQY (SEQ ID NO: 292) of H68654_1_P14 (SEQ ID NO: 40) A polypeptide comprising an amino acid sequence that is preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95, 96, 97, 98, or 99% homologous.

  2. Comparative report between H68654_1_P14 (SEQ ID NO: 40) and the known protein NP_001034461 (SEQ ID NO: 35) (Figure 30H):

  A. An isolated chimeric polypeptide encoding H68665_1_P14 (SEQ ID NO: 40), wherein the polypeptide has at least 70 against the polypeptide having the sequence MWLKVFTTFLSAT (SEQ ID NO: 289) corresponding to amino acids 1-14 of H68654_1_P14 (SEQ ID NO: 40) %, Optionally at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95, 96, 97, 98 or 99% homologous to the first amino acid sequence, known GACSGLKVTVPSHTVHGVRGQALYLPVHYGFHT corresponding to amino acids 27-401 of protein NP_001034461 (SEQ ID NO: 35) and corresponding to amino acids 15-389 of H68654_1_P14 (SEQ ID NO: 40) ASDIQIIWLFERPHTMPKYLLGSVNKSVVPDLEYQHKFTMMPPNASLLINPLQFPDEGNYIVKVNIQGNGTLSASQKIQVTVDDPVTKPVVQIHPPSGAVEYVGNMTLTCHVEGGTRLAYQWLKNGRPVHTSSTYSFSPQNNTLHIAPVTKEDIGNYSCLVRNPVSEMESDIIMPIIYYGPYGLQVNSDKGLKVGEVFTVDLGEAILFDCSADSHPPNTYSWIRRTDNTTYIIKHGPRLEVASEKVAQKTMDYVCCAYNNITGRQDETHFTVIITSVGLEKLAQKGKSLSPLASITGISLFLIISMCLLFLWKKYQPYKVIKQKLEGRPETE A second amino acid sequence that is at least 90% homologous to RKAQTFSG and at least 70% for a polypeptide having the sequence FMLAAPSQREEEKKIWQGPLLLLPHPHNPPHYHQY (SEQ ID NO: 292) corresponding to amino acids 390-423 of H68654_1_P14 (SEQ ID NO: 40), optional A third amino acid sequence that is optionally at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95, 96, 97, 98, or 99% homologous, wherein The chimeric polypeptide wherein the first amino acid sequence, the second amino acid sequence, and the third amino acid sequence are consecutive and in the order described.

  B. An isolated polypeptide encoding the head of H68654_1_P14 (SEQ ID NO: 40), wherein the polypeptide is at least 70%, optionally at least about 80%, relative to the sequence MWLKVFTFLSFAT (SEQ ID NO: 289) of H68654_1_P14 (SEQ ID NO: 40); Preferably a polypeptide comprising a polypeptide that is at least about 85%, more preferably at least about 90%, and most preferably at least about 95, 96, 97, 98, or 99% homologous.

  C. An isolated polypeptide encoding an end of H68665_1_P14 (SEQ ID NO: 40), wherein the polypeptide is at least 70%, optionally at least about 80% relative to the sequence FMLAAPSQREEEKKIWQGPLLLLPHCHNPPHYHQY (SEQ ID NO: 292) of H68654_1_P14 (SEQ ID NO: 40) A polypeptide comprising an amino acid sequence that is preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95, 96, 97, 98, or 99% homologous.

  The localization of this mutant protein was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, this mutant protein is thought to be localized next: membrane.

  Mutant protein H68654_1_P14 (SEQ ID NO: 40) has the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 85. (Substituted amino acids were listed in the light of their position on the amino acid sequence (SEQ ID NO: 40).)

Table 85: Amino acid mutations

  The mutant protein has the following regions as determined using InterPro. The region is shown in Table 86:

Table 86: InterPro area

  Mutant protein H68654_1_P14 (SEQ ID NO: 40) is encoded by transcript H68654_1_T20 (SEQ ID NO: 34), the coding portion of which begins at position 102 and ends at position 1370. This transcript also has the following SNPs listed in Table 87. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 87: Nucleic acid SNPs

  As described above, cluster H68654 features the three intercepts listed in Table 63. These sections are part of the nucleic acid sequences described elsewhere herein because they are of particular interest. Here, each section of the present invention will be described.

  The section cluster H68654_1_N3 (SEQ ID NO: 123) of the present invention is supported by 24 libraries. The number of libraries was determined as described above. This section is found in the following transcripts: H68654_1_T0 (SEQ ID NO: 25), H68654_1_T15 (SEQ ID NO: 29), H68654_1_T16 (SEQ ID NO: 30), H68665_1_T17 (SEQ ID NO: 31), H68654_1_T18 (SEQ ID NO: 32), H68654_1_T19 (SEQ ID NO: 32) No. 33), H68654_1_T20 (SEQ ID NO: 34), H68654_1_T4 (SEQ ID NO: 26), H68654_1_T5 (SEQ ID NO: 27), and H68654_1_T8 (SEQ ID NO: 28). Table 88 below lists the start and end positions of this section on each transcript.

Table 88: Position of the section on the transcript

  The section cluster H68654_1_N7 (SEQ ID NO: 124) of the present invention is supported by 20 libraries. The number of libraries was determined as described above. This section is found in the following transcripts: H68654_1_T0 (SEQ ID NO: 25), H68654_1_T15 (SEQ ID NO: 29), H68654_1_T16 (SEQ ID NO: 30), H68665_1_T17 (SEQ ID NO: 31), H68654_1_T18 (SEQ ID NO: 32), H68654_1_T19 (SEQ ID NO: 32) No. 33), H68654_1_T20 (SEQ ID NO: 34), H68654_1_T4 (SEQ ID NO: 26), H68654_1_T5 (SEQ ID NO: 27), and H68654_1_T8 (SEQ ID NO: 28). Table 89 below lists the start and end positions of this section on each transcript.

Table 89: Location of sections on transcripts

  The section cluster H68654_1_N12 (SEQ ID NO: 125) of the present invention is supported by 18 libraries. The number of libraries was determined as described above. This section is found in the following transcripts: H68654_1_T0 (SEQ ID NO: 25), H68654_1_T15 (SEQ ID NO: 29), H68654_1_T16 (SEQ ID NO: 30), H68665_1_T17 (SEQ ID NO: 31), H68654_1_T18 (SEQ ID NO: 32), H68654_1_T19 (SEQ ID NO: 32) No. 33), H68654_1_T20 (SEQ ID NO: 34), H68654_1_T4 (SEQ ID NO: 26), H68654_1_T5 (SEQ ID NO: 27), and H68654_1_T8 (SEQ ID NO: 28). Table 90 below lists the start and end positions of this section on each transcript.

Table 90: Position of the section on the transcript

  Expression of the hypothesis-based protein LOC253012 H68654 transcript that is detectable by normal and cancerous lung tissue, as well as in different normal tissues, by the amplicon represented by SEQ ID NO: H68654_seg3WTF2R2 (SEQ ID NO: 226)

  Expression of the lung-based protein LOC2530102 transcript is detectable by or in accordance with seg3F2R2-H68654_seg3WTF2R2 (SEQ ID NO: 226) amplicon and primers H68654_seg3WTF2 (SEQ ID NO: 224) and H68654_seg3WTR2 (SEQ ID NO: 225), And measured by real-time PCR on a normal panel. Details of the samples used are shown in Table 3 and Table 2 above, respectively. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1.

  Lung Panel—The normalized amount of each RT sample is then divided by the median value of the normal sample (sample numbers 51-64 and 69-70, Table 3 above), to the median value of the normal sample Thus, the value of how many times each sample was enhanced was obtained.

  FIG. 31 is a histogram showing overexpression of protein LOC2530112 transcript based on the hypothesis shown above in cancerous lung samples compared to normal samples.

  As is clear from FIG. 31, the expression of the protein LOC2530102 transcript based on the hypothesis that it can be detected by the above amplicons in small cell carcinoma samples is shown in non-cancerous samples (sample numbers 51-64 and 69-70, It was significantly higher than Table 3) above. In particular, at least 80-fold overexpression was observed in 7 out of 9 small cell carcinoma samples.

  The significance of these results was verified using statistical analysis as described below.

  The P value of the difference in the expression level of the protein LOC2530112 transcript based on the hypothesis that can be detected by the above amplicon in the small cell lung cancer sample with respect to the normal tissue sample was determined by T test as 3.24e-003.

  When checked with Fisher's exact test, the 80-fold overexpression threshold was found to make a difference between small cell carcinoma samples and normal samples with a P value of 7.49e-005.

  The above values indicate that this result is statistically significant.

  Normal Panel—Next, as shown in FIG. 32, the normalized amount of each RT sample is divided by the median of the amounts of lung samples (sample numbers 26, 28, 29, and 30, Table 2 above). The value of relative expression of each sample relative to the median value of lung samples was obtained.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as just one example to serve as a non-limiting illustration of a suitable primer pair: H68654_seg3WTF2 (SEQ ID NO: 224) forward primer; and H68654_seg3WTR2 (SEQ ID NO: 225) reverse direction Primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to serve as a non-limiting illustration of a suitable amplicon: H68654_seg3WTF2R2 (SEQ ID NO: 226)

  Forward primer> H68654_seg3WTF2 (SEQ ID NO: 224): ATCACACACTGTCCATGGGCGT

  Reverse primer> H68654_seg3WTR2 (SEQ ID NO: 225): GTCTCTCAAATAGCCCATGATCTG

  Amplicon> H68654_seg3WTF2R2 (SEQ ID NO: 226)

  ATCACAACTGTTCCATGGCGTCAGAGGTCAGGCCCTCTACTCACCCGTCCCACTATGGCTTCCCACACTCCAGCATCAGACATCCAGATCATATGGCTATTTGAGAGAC

  Expression of the hypothesis-based protein LOC253012 H68654 transcript that is detectable by normal and cancerous lung tissue, as well as in different normal tissues, by the amplicon represented by SEQ ID NO: H68654_seg7-12WT (SEQ ID NO: 223)

  seg7-12-H68654_seg7-12WT (SEQ ID NO: 223) amplicon and the hypothetical protein LOC253012 that can be detected by or in accordance with primers H68654_seg7-12WTF1 (SEQ ID NO: 221) and H68654_seg7-12WTR1 (SEQ ID NO: 222) Transcript expression was measured by real-time PCR in lung and normal panels. Details of the samples used are shown in Table 3 and Table 2 above, respectively. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1.

  Lung panel—Non-detected samples (sample numbers 30, 41, 78, and 92, Table 3) were calculated with a Ct value of 41. Next, the normalized amount of each RT sample is divided by the intermediate value of the amount of normal samples (sample numbers 51-64 and 69-70, Table 3 above), and each sample relative to the intermediate value of the normal sample The value of how much was increased.

  FIG. 33 is a histogram showing overexpression of the protein LOC2530112 transcript based on the hypothesis shown above in cancerous lung samples compared to normal samples.

  As is apparent from FIG. 33, the expression of the protein LOC2530112 transcript based on the hypothesis that it can be detected by the amplicon in small cell carcinoma samples is shown in non-cancerous samples (sample numbers 51-64 and 69-70, It was significantly higher than in Table 3) above and higher than several squamous cell carcinoma samples. In particular, at least 25-fold overexpression was seen in 6 of the 9 small cell carcinoma samples and 4 of the 24 squamous cell carcinoma samples.

  The significance of these results was verified using statistical analysis as described below. The P value of the difference between the expression level of the protein LOC2530112 transcript based on the hypothesis that can be detected by the above amplicon in the small cell lung cancer sample and the squamous cell lung cancer sample from the normal tissue sample, respectively, is 1.24e −. Determined by T test as 002 and 2.97e-002.

  When checked with Fisher's exact test, it was found that the 25-fold overexpression threshold produced a difference between small cell carcinoma samples and normal samples with a P value of 4.74e-004.

  The above values indicate that this result is statistically significant.

  A normal panel-non-detected sample (sample number 16) was set to a Ct value of 41 and calculated accordingly. Next, as shown in FIG. 34, the normalized amount of each RT sample is divided by the intermediate value of the amount of lung samples (sample numbers 26, 28, 29, and 30, Table 2 above) to obtain a lung sample. The relative expression value of each sample relative to the intermediate value of was obtained.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as an example to serve as a non-limiting illustration of a suitable primer pair: H68654_seg7-12WTF1 (SEQ ID NO: 221) forward primer; and H68654_seg7-12WTR1 (SEQ ID NO: 222) Reverse primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to serve as a non-limiting illustration of a suitable amplicon: H68654_seg7-12WT (SEQ ID NO: 223)

  Forward primer> H68654_seg7-12WTF1 (SEQ ID NO: 221): ATGGGCCTCGCTTAGAAGTTG

  Reverse primer> H68654_seg7-12WTR1 (SEQ ID NO: 222): TTCTGTGCAAGCTTCTCCCAGTC

  Amplicon> H68654_seg7-12WT (SEQ ID NO: 223): ATGGGCCTCGCTTAGAAGTTGCCATCTGAGAAAGTAGCCCAGAAGACATGGCATCATGATGACCTGCATGCATGATCGATGCT

  Expression of the hypothetical protein LOC253012 H68654 transcript that is detectable by the amplicon shown in SEQ ID NO: H68654_seg3WTF2R2 (SEQ ID NO: 226) in the blood specific panel

  The expression of the LOC25303012 transcript, which can be detected by or according to the seg3-H68654seg3F2R2 (SEQ ID NO: 226) amplicon, and primers H68654seg3F2 (SEQ ID NO: 224) and H68654seg3R2 (SEQ ID NO: 225) is measured by real-time PCR in the blood panel did. Details of the used samples are shown in Table 1 above. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1. Next, the normalized amount of each RT sample is divided by the median of the amount of normal kidney samples (sample numbers 65-67, Table 1 above) and the relative expression of each sample relative to the median value of normal kidney samples. Was obtained.

  The result of this analysis is shown in the histogram of FIG. 35A. The expression of the LOC253012 transcript described above is high in normal kidney, normal colon, and normal small intestine samples, unlike most blood samples evaluated.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pairs were used as just one example to serve as a non-limiting illustration of a suitable primer pair: H68654_seg3WTF2 forward primer; and H68654_seg3WTR2 reverse primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to serve as a non-limiting illustration of a suitable amplicon: H68654_seg3WTF2R2

  Forward primer> H68654_seg3WTF2: ATCACACACTGTCCATGGCGT (SEQ ID NO: 224)

  Reverse primer> H68654_seg3WTR2: GTCTCTCAAATAGCCATATATCTGG (SEQ ID NO: 225)

  Amplicon> H68654_seg3WTF2R2 (SEQ ID NO: 226)

  ATCACAACTGTTCCATGGCGTCAGAGGTCAGGCCCTCTACTCACCCGTCCCACTATGGCTTCCCACACTCCAGCATCAGACATCCAGATCATATGGCTATTTGAGAGAC

  Expression of the LOC253012 H68654 transcript detectable by the amplicon shown in SEQ ID NO: H68654seg7-12 (SEQ ID NO: 223) in the blood specific panel

  The expression of the LOC25303012 transcript, which is detectable by or according to the seg7-12-H68654seg7-12WTF1R1 (SEQ ID NO: 223) amplicon, and the primers H68654seg7-12WTF1 (SEQ ID NO: 221) and H68654seg7-12WTR1 (SEQ ID NO: 222) Measured by real-time PCR on a blood panel. Details of the used samples are shown in Table 1 above. For each RT sample, the expression of the amplicon was normalized to the normalization factor calculated from the expression of multiple housekeeping genes described in Example 1. Next, the normalized amount of each RT sample is divided by the median of the amount of normal kidney samples (sample numbers 65-67, Table 1 above) and the relative expression of each sample relative to the median value of normal kidney samples. Was obtained.

  The result of this analysis is shown in the histogram of FIG. 35B. The expression of the LOC253012 transcript described above is higher in normal kidney, normal colon, and normal small intestine samples compared to the different blood samples evaluated.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as an example to serve as a non-limiting illustration of a suitable primer pair: seg7-12WTF1 forward primer (SEQ ID NO: 221); and seg7-12WTR1 reverse primer (SEQ ID NO: 222)

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as an example to serve as a non-limiting illustration of a suitable amplicon: seg7-12WTF1R1 (SEQ ID NO: 223)

  Forward primer> H68654_seg7-12WTF1 (SEQ ID NO: 221)

  ATGGGCCTCGCTTAGAAGTTG

  Reverse primer> H68654_seg7-12WTR1 (SEQ ID NO: 222)

  TTCTGTGCAAGCTTCTCAGTC

  Amplicon> H68654_seg7-12WT (SEQ ID NO: 223)

  ATGGGCCTCGCTTAGAAGTTGCCATCTGAGAAAGTAGCCCAGAAGACATGGGACTATTGTGCTTGTCTACAACAACATAACCCGGCAGCAAGATGAAACTCATTTTCACAGTTATCATCACTTCCCTAGGAGCTGGAGAATCTGCAGA

  Expression of the hypothetical protein LOC253012 H68654 transcript detectable in normal and cancerous lung tissue by the amplicon shown in SEQ ID NO: H68654_seg0-3 (SEQ ID NO: 229)

  The expression of LOC253012 transcript, detectable by or according to seg0-3-H68654_seg0-3 (SEQ ID NO: 229) amplicon, and primers H68654_seg0-3F1 (SEQ ID NO: 227) and H68654_seg0-3R1 (SEQ ID NO: 228) is Measured by real-time PCR on lung panels. Details of the used samples are shown in Table 3 above. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1. Next, the normalized amount of each RT sample is divided by the median value of the normal sample (sample numbers 51-54, 56-64, 69, and 70, Table 3 above), and the normal value of the normal sample is divided. The value of how many times each sample was enhanced was obtained.

  FIG. 36 is a histogram showing overexpression of the LOC253012 transcript shown above in cancerous lung samples compared to normal samples.

  As is clear from FIG. 36, in the squamous cell carcinoma sample and the small cell carcinoma sample, the expression of the LOC2530112 transcript that can be detected by the above amplicon is shown in non-cancerous samples (sample numbers 51 to 54, 56 to 64, 69 and 70, significantly higher than Table 3) above. In particular, at least 5-fold overexpression was observed in 6 of 21 squamous cell carcinoma samples and 8 of 9 small cell carcinoma samples.

  The significance of these results was verified using statistical analysis as described below. The P value of the difference in the expression level of the LOC253012 transcript detectable by the above amplicon in the lung squamous cell carcinoma sample and the small cell lung cancer sample from the normal tissue sample was 4.96e-002 and 1.05e, respectively. It was decided by T test as -003.

  When checked with Fisher's exact test, a five-fold overexpression threshold was found between squamous cell carcinoma samples and small cell carcinoma samples and normal samples with P-values of 2.79e-002 and 1.22e-005, respectively. , It was found to make a difference. The above values indicate that this result is statistically significant.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as just one example to serve as a non-limiting illustration of a suitable primer pair: H68654_seg0-3F1 (SEQ ID NO: 227) forward primer; and H68654_seg0-3R1 (SEQ ID NO: 228) Reverse primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to serve as a non-limiting description of a suitable amplicon: H68654_seg0-3F1R1 (SEQ ID NO: 229)

  Forward primer> H68654_seg0-3F1 (SEQ ID NO: 227)

  GCTTTCATGAGGCCCTTCG

  Reverse primer> H68654_seg0-3R1 (SEQ ID NO: 228)

  GCCTGACCTCTGACGCCA

  Amplicon> H68654_seg0-3F1R1 (SEQ ID NO: 229)

  GCTTTCATGGAGCCCTTCGGGTGACACTCTGGGGTCTTTCAGTGCAAAATACACTCCCTTCTCTCCGGTGCTGTCCTGGGGCTGAAGGTGACGTGCCATCACACTGTCCCATGGCGTCAGGC

  Expression of the hypothetical protein LOC253012 H68654 transcript detectable in normal and cancerous lung tissue by the amplicon set forth in SEQ ID NO: H68654_seg2-3 (SEQ ID NO: 232)

  The expression of the LOC25303012 transcript that is detectable by or in accordance with the seg2-3-H68654_seg2-3 (SEQ ID NO: 232) amplicon and the primers H68654_seg2-3F1 (SEQ ID NO: 230) and H68654_seg2-3R1 (SEQ ID NO: 231) Measured by real-time PCR on lung panels. Details of the used samples are shown in Table 3 above. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1. Next, the normalized amount of each RT sample is divided by the median value of the normal sample (sample numbers 51, 52, 54-64, 69, and 70, Table 3 above), and the normal value of the normal sample The value of how many times each sample was enhanced was obtained.

  FIG. 37 is a histogram showing overexpression of the LOC253012 transcript shown above in cancerous lung samples compared to normal samples.

  As is clear from FIG. 37, in the small cell carcinoma sample, the expression of the LOC25303012 transcript, which can be detected by the amplicon, is shown in non-cancerous samples (sample numbers 51, 52, 54-64, 69, and 70, It was significantly higher than Table 3) above. In particular, at least 8-fold overexpression was seen in 7 out of 9 small cell carcinoma samples.

  The significance of these results was verified using statistical analysis as described below. The P value of the difference in the expression level of the LOC25303012 transcript, which can be detected by the above amplicon, from the normal tissue sample in the small cell lung cancer sample was determined by T test as 1.93e-002.

  When checked with Fisher's exact test, it was found that the 8-fold overexpression threshold produced a difference between small cell carcinoma samples and normal samples with a P value of 1.04e-004.

  The above values indicate that this result is statistically significant.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as an example to serve as a non-limiting illustration of a suitable primer pair: H68654_seg2-3F1 (SEQ ID NO: 230) forward primer; and H68654_seg2-3R1 (SEQ ID NO: 231) Reverse primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to serve as a non-limiting description of a suitable amplicon: H68654_seg2-3F1R1 (SEQ ID NO: 232)

  Forward primer> H68654_seg2-3F1 (SEQ ID NO: 230)

  CTCTGCATTTGCCCCTTTAGA

  Reverse primer> H68654_seg2-3R1 (SEQ ID NO: 231)

  GATGGCACTGTCACCTCCAGC

  Amplicon> H68654_seg2-3F1R1 (SEQ ID NO: 232)

  CTCTGCATTGTCCCCTTTAGATTGTGAAATGTGGCTCAAGGTCTTCACAACTTTCCTTTCCTTGCAACAGGTGCTGTCCTGGGGCTGAAGGTGACAGTGCCATC

Example 6: Description of Cluster H19011_1 The present invention relates to C1ORF32 polypeptides, novel splice variants, and diagnostic and therapeutic agents based on them.

  According to the present invention, cluster H19011_1 (internal ID 743282827) features two target transcripts and five target sections, the names of which are shown in Tables 91 and 92, respectively. Selected protein variants are shown in Table 93.

Table 91: Target transcripts

Table 92: Target sections

Table 93: Target proteins

  These sequences are known proteins referred to herein as known proteins, hypothetical protein LOC387597 (RefSeq accession number NP — 955383 (SEQ ID NO: 47), aka: C1ORF32, NP — 955383; LISCH-like; RP4-782G3.2 DJ782G3.1) variant.

  C1ORF32 is a protein based on a hypothesis discovered by calculation during annotation of chromosome 1 (Gregory SG et al. 2006, Nature 441 (7091) 315-321). Its closest annotated homolog belongs to the LISCH7 family, a subfamily of the immunoglobulin superfamily. One annotated member of this family is the lipolysis-stimulated lipoprotein receptor that is thought to have a role in the clearance of triglyceride-rich lipoproteins from the blood (Swissprot with accession number Q86X29). Annotation).

  According to the present invention, C1ORF32, like other known B7 members, was expected to be a novel B7 / CD28 member based on the presence of the IgV region in addition to being a type I membrane protein. . In addition, two alternatively spliced variants of the invention (H19011_1_P8 (SEQ ID NO: 48) and H19011_1_P9 (SEQ ID NO: 50)) that only share the first five exons with wild type C1ORF32. Are similar to known B7 family members with respect to their exon size and the location of IgV and transmembrane regions within these exons. Furthermore, C1ORF32 has been shown to be overexpressed in the present invention in small cell lung cancer.

  As mentioned above, cluster H19011 features the two transcripts listed in Table 91 above. These transcripts encode a protein, a protein that is a variant of the hypothetical protein LOC387597 (SEQ ID NO: 47). Here, each mutant protein of the present invention will be described.

  The mutant protein H19011_1_P8 (SEQ ID NO: 48) of the present invention has an amino acid sequence encoded by the transcript H19011_1_T8 (SEQ ID NO: 45). An alignment to one or more published protein sequences is shown in FIG. 38A. A brief description of the relationship of the mutant protein of the invention to each of its aligned proteins is as follows:

  Comparative report between H19011_1_P8 (SEQ ID NO: 48) and the known proteins Q71H61_HUMAN and NP_955383 (SEQ ID NO: 47) (FIG. 38A):

  A. An isolated chimeric polypeptide encoding H19011_1_P8 (SEQ ID NO: 48), corresponding to amino acids 1 to 158 of the known proteins Q71H61_HUMAN and NP_955383 (SEQ ID NO: 47), and amino acids 1 to 1 of H19011_1_P8 (SEQ ID NO: 48) a first amino acid sequence that is at least 90% homologous to MDRVLLRWISLFWLTAMVEGLQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRGREITIVHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE also corresponding to 158, H19011_1_P8 (distribution No. 48), the bridged amino acid G corresponding to amino acid 159, and amino acids 160-160 of known proteins Q71H61_HUMAN and NP_955383 (SEQ ID NO: 47), and also corresponding to amino acids 160-160 of H19011_1_P8 (SEQ ID NO: 48) A second amino acid sequence that is at least 90% homologous to, a bridging amino acid LG corresponding to amino acids 161-162 of H19011_1_P8 (SEQ ID NO: 48), and amino acids 163-3 of known proteins Q71H61_HUMAN and NP_955383 (SEQ ID NO: 47) LLVLGRTGLLADLLLPSFAVEIMPEWWVFVGLVLGLVFLFFVLV corresponding to 229 and also corresponding to amino acids 163 to 229 of H19011_1_P8 (SEQ ID NO: 48) ICWCQCCPHSCCCCYVRCPCCPPDSC third amino acid sequence that is at least 90% homologous, bridged amino acid W corresponding to amino acid 230 of H19011_1_P8 (SEQ ID NO: 48), and amino acids 231 to 234 of known protein Q71H61_HUMAN and NP_955383 (SEQ ID NO: 47) Corresponding to amino acids 235 to 254 of H19011_1_P8 (SEQ ID NO: 48) and a fourth amino acid sequence that is at least 90% homologous to CPQA corresponding to amino acids 231 to 234 of H19011_1_P8 (SEQ ID NO: 48) At least 70%, optionally at least 80%, preferably at least 8 for a polypeptide having the sequence CEYSDRWGDRAIERNVYLST (SEQ ID NO: 293) A fifth amino acid sequence that is 5%, more preferably at least 90%, and most preferably at least 95, 96, 97, 98, or 99% homologous, wherein said first amino acid sequence, bridge A chimeric polypeptide wherein the amino acid, the second amino acid sequence, the cross-linked amino acid, the third amino acid sequence, the cross-linked amino acid, the fourth amino acid sequence, and the fifth amino acid sequence are in the order noted.

  B. An isolated polypeptide encoding an end of H19011_1_P8 (SEQ ID NO: 48), wherein the polypeptide is at least 70%, optionally at least about 80% relative to the sequence CEYSDRWGDRAIERNVYLST (SEQ ID NO: 293) of H19011_1_P8 (SEQ ID NO: 48) A polypeptide comprising an amino acid sequence that is preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95, 96, 97, 98, or 99% homologous.

  The localization of this mutant protein was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, this mutant protein is thought to be localized next: membrane.

  Moreover, mutant protein H19011_1_P8 (SEQ ID NO: 48) has the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 94. (The alternative amino acids are listed in the light of their position on the amino acid sequence (SEQ ID NO: 48).) Examples of such putative sequences made with alternative amino acids (using some of the following SNPs) are H19011_1_P8_V1 This is indicated by the name (SEQ ID NO: 49).

Table 94: Amino acid mutations

  The mutant protein has the following regions as determined using InterPro. The region is shown in Table 95:

Table 95: InterPro area

  Mutant protein H19011_1_P8 (SEQ ID NO: 48) is encoded by transcript H19011_1_T8 (SEQ ID NO: 45), the coding portion of which begins at position 181 and ends at position 942. This transcript also has the following SNPs listed in Table 96. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 96: Nucleic acid SNPs

  As shown in Table 97, the genomic structure of protein H19011_1_P8 (SEQ ID NO: 48) (number of exons associated with the extracellular region of the protein, length of these exons, the frame of codons into which introns were inserted, and within the gene structure The properties of the proteins and the location of the regions are characteristic of the B7 / costimulatory protein family of ligands.

Table 97: Genomic structure and protein properties

  The mutant protein H19011_1_P9 (SEQ ID NO: 50) of the present invention has an amino acid sequence encoded by the transcript H19011_1_T9 (SEQ ID NO: 46). An alignment to one or more published protein sequences is shown in FIG. 38B. A brief description of the relationship between the mutant proteins of the invention and each of the aligned proteins is as follows:

  Comparative report between H19011_1_P9 (SEQ ID NO: 50) and the known proteins Q71H61_HUMAN and NP_955383 (SEQ ID NO: 47) (FIG. 38B):

  A. An isolated chimeric polypeptide encoding H19011_1_P9 (SEQ ID NO: 50), which corresponds to amino acids 1 to 158 of known proteins Q71H61_HUMAN and NP_955383 (SEQ ID NO: 47), and amino acids 1 to 1 of H19011_1_P9 (SEQ ID NO: 50) a first amino acid sequence that is at least 90% homologous to MDRVLLRWISLFWLTAMVEGLQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRGREITIVHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE also corresponding to 158, H19011_1_P9 (distribution No. 50), the bridged amino acid G corresponding to amino acid 159, and amino acids 160 to 160 of known proteins Q71H61_HUMAN and NP_955383 (SEQ ID NO: 47), and also corresponding to amino acids 160 to 160 of H19011_1_P9 (SEQ ID NO: 50) A second amino acid sequence that is at least 90% homologous to, a bridging amino acid LG corresponding to amino acids 161-162 of H19011_1_P9 (SEQ ID NO: 50), and amino acids 163-3 of known proteins Q71H61_HUMAN and NP_955383 (SEQ ID NO: 47) A third amino acid sequence that is at least 90% homologous to LLVL corresponding to amino acids 163 to 166 of H19011_1_P9 (SEQ ID NO: 50), and a known protein Q7 A fourth 190th amino acid sequence that is at least 90% homologous to EWVFVGLVLGLVFLFFVLVGICWCQCCCPPHSCCYVRCPCCPPDSC corresponding to amino acids 186 to 229 of H61_HUMAN and NP_955383 (SEQ ID NO: 47) and also corresponding to amino acids 167 to 210 of H19011_1_P9 (SEQ ID NO: 50) (Corresponding to amino acids 211 to 234 of the known protein Q71H61_HUMAN and NP_955383 (SEQ ID NO: 47), corresponding to amino acids 212 to 215 of H19011_1_P9 (SEQ ID NO: 50) A fifth amino acid sequence that is at least 90% homologous to CPQA, and H19011_1_P9 At least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% relative to a polypeptide having the sequence CEYSDRWGDRAIERNVYLST (SEQ ID NO: 293) corresponding to amino acids 216-235 of (SEQ ID NO: 50) And most preferably at least 95, 96, 97, 98, or 99% homologous amino acid sequence, wherein said first amino acid sequence, bridging amino acid, second amino acid sequence, bridging A chimeric polypeptide wherein the amino acid, third amino acid sequence, fourth amino acid sequence, bridged amino acid, fifth amino acid sequence, and sixth amino acid sequence are in the order noted.

  B. An isolated chimeric polypeptide encoding an end of H19011_1_P9 (SEQ ID NO: 50), comprising a polypeptide having a length “n”, wherein n is at least about 10 amino acids in length, optionally At least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length, and most preferably at least about 50 amino acids in length, wherein at least 2 amino acids are LE And starts with any amino acid number from 166-x to 166 and ends with any amino acid number of 167 + ((n−2) −x), where x is from 0 to n− A chimeric polypeptide having a sequence that varies up to 2).

  C. An isolated polypeptide encoding an end of H19011_1_P9 (SEQ ID NO: 50), wherein the polypeptide is at least 70%, optionally at least about 80% relative to the sequence CEYSDRWGDRAIERNVYLST (SEQ ID NO: 293) of H19011_1_P9 (SEQ ID NO: 50) A polypeptide comprising an amino acid sequence that is preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95, 96, 97, 98, or 99% homologous.

  The localization of this mutant protein was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, this mutant protein is thought to be localized next: membrane.

  Mutant protein H19011_1_P9 (SEQ ID NO: 50) has the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 98. (Substituted amino acids were listed in the light of their position on the amino acid sequence (SEQ ID NO: 50).)

Table 98: Amino acid mutations

  Mutant protein H19011_1_P9 (SEQ ID NO: 50) is encoded by transcript H19011_1_T9 (SEQ ID NO: 46), the coding portion of which begins at position 181 and ends at position 885. This transcript also has the following SNPs listed in Table 99. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 99: Nucleic acid SNPs

  As described above, cluster H19011 features the five intercepts listed in Table 92 above. These sections are part of the nucleic acid sequences described elsewhere herein because they are of particular interest. Here, each section of the present invention will be described.

  The section cluster H19011_1_N13 (SEQ ID NO: 129) of the present invention is supported by three libraries. The number of libraries was determined as described above. This section is found in the following transcripts: H19011_1_T8 (SEQ ID NO: 45) and H19011_1_T9 (SEQ ID NO: 46). Table 100 below lists the start and end positions of this section on each transcript.

Table 100: Position of the section on the transcript

  According to an optional embodiment of the invention, short sections associated with the above clusters are also provided. These sections are up to about 120 bp in length and are included in another specification.

  The section cluster H19011_1_N8 (SEQ ID NO: 130) of the present invention is supported by four libraries. The number of libraries was determined as described above. This section is found in the following transcript: H19011_1_T8 (SEQ ID NO: 45). Table 101 below lists the start and end positions of this section on each transcript.

Table 101: Position of the section on the transcript

  The section cluster H19011_1_N10 (SEQ ID NO: 131) of the present invention is supported by four libraries. The number of libraries was determined as described above. This section is found in the following transcripts: H19011_1_T8 (SEQ ID NO: 45) and H19011_1_T9 (SEQ ID NO: 46). Table 102 below lists the start and end positions of this section on each transcript.

Table 102: Position of the section on the transcript

  The section cluster H19011_1_N11 (SEQ ID NO: 132) of the present invention is supported by three libraries. The number of libraries was determined as described above. This section is found in the following transcripts: H19011_1_T8 (SEQ ID NO: 45) and H19011_1_T9 (SEQ ID NO: 46). Table 103 below lists the start and end positions of this section on each transcript.

Table 103: Position of the section on the transcript

  The section cluster H19011_1_N12 (SEQ ID NO: 133) of the present invention is supported by 5 libraries. The number of libraries was determined as described above. This section is found in the following transcripts: H19011_1_T8 (SEQ ID NO: 45) and H19011_1_T9 (SEQ ID NO: 46). Table 104 below lists the start and end positions of this section on each transcript.

Table 104: Section location on transcript

  C1ORF32, Chromosome 1 open reading frame 32, H19011, detectable in normal and cancerous colon tissue, normal and cancerous lung tissue, and in different normal tissues by the amplicon represented by SEQ ID NO: H19011_seg13F2R2 (SEQ ID NO: 235) Transcript expression

  Expression of the C1ORF32, chromosome 1 open reading frame 32 transcript, detectable by or according to seg13-H19011_seg13F2R2 (SEQ ID NO: 235) amplicon and primers H19011_seg13F2 (SEQ ID NO: 233) and H19011_seg13R2 (SEQ ID NO: 234) Measured by real-time PCR on colon, lung, and normal panels. Details of the used samples are shown in Table 5, Table 3, and Table 2, respectively. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1.

  Colon panel—The normalized amount of each RT sample was then divided by the median of the amount of normal samples (sample numbers 42-70, Table 5 above). Then, the reciprocal of this ratio was calculated to obtain a value indicating how many times each sample was controlled downward with respect to the intermediate value of the normal sample.

  FIG. 39 is a histogram showing the down-regulation of the C1ORF32 transcript shown above in a cancerous colon sample compared to a normal sample.

  As is apparent from FIG. 39, the expression of the C1ORF32 transcript detectable in the cancer sample by the amplicon was significantly lower than that of the non-cancerous sample (sample numbers 42 to 70, Table 5 above). In particular, at least 6-fold down-regulation was observed in 17 out of 55 adenocarcinoma samples.

  The significance of these results was verified using statistical analysis as described below. The P value of the difference in the expression level of C1ORF32 transcripts detectable by the above amplicons in colon cancer samples relative to normal tissue samples was determined by T-test as 9.36e-004.

  When checked with Fisher's exact test, it was found that the 6-fold down-regulation threshold produced a difference between cancer and normal samples with a P value of 2.67e-004.

  The above values indicate that this result is statistically significant.

  Lung Panel—The normalized amount of each RT sample is then divided by the median value of the normal sample (sample numbers 51-64 and 69-70, Table 3 above), to the median value of the normal sample Thus, the value of how many times each sample was enhanced was obtained.

  FIG. 40 is a histogram showing overexpression of the C1ORF32 transcript shown above in cancerous lung samples compared to normal samples.

  As is apparent from FIG. 40, the expression of the C1ORF32 transcript detectable in the small cell cancer sample by the above amplicon is shown in the non-cancerous samples (sample numbers 51 to 64 and 69 to 70, Table 3 above). Was significantly higher. In particular, at least 6-fold overexpression was observed in 9 out of 9 small cell carcinoma samples.

  The significance of these results was verified using statistical analysis as described below.

  The P value of the difference in the expression level of C1ORF32 transcripts detectable by the above amplicons in small cell lung cancer samples from normal tissue samples was determined by T test as 3.43e-003.

  When checked with Fisher's exact test, the 6-fold overexpression threshold was found to produce a difference between small cell carcinoma samples and normal samples with a P value of 4.89e-007.

  The above values indicate that this result is statistically significant.

  Normal panel—Next, as shown in FIG. 41A, the normalized amount of each RT sample is divided by the median value of the amount of colon samples (sample numbers 3, 4, and 5, Table 2 above) A value for the relative expression of each sample relative to the median value of the sample was obtained. Next, as shown in FIG. 41B, the normalized amount of each RT sample is divided by the median value of the amount of lung samples (sample numbers 26, 28, 29, and 30, Table 2 above) to obtain a lung sample. The relative expression value of each sample relative to the intermediate value of was obtained.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pairs were used as just one example to serve as a non-limiting illustration of suitable primer pairs: H19011_seg13F2 (SEQ ID NO: 233) forward primer; and H19011_seg13R2 (SEQ ID NO: 234) reverse direction Primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to serve as a non-limiting illustration of a suitable amplicon: H19011_seg13F2R2 (SEQ ID NO: 235)

  Forward primer> H19011_seg13F2 (SEQ ID NO: 233): GTGAGTACAGTGACCGCTGGG

  Reverse primer> H19011_seg13R2 (SEQ ID NO: 234): GGAGAAGAGTCTGGAATGACCAA

  Amplicon> H19011_seg13F2R2 (SEQ ID NO: 235)

  GTGAGTACAGGTGACCGCTGGGGGAGACAGAGCGGATCGAGAGAAAATGTCTACTCTCTTACCTGACAGCTGTGTGCGCTGGGTTCCTCCTCCCACCTCCTGTCCTGCCACCCCCCAAATTGGTCACTTCTCAGACTCTTCTCC

  Expression of C1ORF32, Chromosome 1 open reading frame 32, H19011 transcript, detectable by the amplicon shown in SEQ ID NO: H19011_seg8-13F1R1 (SEQ ID NO: 238) in normal and cancerous lung tissue

  seg8-13F1R1-H19011_seg8-13F1R1 (SEQ ID NO: 238) amplicon and C1ORF32, chromosome 1 open, detectable by or in accordance with primers H19011_seg8-13F1 (SEQ ID NO: 236) and H19011_seg8-13R1 (SEQ ID NO: 237) Reading frame 32, transcript expression was measured by real-time PCR on lung panels. Details of the used samples are shown in Table 3 above. Samples that did not show amplicon detection (Sample Nos. 1, 2, 4 to 10, 12 to 27, 29 to 35, 37 to 41, 51 to 64, and 69 to 70, Table 3) were assigned Ct values of 41. And calculated accordingly. These samples showed a primer-dimer product with a characteristic dissociation curve and a significantly lower TM (this artifact was identified by appearing in a negative control without RT sample). For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1. Next, the normalized amount of each RT sample is divided by the intermediate value of the amount of normal samples (sample numbers 51-64 and 69-70, Table 3 above), and each sample relative to the intermediate value of the normal sample The value of how much was increased.

  FIG. 42 is a histogram showing overexpression of the C1ORF32 transcript shown above in cancerous lung samples compared to normal samples.

  As is clear from FIG. 42, the expression of the C1ORF32 transcript detectable in the small cell cancer sample by the above amplicon is shown in the non-cancerous samples (sample numbers 51-64 and 69-70, Table 3 above). Was significantly higher. In particular, at least 500-fold overexpression was observed in 9 out of 9 small cell carcinoma samples.

  The significance of these results was verified using statistical analysis as described below. The P value of the difference in the expression level of the C1ORF32 transcript detectable by the above amplicon in the small cell lung cancer sample from the normal tissue sample was determined by T test as 6.70e-003.

  When checked with Fisher's exact test, it was found that the 500-fold overexpression threshold produced a difference between small cell carcinoma samples and normal samples with a P value of 4.89e-007.

  The above values indicate that this result is statistically significant.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as just one example to serve as a non-limiting illustration of a suitable primer pair: H19011_seg8-13F1 (SEQ ID NO: 236) forward primer; and H19011_seg8-13R1 (SEQ ID NO: 237) Reverse primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to help in a non-limiting description of a suitable amplicon: H19011_seg8-13F1R1 (SEQ ID NO: 238).

  Forward primer> H19011_seg8-13F1 (SEQ ID NO: 236): GCCCAGTTTTGCTGTGGAGA

  Reverse primer> H19011_seg8-13R1 (SEQ ID NO: 237): GGTAGACATTTCTCTCGATCGCTC

  Amplicon> H19011_seg8-13F1R1 (SEQ ID NO: 238)

  GCCCAGTTTTGCTGTGGAGATTATGCCAGAGTGGGTGTTTGTTGGCCTGGTGCTCCTGGGCGTCTTCCTCTTCTTCGTCCTGGTGGGGATCTGCTGGTGCCAGTGCTGCCCTCACAGCTGCTGCTGCTATGTCCGCTGCCCATGCTGCCCAGATTCCTGCTGGTGCCCTCAAGCCTGTGAGTACAGTGACCGCTGGGGAGACAGAGCGATCGAGAGAAATGTCTACC

  C1ORF32, No. 1, detectable in normal and cancerous lung tissue, normal and cancerous colon tissue, different normal tissues, and blood-specific panels by the amplicon shown in SEQ ID NO: H19011_junc8-10seg13 (SEQ ID NO: 241) Chromosome open reading frame 32, expression of H19011 transcript

  Junc8-10seg13-H19011_junc8-10seg13 (SEQ ID NO: 241) amplicon and primers H19011_junc8-10seg13F1 (SEQ ID NO: 239) and H19011_junc8-10seg13R1 (SEQ ID NO: 240), or detectable according to these C1 ORF32 transcript expression Measured by real-time PCR on lung, colon, normal, and blood panels. Details of the used samples are shown in Table 3, Table 5, Table 2, and Table 1, respectively. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1.

  For the lung panel-the non-detected sample (Sample No. 69, Table 3) was Ct value 41 and calculated accordingly. Next, the normalized amount of each RT sample is the intermediate value of the amount of normal samples (sample numbers 51, 53, 54, 56, 57, 59, 61, 62, 64, and 70, Table 3 above). The value of how many times each sample was enhanced relative to the intermediate value of the normal sample was obtained.

  FIG. 43 is a histogram showing overexpression of the C1ORF32 transcript shown above in cancerous lung samples compared to normal samples.

  As is clear from FIG. 43, the expression of the C1ORF32 transcript that can be detected by the amplicon in the small cell carcinoma sample is shown in the non-cancerous samples (sample numbers 51, 53, 54, 56, 57, 59, 61). 62, 64, and 70, significantly higher than Table 3) above. In particular, at least 7-fold overexpression was observed in 9 out of 9 small cell carcinoma samples.

  The significance of these results was verified using statistical analysis as described below.

  The P value of the difference in the expression level of the C1ORF32 transcript detectable by the amplicon in the small cell lung cancer sample from the normal tissue sample was determined by T test as 2.34e-003.

  When checked with Fisher's exact test, it was found that the 7-fold overexpression threshold produced a difference between small cell carcinoma samples and normal samples with a P value of 1.08e-005.

  The above values indicate that this result is statistically significant.

  For the colon panel-a non-detected sample (Sample No. 79, Table 5) was calculated with a Ct value of 41. Next, the normalized amount of each RT sample was divided by the median value of the amount of normal samples (sample numbers 42-62 and 65-70, Table 5 above). Next, the reciprocal of this ratio was calculated to obtain a value indicating how many times each sample was controlled downward relative to the intermediate value of the normal sample.

  FIG. 44 is a histogram showing the down-regulation of the C1ORF32 transcript shown above in a cancerous colon sample compared to a normal sample.

  As is clear from FIG. 44, the expression of the C1ORF32 transcript detectable in the cancer sample by the amplicon is higher than that in the non-cancerous samples (sample numbers 42 to 62 and 65 to 70, Table 5 above). It was very low. In particular, at least 5 times down-regulation was seen in 15 of 36 adenocarcinoma samples.

  The significance of these results was verified using statistical analysis as described below. When checked with Fisher's exact test, it was found that the 5-fold down-regulation threshold produced a difference between cancer and normal samples with a P value of 4.29e-004.

  The above values indicate that this result is statistically significant.

  For normal panels-Non-detected samples (Sample Nos. 42 and 49, Table 2) were Ct values 41 and calculated accordingly. Next, as shown in FIG. 45A, the normalized amount of each RT sample is divided by the median value of the colon sample (sample numbers 4 and 5, Table 2 above), and Sample relative expression values were obtained. Next, as shown in FIG. 45B, the normalized amount of each RT sample is divided by the median value of the amount of lung samples (sample numbers 26, 29, and 30, Table 2 above) to obtain a median of lung samples. The value of the relative expression of each sample relative to the value was obtained.

  For the blood panel-the normalized amount of each RT sample is divided by the median value of the normal kidney sample (sample numbers 65-67, Table 1 above) and the relative value of each sample to the median value of the normal kidney sample Expression values were obtained.

  The result of this analysis is shown in the histogram of FIG. The expression of the above-mentioned C1ORF32 transcript is high in one lymphoma sample (sample number 33, Table 1), but is also high in normal brain samples.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pairs were used as just one example to help non-limiting description of suitable primer pairs: H19011_junc8-10seg13F1 (SEQ ID NO: 239) forward primer; and H19011_junc8-10seg13R1 (SEQ ID NO: 240) Reverse primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to serve as a non-limiting description of a suitable amplicon: H19011_junc8-10seg13F1R1 (SEQ ID NO: 241)

  Forward primer> H19011_junc8-10seg13F1 (SEQ ID NO: 239)

  TGTGGAGATATTGCCCAGAGTG

  Reverse primer> H19011_junc8-10seg13R1 (SEQ ID NO: 240)

  GACATTTCTCTCGATCGCTCTGT

  Amplicon> H19011_junc8-10seg13F1R1 (SEQ ID NO: 241)

  TGTGGAGATTATGCCAGAGTGGGTGTTTGTTGGCCTGGTGCTCCTGGGCGTCTTCCTCTTCTTCGTCCTGGTGGGGATCTGCTGGTGCCAGTGCTGCCCTCACAGCTGCTGCTGCTATGTCCGCTGCCCATGCTGCCCAGATTCCTGCTGGTGCCCTCAAGCCTGTGAGTACAGTGACCGCTGGGGAGACAGAGCGATCGAGAGAAATGTC

  C1ORF32, Chromosome 1 open reading frame 32, H19011 transcription, detectable in normal and cancerous lung tissue, and in normal and cancerous colon tissue, by the amplicon represented by SEQ ID NO: H19011_junc6-10 (SEQ ID NO: 244) Product expression

  The expression of the C1ORF32 transcript, which is detectable by or according to the junc6-10-H19011_junc6-10F1R1 (SEQ ID NO: 244) amplicon and the primers H19011_junc6-10F1 (SEQ ID NO: 242) and H19011_junc6-10R1 (SEQ ID NO: 243), Measured by real-time PCR on lung and colon panels. Details of the used samples are shown in Tables 3 and 5 above, respectively. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1.

  Lung panel—The normalized amount of each RT sample was then divided by the median of the amount of normal samples (sample numbers 51-64, 69, and 70, Table 3 above). Next, the reciprocal of this ratio was calculated to obtain a value indicating how many times each sample was controlled downward relative to the intermediate value of the normal sample.

  FIG. 47 is a histogram showing downregulation of the C1ORF32 transcript shown above in cancerous lung samples compared to normal samples.

  As is clear from FIG. 47, the expression of the C1ORF32 transcript detectable by the above amplicon in non-small cell cancer samples, adenocarcinoma and squamous cell carcinoma is shown in non-cancerous samples (sample numbers 51 to 64, 69). , And 70, significantly lower than Table 3) above. In particular, at least 5-fold down-regulation was seen in 23 of 39 non-small cell cancer samples, especially in 8 samples of 17 adenocarcinoma samples and 12 samples of 16 squamous cell carcinoma samples.

  The significance of these results was verified using statistical analysis as described below. The P value of the difference in the expression level of the C1ORF32 transcript detectable by the above amplicon in the non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma samples from the normal tissue sample, respectively, was 1.18e − Determined by T test as 003, 2.87e-002, and 3.55e-004.

  When checked by Fisher's exact test, a down-regulation 5-fold threshold was 1.59e-003, 3.3 between non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma samples and normal samples, respectively. Differences were found to occur at P values of 54e-002 and 4.78e-004.

  The above values indicate that this result is statistically significant.

  Colon panel—The normalized amount of each RT sample was then divided by the median of the amount of normal samples (sample numbers 42-70, Table 5 above). Next, the reciprocal of this ratio was calculated to obtain a value indicating how many times each sample was controlled downward relative to the intermediate value of the normal sample.

  FIG. 48 is a histogram showing down-regulation of the C1ORF32 transcript shown above in a cancerous colon sample compared to a normal sample.

  As is clear from FIG. 48, the expression of the C1ORF32 transcript detectable in the cancer sample by the amplicon was significantly lower than that in the non-cancerous sample (sample numbers 42 to 70, Table 5 above). In particular, at least 9-fold down-regulation was observed in 23 of 55 adenocarcinoma samples.

  The significance of these results was verified using statistical analysis as described below.

  When checked with Fisher's exact test, it was found that a 9-fold down-regulation threshold produced a difference between cancer and normal samples with a P value of 7.39e-006.

  The above values indicate that this result is statistically significant.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as just one example to serve as a non-limiting illustration of a suitable primer pair: H19011_junc6-10F1 (SEQ ID NO: 242) forward primer; and H19011_junc6-10R1 (SEQ ID NO: 243) Reverse primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to serve as a non-limiting description of a suitable amplicon: H19011_junc6-10F1R1 (SEQ ID NO: 244)

  Forward primer> H19011_junc6-10F1 (SEQ ID NO: 242)

  ACTCTATTACTGTATTATTACCACCACCAG

  Reverse primer> H19011_junc6-10R1 (SEQ ID NO: 243)

  CCCAACAAACACCCCACTCCAAC

  Amplicon> H19011_junc6-10F1R1 (SEQ ID NO: 244)

  ACTCTATTACTGTATTATACCACCACCCCAGATGACCTGGAGGGGAAAAATGAGGGCTCACTGGGACTGCTGGTGTTGGAGTGGGTGTTTGTTG

Example 7: Description of Cluster R31375 The present invention relates to the specific antigen FXYD3 and related diagnostics and therapeutics based thereon.

  According to the present invention, cluster R31375 (internal ID 72360301) features 19 target transcripts and 4 target sections, the names of which are shown in Tables 105 and 106, respectively. Selected protein variants are shown in Table 107.

Table 105: Target transcripts

Table 106: Target sections

Table 107: Proteins of interest

  These sequences include the known protein FXYD domain-containing ion transport regulator 3 precursor (SwissProt accession number FXYD3_HUMAN (SEQ ID NO: 70); chlorine conductance inducer cnc Mat-8; Mammary tumor 8 kDa protein; also known as Phospholeman-like, referred to herein as a known protein.

  FXYD3 was previously identified within a gene cluster induced by the neu or Ha-Ras oncogene in mouse mammary tumors and was named Mat-8 (breast tumor, 8 kDa) (Morrison et al 1995). In normal tissues, FXYD3 is mainly expressed in the uterus, stomach, colon, and skin (Morrison et al 1995). Its expression was found to be elevated in human primary breast tumors, as well as prostate and pancreatic ductal adenocarcinoma (Grzmil et al 2004, Kayed et al 2006). A specific inhibition of its expression in prostate cancer cell lines by siRNA suggests a role in cell proliferation (Grzmil et al 2004).

  FXYD3 belongs to the FXYD family protein. All seven known members of this family are small membrane proteins with 6 amino acids in common that contain the FXYD motif. Most FXYD proteins, including human FXYD3, are type I membrane proteins with a transmembrane domain and a cleavable signal peptide. However, the signal peptide of mouse FXYD3 is not cleaved and this signal peptide can act as a second transmembrane domain (Crambert et al 2005, Geering 2006). Like other members of the FXYD family, FXYD3 interacts with Na / K-ATPase and regulates its activity in a tissue-specific manner (Crambert et al 2005, Arimochi et al 2007, Geering 2006). ).

  Two splice variant isoforms of FXYD3 have been previously reported (Bibert et al 2006). They differ by 26 amino acids in the frame insertion after the transmembrane domain and are differentially expressed during cell division. Furthermore, both isoforms can stably associate with Na / K-ATPase and play different functional roles in the control of the activity of this ATPase (Bibert et al 2006).

  In addition, WO2003101283 may be relevant to the present invention. This PCT application is a differential encoding that encodes a protein sequence that R31375_P0 (SEQ ID NO: 70) (wild-type FXYD3 nucleic acid coding sequence reported herein) can be used as a diagnostic marker for human lung cancer. Claims to disclose that the nucleic acid is expressed.

  Furthermore, WO2003000012 claims to disclose a protein associated with human breast cancer, referred to as protein # 12, which appears to correspond to R31375-P0 (wild type) disclosed herein. Furthermore, US Pat. No. 7,189,507 discloses a gene called MAT8 that appears to correspond to R31375_P0 (SEQ ID NO: 70) in a long table of gene sequences. This table appears to suggest that this gene can be expressed in ovarian cancer.

  The protein FXYD domain-containing ion transport regulator 3 precursor (SEQ ID NO: 70) is known or believed to have the following functions: when expressed in Xenopus oocytes Next, a hyperpolarization-activated chloride current is induced. It can be a modifier with the ability to activate endogenous oocyte channels. Known polymorphisms for this sequence are shown in Table 108.

Table 108: Amino acid mutations for known proteins

  The localization of the protein FXYD domain-containing ion transport regulator 3 precursor (SEQ ID NO: 70) is thought to be a membrane; a type I membrane protein (potential) that penetrates the membrane once.

  The following GO annotations apply to known proteins. The following annotations were discovered: chloride ion transport, which is related to biological processes; chloride channel activity, which is related to molecular function; and integration into the cell membrane, which is related to cellular components Annotation.

  This GO assignment is <http: // www. expasy. SwissProt / TremBl protein knowledge base available from ch / sprot />; or <http: // www. ncbi. nlm. nih. Based on information from one or more of Locuslinks available from gov / projects / LocusLink />.

  According to the present invention, a novel FXYD3 splice variant has been identified as a membrane-bound protein that is expected to be overexpressed in cancer. FXYD3 is a type I membrane-bound protein (Bibert et. Al Al 2006), and according to the present invention, three novel splice variants of this protein are provided. A novel splice variant referred to herein as R31375_P14 (SEQ ID NO: 72) has an additional in-frame exon within its extracellular region. The addition of this exon increases the length of the extracellular region by 298 amino acids, which is an important addition to the relatively short extracellular domain of 18 amino acids of the wild type protein. A novel splice variant, referred to herein as R31375_P31 (SEQ ID NO: 73), has an additional in-frame exon in the near-membrane domain of FXYD3, which adds 26 new amino acids to the intracellular region. A novel splice variant referred to herein as R31375_P33 (SEQ ID NO: 74) has the third coding exon of wild type FXYD3 skipped and, like R31375_P31 (SEQ ID NO: 73), within the near membrane domain Has an additional in-frame exon. This causes a deletion of 8 amino acids in the ectodomain.

  According to the present invention, FXYD3 and R31375_P14 (SEQ ID NO: 72) were shown to be overexpressed in ovarian cancer.

  As mentioned above, cluster R31375 features 19 transcripts and these are shown in Table 105 above. These transcripts encode proteins that are variants of the protein FXYD domain containing ion transport regulator 3 precursor (SEQ ID NO: 70). Each mutant protein of the present invention will be described.

  Mutant proteins R31375_P0 (SEQ ID NO: 70) of the present invention are transcripts R31375_T0 (SEQ ID NO: 51), R31375_T1 (SEQ ID NO: 52), R31375_T10 (SEQ ID NO: 61), R31375_T11 (SEQ ID NO: 62), R31375_T12 (SEQ ID NO: 63). R31375_T13 (SEQ ID NO: 64), R31375_T2 (SEQ ID NO: 53), R31375_T3 (SEQ ID NO: 54), R31375_T4 (SEQ ID NO: 55), R31375_T5 (SEQ ID NO: 56), R31375_T6 (SEQ ID NO: 57), R31375_T7 (SEQ ID NO: 58), It has an amino acid sequence encoded by R31375_T8 (SEQ ID NO: 59) and R31375_T9 (SEQ ID NO: 60).

  Mutant protein localization was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, mutant proteins are thought to be localized next: membranes.

  In addition, mutant protein R31375_P0 (SEQ ID NO: 70) listed in Table 109 listed the following non-silent SNPs (single nucleotide polymorphisms) (alternative amino acids in light of the positions on the amino acid sequence (SEQ ID NO: 70)). ).)

Table 109: Amino acid mutations

  The coding part of the transcript R31375_T0 (SEQ ID NO: 51) starts at position 491 and ends at position 751. The transcript also has the following SNPs listed in Table 110. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 110: Nucleic acid SNPs

  The coding part of the transcript R31375_T1 (SEQ ID NO: 52) starts at position 795 and ends at position 1055. The transcript also has the following SNPs listed in Table 111. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 111: Nucleic acid SNPs

  The coding part of the transcript R31375_T10 (SEQ ID NO: 61) starts at position 1826 and ends at position 2086. The transcript also has the following SNPs listed in Table 112. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 112: Nucleic acid SNPs

  The coding part of the transcript R31375_T11 (SEQ ID NO: 62) starts at position 613 and ends at position 873. The transcript also has the following SNPs listed in Table 113: (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 113: Nucleic acid SNPs

  The coding part of the transcript R31375_T12 (SEQ ID NO: 63) starts at position 711 and ends at position 971. The transcript also has the following SNPs listed in Table 114. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 114: Nucleic acid SNPs

  The coding part of the transcript R31375_T13 (SEQ ID NO: 64) starts at position 1015 and ends at position 1275. The transcript also has the following SNPs listed in Table 115. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 115: Nucleic acid SNPs

  The coding part of the transcript R31375_T2 (SEQ ID NO: 53) starts at position 678 and ends at position 938. The transcript also has the following SNPs listed in Table 116. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 116: Nucleic acid SNPs

  The coding part of the transcript R31375_T3 (SEQ ID NO: 54) starts at position 572 and ends at position 832. The transcript also has the following SNPs listed in Table 117: (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 117: Nucleic acid SNPs

  The coding part of the transcript R31375_T4 (SEQ ID NO: 55) starts at position 575 and ends at position 835. The transcript also has the following SNPs listed in Table 118. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 118: Nucleic acid SNPs

  The coding part of the transcript R31375_T5 (SEQ ID NO: 56) starts at position 656 and ends at position 916. The transcript also has the following SNPs listed in Table 119. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 119: Nucleic acid SNPs

  The coding part of the transcript R31375_T6 (SEQ ID NO: 57) starts at position 697 and ends at position 957. The transcript also has the following SNPs listed in Table 120. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 120: Nucleic acid SNPs

  The coding part of the transcript R31375_T7 (SEQ ID NO: 58) starts at position 2475 and ends at position 2735. The transcript also has the following SNPs listed in Table 121. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 121: Nucleic acid SNPs

  The coding part of the transcript R31375_T8 (SEQ ID NO: 59) starts at position 1329 and ends at position 1589. The transcript also has the following SNPs listed in Table 122: (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 122: Nucleic acid SNPs

  The coding part of the transcript R31375_T9 (SEQ ID NO: 60) starts at position 2586 and ends at position 2846. The transcript also has the following SNPs listed in Table 123. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 123: Nucleic acid SNPs

  The mutant protein R31375_P14 (SEQ ID NO: 72) of the present invention has the amino acid sequence encoded by the transcripts R31375_T19 (SEQ ID NO: 65), R31375_T25 (SEQ ID NO: 66), and R31375_T26 (SEQ ID NO: 67). An alignment to one or more published protein sequences is shown in FIG. 49A. A brief description of the relationship of the mutant protein of the present invention to each of the aligned proteins is as follows.

  1. Comparative report between R31375_P14 (SEQ ID NO: 72) and the known proteins FXYD3_HUMAN, NP_005962, and Q6IB59_HUMAN (SEQ ID NO: 70) (FIG. 49A)

  A. An isolated chimeric polypeptide encoding R31375_P14 (SEQ ID NO: 72), corresponding to amino acids 1 to 32 of the known proteins FXYD3_HUMAN, NP_005962, and Q6IB59_HUMAN (SEQ ID NO: 70), of R31375_P14 (SEQ ID NO: 72) A first amino acid sequence at least 90% homologous to MQKVTLGLVFLAGFPVLDANDLEDKNSPFYY, which also corresponds to amino acids 1-32, and a sequence GAPYIFVKRMGQQKRTQAGTEVPSTFLL (SEQ ID NO: 294) corresponding to amino acids 33-61 of R31375_P14 (SEQ ID NO: 72) At least 70%, optionally at least 80%, preferably at least 85%, more preferably at least Corresponding to amino acids 33-87 of the second amino acid sequence 90%, and most preferably at least 95, 96, 97, 98, or 99% homologous, and the known proteins FXYD3_HUMAN, NP_005962, and Q6IB59_HUMAN (SEQ ID NO: 70) And a third amino acid sequence that is at least 90% homologous to DWHSLQVGGLICAGVLCAMGGIIIVMSACKCKFGFGKSFGHPGETPPLITPGSAQS corresponding to amino acids 62-116 of R31375_P14 (SEQ ID NO: 72), and the first amino acid sequence, the second amino acid sequence, and A third amino acid sequence is a polypeptide that is contiguous and in the order noted herein.

  B. An isolated polypeptide encoding an end of R31375_P14 (SEQ ID NO: 72), wherein the polypeptide is at least 70%, optionally at least about 80% relative to the sequence GAPYIFVKRMGGGQKRTQAGTEVPSTFLL (SEQ ID NO: 294) of R31375_P14 (SEQ ID NO: 72) A polypeptide comprising an amino acid sequence that is preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95, 96, 97, 98, or 99% homologous.

  The localization of this mutant protein was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, this mutant protein is thought to be localized next: membrane.

  Mutant protein R31375_P14 (SEQ ID NO: 72) has the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 124. (Substituted amino acids were listed in the light of their position on the amino acid sequence (SEQ ID NO: 72).)

Table 124: Amino acid mutations

  The coding part of the transcript R31375_T19 (SEQ ID NO: 65) starts at position 491 and ends at position 838. This transcript also has the following SNPs listed in Table 125. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 125: Nucleic acid SNPs

  The coding part of the transcript R31375_T25 (SEQ ID NO: 66) starts at position 575 and ends at position 922. This transcript also has the following SNPs listed in Table 126. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 126: Nucleic acid SNPs

  The coding part of the transcript R31375_T26 (SEQ ID NO: 67) starts at position 1443 and ends at position 1790. This transcript also has the following SNPs listed in Table 127. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 127: Nucleic acid SNPs

  The mutant protein R31375_P31 (SEQ ID NO: 73) of the present invention has an amino acid sequence encoded by the transcription product R31375_T29 (SEQ ID NO: 68). Alignments to one or more published protein sequences are shown in FIGS. 49B and 49C. A brief description of the relationship between the mutant proteins of the invention and each of the aligned proteins is as follows:

  1. Comparative report between R31375_P31 (SEQ ID NO: 73) and the known proteins FXYD3_HUMAN, NP_005962, and Q6IB59_HUMAN (SEQ ID NO: 70) (FIG. 49B):

  A. An isolated chimeric polypeptide encoding R31375_P31 (SEQ ID NO: 73), which corresponds to amino acids 1 to 58 of the known proteins FXYD3_HUMAN, NP_005962, and Q6IB59_HUMAN (SEQ ID NO: 70), of R31375_P31 (SEQ ID NO: 73) MQKVTLGLLVFLAGFPVLDANDLEDKNSPFYYDWHSLQVGGLICAGGVLCAMMGIIIVMS corresponding to amino acids 1 to 58 and a sequence EWRSSGEQAGRGWSPTTGPTQ2TGPTGSPTGS2TGPT peptide (SEQ ID NO: 73) corresponding to amino acids 59 to 84 of R31375_P31 (SEQ ID NO: 73) At least 70%, optionally at least 80% A second amino acid sequence that is preferably at least 85%, more preferably at least 90%, and most preferably at least 95, 96, 97, 98, or 99% homologous to the known proteins FXYD3_HUMAN, NP_005962, and Q6IB59_HUMAN ( A third amino acid sequence that is at least 90% homologous to AKCKCKFGQKSG corresponding to amino acids 59-70 of SEQ ID NO: 70) and also corresponding to amino acids 85-96 of R31375_P31 (SEQ ID NO: 73), wherein The chimeric polypeptide wherein the first amino acid sequence, the second amino acid sequence, and the third amino acid sequence are consecutive and in the order described.

  B. An isolated polypeptide encoding an end of R31375_P31 (SEQ ID NO: 73), wherein the polypeptide is at least 70%, optionally at least about 80% relative to the sequence EWRSSGEQAGRGGWSPPLTTQLSPTG (SEQ ID NO: 295) of R31375_P31 (SEQ ID NO: 73) A polypeptide comprising an amino acid sequence that is preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95, 96, 97, 98, or 99% homologous.

  2. Comparative report between R31375_P31 (SEQ ID NO: 73) and the known protein NP_068710 (SEQ ID NO: 71) (FIG. 49C):

  A. An isolated chimeric polypeptide encoding R31375_P31 (SEQ ID NO: 73), corresponding to amino acids 1 to 96 of the known protein NP_068710 (SEQ ID NO: 71), and amino acids 1 to 96 of R31375_P31 (SEQ ID NO: 73) The corresponding MQKVTLGLLVFLAGFPVLDANDLEDKNSPFYYDWHSLQVGGLICAGGVLCAMMGIIIVMSEWSRSSGEQAGRGGWSPPLTTQLSPTGAKCKCK is an amino acid sequence that is at least 90% homologous to the sequence, wherein said first amino acid sequence

  The localization of this mutant protein was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, this mutant protein is thought to be localized next: membrane.

  Mutant protein R31375_P31 (SEQ ID NO: 73) has the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 128. (Substituted amino acids were listed in the light of their position on the amino acid sequence (SEQ ID NO: 73).)

Table 128: Amino acid mutations

  Mutant protein R31375_P31 (SEQ ID NO: 73) is encoded by transcript R31375_T29 (SEQ ID NO: 68), the coding portion of which begins at position 491 and ends at position 778. This transcript also has the following SNPs listed in Table 129. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 129: Nucleic acid SNPs

  The mutant protein R31375_P33 (SEQ ID NO: 74) of the present invention has the amino acid sequence encoded by the transcript R31375_T39 (SEQ ID NO: 69). Alignments to one or more published protein sequences are shown in FIGS. 49D and 49E. A brief description of the relationship between the mutant proteins of the invention and each of the aligned proteins is as follows:

  1. Comparative report between R31375_P33 (SEQ ID NO: 74) and the known proteins FXYD3_HUMAN, NP_005962, and Q6IB59_HUMAN (SEQ ID NO: 70) (FIG. 49D):

  A. An isolated chimeric polypeptide encoding R31375_P33 (SEQ ID NO: 74), which corresponds to amino acids 1 to 24 of the known proteins FXYD3_HUMAN, NP_005962, and Q6IB59_HUMAN (SEQ ID NO: 70), of R31375_P33 (SEQ ID NO: 74) The first amino acid sequence that is at least 90% homologous to MQKVTLGLLVFLAGFPVLDANDLE, which also corresponds to amino acids 1 to 24, corresponds to amino acids 33 to 58 of the known proteins FXYD3_HUMAN, NP_005962, and Q6IB59_HUMAN (SEQ ID NO: 70), R31375_P33 ( At least 9 relative to DWHSLQVGGLICAGVLCAMGIIIVMS, which also corresponds to amino acids 25-50 of SEQ ID NO: 74) At least 70%, optionally at least 80% relative to a polypeptide having a second amino acid sequence that is% homologous and the sequence EWRSSGEQAGRGGWSPPLTTQLSPTG (SEQ ID NO: 295) corresponding to amino acids 51-76 of R31375_P33 (SEQ ID NO: 74); A third amino acid sequence that is preferably at least 85%, more preferably at least 90%, and most preferably at least 95, 96, 97, 98, or 99% homologous to the known proteins FXYD3_HUMAN, NP_005962, and Q6IB59_HUMAN (sequence No. 70) corresponding to amino acids 59 to 70 and at least 90% homologous to AKCKCKFGQKSG corresponding to amino acids 77 to 88 of R31375_P33 (SEQ ID NO: 74) Wherein the first amino acid sequence, the second amino acid sequence, the third amino acid sequence, and the fourth amino acid sequence are consecutive and in the stated order. A chimeric polypeptide.

  B. An isolated chimeric polypeptide encoding an end of R31375_P33 (SEQ ID NO: 74), comprising a polypeptide having a length “n”, wherein n is at least about 10 amino acids in length, optionally At least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length, and most preferably at least about 50 amino acids in length, wherein at least two amino acids are ED And starts with any amino acid number from 24-x to 24 and ends with any amino acid number of 25 + ((n−2) −x), where x is from 0 to n− A chimeric polypeptide having a sequence that varies up to 2).

  C. An isolated polypeptide encoding an end of R31375_P33 (SEQ ID NO: 74), wherein the polypeptide is at least 70%, optionally at least about 80% relative to the sequence EWRSSGEQAGRGGWSPPLTTQLSPTG (SEQ ID NO: 295) of R31375_P33 (SEQ ID NO: 74) A polypeptide comprising an amino acid sequence that is preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95, 96, 97, 98, or 99% homologous.

  2. Comparative report between R31375_P33 (SEQ ID NO: 74) and the known protein NP_068710 (SEQ ID NO: 71) (FIG. 49E):

  A. An isolated chimeric polypeptide encoding R31375_P33 (SEQ ID NO: 74), which corresponds to amino acids 1-24 of the known protein NP_068710 (SEQ ID NO: 71), and amino acids 1-24 of R31375_P33 (SEQ ID NO: 74) Corresponds to the first amino acid sequence that is at least 90% homologous to the corresponding MQKVTLGLLVFLAGFPVLDANDLE, and corresponds to amino acids 33 to 96 of the known protein NP_068710 (SEQ ID NO: 71), and to amino acids 25 to 88 of R31375_P33 (SEQ ID NO: 74) A second amino acid sequence that is at least 90% homologous to the corresponding DWHSLQVGGLICAGVLCAMGIIIVMSEWSRSSGEGESGWGPLPLTTQLSPTGAKCKCKFGQKSG; Wherein, wherein said first amino acid sequence and a second amino acid sequence is a continuous, it is this marked the order, chimeric polypeptides.

  B. An isolated chimeric polypeptide encoding an end of R31375_P33 (SEQ ID NO: 74), comprising a polypeptide having a length “n”, wherein n is at least about 10 amino acids in length, optionally At least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length, and most preferably at least about 50 amino acids in length, wherein at least two amino acids are ED And starts with any amino acid number from 24-x to 24 and ends with any amino acid number of 25 + ((n−2) −x), where x is from 0 to n− A chimeric polypeptide having a sequence that varies up to 2).

  The localization of this mutant protein was determined according to the results from a number of different software programs and analyses, including analysis by SignalP and other special programs. With respect to cells, this mutant protein is thought to be localized: secretion.

  Mutant protein R31375_P33 (SEQ ID NO: 74) has the following non-silent SNPs (single nucleotide polymorphisms) listed in Table 130. (Substituted amino acids were listed in the light of their position on the amino acid sequence (SEQ ID NO: 74).)

Table 130: Amino acid mutations

  The coding part of the transcript R31375_T39 (SEQ ID NO: 69) starts at position 491 and ends at position 754. This transcript also has the following SNPs listed in Table 131. (The alternative nucleic acids were listed in light of their position on the nucleotide sequence.)

Table 131: Nucleic acid SNPs

  According to an optional embodiment of the invention, short sections associated with the above clusters are also provided. These sections are up to about 120 bp in length and are included in another specification.

  The section cluster R31375_N30 (SEQ ID NO: 134) of the present invention is supported by 7 libraries. The number of libraries was determined as described above. This section is found in the following transcripts: R31375_T19 (SEQ ID NO: 65), R31375_T25 (SEQ ID NO: 66), and R31375_T26 (SEQ ID NO: 67). Table 132 below lists the start and end positions of this section on each transcript.

Table 132: Section location on transcript

  The section cluster R31375_N33 (SEQ ID NO: 135) of the present invention is supported by a library of 278. The number of libraries was determined as described above. This section is found in the following transcripts: R31375_T0 (SEQ ID NO: 51), R31375_T1 (SEQ ID NO: 52), R31375_T10 (SEQ ID NO: 61), R31375_T11 (SEQ ID NO: 62), R31375_T12 (SEQ ID NO: 63), R31375_T13 (SEQ ID NO: 63) No. 64), R31375_T19 (SEQ ID NO: 65), R31375_T2 (SEQ ID NO: 53), R31375_T25 (SEQ ID NO: 66), R31375_T26 (SEQ ID NO: 67), R31375_T29 (SEQ ID NO: 68), R31375_T3 (SEQ ID NO: 54), R31375_T39 (SEQ ID NO: 69), R31375_T4 (SEQ ID NO: 55), R31375_T5 (SEQ ID NO: 56), R31375_T6 (SEQ ID NO: 57), R31375_T7 (SEQ ID NO: 58). R31375_T8 (SEQ ID NO: 59), and R31375_T9 (SEQ ID NO: 60). Table 133 below lists the start and end positions of this section on each transcript.

Table 133: Position of the section on the transcript

  The segment cluster R31375_N34 (SEQ ID NO: 136) of the present invention is supported by a library of 275. The number of libraries was determined as described above. This section is found in the following transcripts: R31375_T0 (SEQ ID NO: 51), R31375_T1 (SEQ ID NO: 52), R31375_T10 (SEQ ID NO: 61), R31375_T11 (SEQ ID NO: 62), R31375_T12 (SEQ ID NO: 63), R31375_T13 (SEQ ID NO: 63) No. 64), R31375_T19 (SEQ ID NO: 65), R31375_T2 (SEQ ID NO: 53), R31375_T25 (SEQ ID NO: 66), R31375_T26 (SEQ ID NO: 67), R31375_T29 (SEQ ID NO: 68), R31375_T3 (SEQ ID NO: 54), R31375_T39 (SEQ ID NO: 69), R31375_T4 (SEQ ID NO: 55), R31375_T5 (SEQ ID NO: 56), R31375_T6 (SEQ ID NO: 57), R31375_T7 (SEQ ID NO: 58). R31375_T8 (SEQ ID NO: 59), and R31375_T9 (SEQ ID NO: 60). Table 134 below lists the start and end positions of this section on each transcript.

Table 134: Position of section on transcript

  The section cluster R31375_N37 (SEQ ID NO: 137) of the present invention is supported by 254 libraries. The number of libraries was determined as described above. This section is found in the following transcripts: R31375_T0 (SEQ ID NO: 51), R31375_T1 (SEQ ID NO: 52), R31375_T10 (SEQ ID NO: 61), R31375_T11 (SEQ ID NO: 62), R31375_T12 (SEQ ID NO: 63), R31375_T13 (SEQ ID NO: 63) No. 64), R31375_T19 (SEQ ID NO: 65), R31375_T2 (SEQ ID NO: 53), R31375_T25 (SEQ ID NO: 66), R31375_T26 (SEQ ID NO: 67), R31375_T29 (SEQ ID NO: 68), R31375_T3 (SEQ ID NO: 54), R31375_T39 (SEQ ID NO: 69), R31375_T4 (SEQ ID NO: 55), R31375_T5 (SEQ ID NO: 56), R31375_T6 (SEQ ID NO: 57), R31375_T7 (SEQ ID NO: 58). R31375_T8 (SEQ ID NO: 59), and R31375_T9 (SEQ ID NO: 60). Table 135 below lists the start and end positions of this section on each transcript.

Table 135: Position of sections on transcripts

  Expression of FXYD3 region-containing ion transport regulator 3 R31375 transcript detectable in normal and cancerous ovarian tissues, as well as in different normal tissues, by the amplicon represented by SEQ ID NO: R31375_junc30-33 (SEQ ID NO: 247)

  Junc30-33-R31375_junc30-33 (SEQ ID NO: 247) amplicon, and FXYD3 region-containing ion transport control detectable by or in accordance with primers R31375_junc30-33F1 (SEQ ID NO: 245) and R31375_junc30-33R1 (SEQ ID NO: 246) Factor 3 transcript expression was measured by real-time PCR in ovarian and normal panels. Details of the used samples are shown in Tables 4 and 2 above. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1.

  Ovarian panel—Non-detected samples (sample numbers 33 and 53, Table 4) were Ct values 41 and calculated accordingly. Next, the normalized amount of each RT sample is divided by the median value of the normal sample (sample numbers 52-78, Table 4 above), and each sample is increased by several times with respect to the median value of the normal sample. The value of what was obtained.

  FIG. 50 is a histogram showing overexpression of the FXYD3 region-containing ion transport regulator 3 transcript shown above in cancerous ovary samples compared to normal samples.

  As is clear from FIG. 50, the expression of the FXYD3 region-containing ion transport regulator 3 transcript detectable by the amplicon in adenocarcinoma samples, particularly mucinous cancer samples and endometrioid cancer samples, is non- It was significantly higher than the cancerous samples (sample numbers 52-78, Table 4 above). In particular, at least 18-fold overexpression was seen in 13 of the 37 adenocarcinoma samples, especially 7 of the 9 mucinous cancer samples and 4 of the class 10 endometrial cancer samples.

  The significance of these results was verified using statistical analysis as described below. The P value of the difference in the expression level of the FXYD3 region-containing ion transport control factor 3 transcript, which can be detected by the above amplicon, in the ovarian adenocarcinoma sample, mucinous cancer sample, and endometrioid cancer sample , Determined by T test as 9.75e-004, 1.92e-002 and 1.55e-002, respectively.

  When checked with Fisher's exact test, an 18-fold overexpression threshold was observed between adenocarcinoma, mucinous cancer sample, endometrioid cancer sample and normal sample, respectively, 2.71e-004, 4.31e. Differences were found to occur at P-values of -006 and 3.18e-003.

  The above values indicate that this result is statistically significant.

  Normal panels—non-detected samples (sample numbers 50 and 54, Table 2) were Ct values 41 and calculated accordingly. Next, as shown in FIG. 51, the normalized amount of each RT sample is divided by the median value of the amount of ovary samples (sample numbers 31-34, Table 2 above) and Sample relative expression values were obtained.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as an example to serve as a non-limiting illustration of a suitable primer pair: R31375_junc30-33F1 (SEQ ID NO: 245) forward primer; and R31375_junc30-33R1 (SEQ ID NO: 246) Reverse primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as an example to serve as a non-limiting description of a suitable amplicon: R31375_junc30-33 (SEQ ID NO: 247)

  Forward primer> R31375_junc30-33F1 (SEQ ID NO: 245): GTGCTCCATATAATTTTGCAAGAGAATG

  Reverse primer> R31375_junc30-33R1 (SEQ ID NO: 246): GGGGCTGTGCCAGTCTAGGG

  Amplicon> R31375_junc30-33 (SEQ ID NO: 247): GTGCTCCATATATATTTTCACAGAGATGGGGGGACAGATAGGAAGAGGACACAGGCTGGCACTGAGGTCCCCTCCACTTTCCTCTCTAGACTGGCACAGCCCTC

  Expression of FXYD3 region-containing ion transport regulator 3 R31375 transcript detectable in normal and cancerous ovarian tissues, as well as in different normal tissues, by the amplicon represented by SEQ ID NO: R31375_seg33junc34-37 (SEQ ID NO: 250)

  An ion transport containing FXYD3 region that is detectable by or in accordance with seg33junc34-37-R31375_seg33junc34-37 (SEQ ID NO: 250) amplicon and primers R31375_seg33junc34-37F1 (SEQ ID NO: 248) and R31375_seg33junc34-37R1 (SEQ ID NO: 249) Factor 3 transcript expression was measured by real-time PCR in ovarian and normal panels. Details of the used samples are shown in Tables 4 and 2 above. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1.

  Ovarian panel—non-detected samples (sample numbers 52, 61, and 70, Table 4) were assigned a Ct value of 41 and calculated accordingly. Next, the normalized amount of each RT sample is divided by the median value of the normal sample (sample numbers 52-78, Table 4 above), and each sample is increased by several times with respect to the median value of the normal sample. The value of what was obtained.

  FIG. 52 is a histogram showing overexpression of the FXYD3 region-containing ion transport regulator 3 transcript shown above in cancerous ovary samples compared to normal samples.

  As is clear from FIG. 52, the expression of the FXYD3 region-containing ion transport regulator 3 transcript detectable by the amplicon in adenocarcinoma samples, serous cancers, mucinous cancers, and endometrioid cancers is It was significantly higher than non-cancerous samples (sample numbers 52-78, Table 4 above). In particular, 20 out of 37 adenocarcinoma samples, especially 7 out of 18 serous cancer samples, 8 out of 9 mucinous cancer samples, and 5 out of 10 endometrial cancer samples have at least 85-fold overexpression. It was seen.

  The significance of these results was verified using statistical analysis as described below. Expression of FXYD3 region-containing ion transport regulator 3 transcripts detectable by the above amplicons against normal tissue samples with expression levels in ovarian adenocarcinoma samples, serous cancer samples, mucinous cancer samples, and endometrioid cancer samples The P value of the difference was determined by T test as 1.61e-004, 5.40e-003, 1.49e-002, and 9.08e-003, respectively.

  When checked with Fisher's exact test, an 85-fold overexpression threshold was found to be 8.11e-007 among adenocarcinoma, serous cancer, mucinous cancer, and endometrioid cancer, and normal samples, respectively. Differences were found to occur at P values of 7.01e-004, 2.97e-007, and 5.78e-004.

  The above values indicate that this result is statistically significant.

  Normal panel—Next, as shown in FIG. 53, the normalized amount of each RT sample is divided by the intermediate value of the amount of the ovary sample (sample numbers 31-34, Table 2 above) The value of the relative expression of each sample relative to the value was obtained.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pair was used as an example to serve as a non-limiting illustration of a suitable primer pair: R31375_seg33junc34-37F1 (SEQ ID NO: 248) forward primer; and R31375_seg33junc34-37R1 (SEQ ID NO: 249) Reverse primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to serve as a non-limiting description of a suitable amplicon: R31375_seg33junc34-37 (SEQ ID NO: 250)

  Forward primer> R31375_seg33junc34-37F1 (SEQ ID NO: 248)

  ACTGGCACAGCCCTCCAGG

  Reverse primer> R31375_seg33junc34-37R1 (SEQ ID NO: 249)

  CATTTGCATTTTGCACTCATG

  Amplicon> R31375_seg33junc34-37 (SEQ ID NO: 250)

  ACTGGCACAGCCCTCCAGGTTGGCGGGCTCATCTGCGCTGGGGTTCTGTGGCCATGGGGCATCATCATGTCATGAGTGCAAAATGCAAATG

  Expression of FXYD3 region-containing ion transport regulator 3 R31375 transcripts detectable in normal and cancerous ovarian tissues, as well as in different normal tissues, by the amplicon represented by the sequence name R31375_junc20-22seg30F6R6 (SEQ ID NO: 253)

  Junc20-22seg30-R31375_junc20-22seg30F6R6 (SEQ ID NO: 253) amplicon, and primers R31375_junc20-22seg30F6 (SEQ ID NO: 251) and R31375_junc20-22seg30R6 (SEQ ID NO: 252), a region that can be detected in accordance with X Factor 3 transcript expression was measured by real-time PCR in ovarian and normal panels. Details of the used samples are shown in Tables 4 and 2 above. For each RT sample, the above amplicon expression was normalized to the normalization factor calculated from the expression of several housekeeping genes described in Example 1.

  About the ovarian panel-non-detected samples (sample numbers 2, 6, 9, 12, 15, 19, 21, 24, 32, 34, 38, 45, 53, 56-59, 62, 63, 65-67, and 72 -78, Table 4) was calculated according to the Ct value 41. Next, the normalized amount of each RT sample is the intermediate value of the amount of normal samples (sample numbers 52, 53, 56-59, 62, 63, 65-67, and 72-78, Table 4 above). The value of how many times each sample was enhanced relative to the intermediate value of the normal sample was obtained.

  FIG. 54 is a histogram showing overexpression of the FXYD3 transcript shown above in cancerous ovary samples compared to normal samples.

  As is apparent from FIG. 54, the expression of the FXYD3 transcript detectable by the amplicon in the adenocarcinoma sample, serous cancer sample, mucinous cancer sample, and endometrioid cancer sample is non-cancerous sample. (Sample numbers 52, 53, 56-59, 62, 63, 65-67, and 72-78, Table 4 above) were significantly higher. In particular, at least 14-fold overexpression was observed in 21 samples of 33 adenocarcinoma samples, 10 samples of 16 serous cancer samples, 5 samples of 8 mucinous cancer samples, and 6 samples of class 9 endometrial cancer samples. It was.

  The significance of these results was verified using statistical analysis as described below. The P value of the difference in the expression level of the FXYD3 transcript detectable by the above amplicon from the normal tissue sample in the ovarian adenocarcinoma sample was determined by T test as 3.78e-003.

  As checked by Fisher's exact test, a 14-fold overexpression threshold was found to be 4.21e-005 between adenocarcinoma, serous cancer, mucinous cancer, and endometrioid cancer, and normal samples, respectively. Differences were found to occur at P values of 5.17e-004, 4.46e-003, and 1.73e-003.

  The above values indicate that this result is statistically significant.

  Normal panel-non-detected samples (sample numbers 1, 10, 11, 14, 17, 25, 29, 31-34, 38, 39, 46, 47, 49-54, 57, 58, 61-65, 68, 69 , And 73, Table 2) were calculated according to Ct value 41. Next, as shown in FIG. 55, the normalized amount of each RT sample is divided by the median value of the amount of ovary samples (sample numbers 31-34, Table 2 above), and Sample relative expression values were obtained.

  Also, primer pairs are optionally and preferably included within the scope of the present invention. For example, in the above experiment, the following primer pairs were used as just one example to help in non-limiting description of suitable primer pairs: R31375_junc20-22seg30F6 (SEQ ID NO: 251) forward primer; and R31375_junc20-22seg30R6 (SEQ ID NO: 252) Reverse primer

  The invention also preferably includes any amplicon obtained through the use of any suitable primer pair. For example, in the above experiment, the following amplicon was obtained as just one example to serve as a non-limiting illustration of a suitable amplicon: R31375_junc20-22seg30F6R6 (SEQ ID NO: 253)

  Forward primer> R31375_junc20-22seg30F6 (SEQ ID NO: 251)

  TTGTGTTCCTGGGCAGGCTTT

  Reverse primer> R31375_junc20-22seg30R6 (SEQ ID NO: 252)

  TCATCTGTCCCCCCATTTCC

  Amplicon> R31375_junc20-22seg30F6R6 (SEQ ID NO: 253)

  TTGTGTTCCTGGCAGGCTTTCCTGTCCTGACGCCCAATGACCTAGAAGATAAAAAACAGTCCTTTTCACTATGGTCTCCATATATTTTGTCAAGGAATGGGGGGACAGATAGGA

Example 8: Cloning of full-length transcripts encoding VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 fused to EGFP Full-length encoding VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 fused to EGFP Product cloning was performed as described below.

  First, an EGFP expression vector was constructed, and then VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 open reading frame (ORF) was cloned. EGFP was subcloned into pIRESpuro3 (Clontech catalog number 631619) as follows: The EGFP-N1 vector (Clontech catalog number 6085-1) was digested with NheI and NotI to excise the EGFP gene. The EGFP insert was then ligated into pIRESpuro3 (Clontech catalog number 631619) that had been previously digested with the same enzymes, resulting in the EGFP-pIRESpuro3 vector.

  Cloning of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, FXYD3 open reading frame (ORF) was performed using the following steps:

  1. A reverse transcription reaction was performed as follows: 10 μg of purified RNA was mixed with 150 ng of random hexamer primer (Invitrogen, Carlsbad, CA, USA, catalog number: 48190-011) and 500 μM dNTPs, and the total volume was 156 μl. This mixture was incubated at 65 ° C. for 5 minutes and then quenched on ice. Then 50 μl of 5X Superscript II first strand buffer (Invitrogen, catalog number: 18064-014, product number: Y00146), 24 μl of 0.1 M DTT, and 400 units of RNasin (Promega, Milwaukee, Wis., USA, catalog) No. N2511) was added and the mixture was incubated at 25 ° C. for 10 minutes and further incubated at 42 ° C. for 2 minutes. Subsequently, 10 μl (2000 units) Superscript II (Invitrogen, catalog number: 18064-014) was added and the reaction (final volume 250 μl) was incubated at 42 ° C. for 50 minutes and then at 70 ° C. for 15 minutes. The obtained cDNA was diluted 1:20 using TE buffer (10 mM Tris, 1 mM EDTA pH 8).

  2. PCR was performed using Platinum PFX ™ (Invitrogen, Carlsbad, CA, USA, catalog number: 1178-021) under the following conditions: 5 μl Platinum PFX 10 × buffer; 5 μl—cDNA from above; 2 μl-10 mM dNTP (2.5 mM each nucleotide); 0.5 μl-Platinum PFX enzyme; 37 μl-H 2 O; and 1.5 μl of each primer (15 μM) in a total reaction volume of 50 μl; 35 minutes at 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 50 seconds; then 68 ° C for 10 minutes. The primers used include gene specific sequences corresponding to the desired protein and restriction enzyme site coordinates, as well as the Kozak sequence, as listed in Table 136 below. In Table 136, bold type represents specific gene sequences, while the range of restriction sites utilized for cloning purposes is italicized and the Kozak sequence is underlined.

  Table 136 shows the cloning process for ORF targets. For example, FXYD3_T25_P14 and VSIG1_T6_P5 are PCR amplification of two full-length overlapping fragments in step 1, followed by step 2 using both PCR fragments from step 1 as templates for full-length production. Cloned by further PCR. VSIG1_T5_P4 was cloned using both PCR fragments generated in step 1 for digestion and direct ligation, and AI216611_T1_P1 was cloned by performing nested PCR on the PCR product generated from step 1. Load 5 μl of the products of numbers 1, 4, 5, 8, 9, 10, 11, 12, 15, 16, and 17 (Table 136) onto a 1% agarose gel stained with ethidium bromide and at 100V Electrophoresis was performed in 1 × TBE solution and visualized using UV light. After confirmation of the expected size band, the remaining PCR product was treated with a Qiaquick PCR purification kit (Qiagen ™, Valencia, CA, USA, catalog number 28106) for DNA purification. The extracted PCR products were digested with the appropriate restriction enzymes (New England Biolabs, Beverly, Massachusetts, USA) as listed in Table 136. After digestion, DNA was loaded onto a 1% agarose gel as described above. The expected band size was excised and extracted from the gel using the QiaQuick ™ gel extraction kit (Qiagen, catalog number: 28707).

  The digested target ORF DNA was ligated to the EGFP_pIRESpuro3 vector using the LigaFast ™ Rapid DNA Ligation System (Promega, catalog number: M8221). The resulting DNA is transformed into competent E. coli DH5α (RBC Bioscience, Taipei, Taiwan, catalog number: RH816) according to the manufacturer's instructions and then LB-ampicillin agar plates for selection of recombinant plasmids. Seeded up and incubated overnight at 37 ° C.

  The next day, a large number of colonies from each transformation that grew on the selection plate were picked, streaked on another selection plate, and further by PCR using GoTaq ReadyMix (Promega, catalog number: M7122). Analysis was carried out. Screening positive clones were performed by PCR using pIRESpuro3 vector specific primer and gene specific primer (data not shown). After completion of all PCR cycles, half of the reaction was analyzed using a 1% agarose gel as described above. After confirmation of the predicted size band, two positive colonies from each ligation reaction were shaken overnight at 37 ° C. in 5 ml of Terrific Broth supplemented with 100 μg / ml ampicillin. And grew up. Plasmid DNA was isolated from bacterial cultures using the Qiaprep ™ Spin Miniprep Kit (Qiagen, catalog number: 27106). The correctness of the cloning was confirmed by sequencing the insert (Weizmann Institute, Rehovot, Israel). After confirmation of error-free colonies (ie, no mutations in the ORF), the recombinant plasmids were processed for further analysis.

  The resulting VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 full length DNA sequences fused to EGFP are shown in FIGS. 56A-J. In FIGS. 56A-J, the gene-specific sequence corresponding to the target full-length sequence is shown in bold, the EGFP sequence is italicized, not bold, and known SNP / silence mutations are underlined. did. 56A shows the DNA sequence (996 bp) of FXYD3_T0_P0_EGFP (SEQ ID NO: 77); FIG. 56B shows the DNA sequence (1083 bp) (SEQ ID NO: 78) of FXYD3_T25_P14_EGFP; FIG. 56C shows the DNA sequence of AI216611_T0_P0_E71 (p) 56D shows the DNA sequence of AI216611_T1_P1_EGFP (1332 bp) (SEQ ID NO: 80); FIG. 56E shows the DNA sequence of C1ORF32_T8_P8_EGFP (1533 bp) (SEQ ID NO: 81); FIG. 56F shows LOC253030_T4_P FIG. 56G shows the DNA sequence of ILDR1_T0_P3_EGFP (237) (SEQ ID NO: 82). bp) (SEQ ID NO: 83); FIG. 56H shows the DNA sequence of ILDR1_T2_P5_EGFP (2241 bp) (SEQ ID NO: 84); FIG. 56I shows the DNA sequence of VSIG1_T6_P5_EGFP (2082 bp) (SEQ ID NO: 85); Shows the DNA sequence (2004 bp) (SEQ ID NO: 86) of VSIG1_T5_P4_EGFP.

  The resulting VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 full-length amino acid sequences fused to EGFP are shown in FIGS. 57A-J; the gene-specific sequence corresponding to the full-length sequence of the protein is shown in bold. The EGFP sequences are italicized, not bold, and amino acids modified based on known SNPs are underlined. FIG. 57A shows the amino acid sequence of FXYD3_P0_EGFP protein (331aa) (SEQ ID NO: 87); FIG. 57B shows the amino acid sequence of FXYD3_P14_EGFP protein (360aa) (SEQ ID NO: 88); FIG. 57C shows the amino acid sequence of AI216611_P0_EGFP protein (SEQ ID NO: 88). 456aa) (SEQ ID NO: 89); FIG. 57D shows the amino acid sequence of AI216611_P1_EGFP protein (443aa) (SEQ ID NO: 90); FIG. 57E shows the amino acid sequence of C1ORF32_P8_EGFP protein (510aa) (SEQ ID NO: 91); FIG. 57F shows the amino acid sequence (694aa) (SEQ ID NO: 92) of the LOC253012_P5_EGFP protein; FIG. 57G shows the ILDR1_P3_EGFP tag Fig. 57H shows the amino acid sequence of ILDR1_P5_EGFP protein (746aa) (SEQ ID NO: 94); Fig. 57I shows the amino acid sequence of VSIG1_P5_EGFP protein (693aa) (sequence) FIG. 57J shows the amino acid sequence (667aa) (SEQ ID NO: 96) of the VSIG1_P4_EGFP protein.

Table 136: Full length cloning details

Example 9: Measurement of cellular localization of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, and FXYD3 In order to determine the cellular localization of protein targets, they were cloned as EGFP (sensitive green fluorescent protein) fusion proteins. . Protein localization was observed using confocal microscopy during transient transfection (Chen et al., Molecular vision 2002; 8; 372-388). Cells were observed for the presence of fluorescent products 48 hours after transfection.

  Measurement of cellular localization of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, and FXYD3 was performed by transient transfection of the recombinant ORF-EGFP construct described above.

  Subsequently, VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, and FXYD3-EGFP pIRESpuro3 construct were transiently transfected into HEK-293T cells as follows:

  HEK-293T (ATCC, CRL-11268) cells were treated with 2 ml of pre-warmed DMEM [Dulbecco's Modified Eagle Medium, Biological Industries (Beit Ha'Emek, Israel), catalog number: 01-055-1A]. + 10% FBS (fetal bovine serum) +4 mM L-glutamine was seeded onto a 13 mm diameter sterile cover glass (Marienfeld, catalog number: 01 115 30) placed in a 6-well plate. 500,000 cells per well were transfected with 2 μg of DNA construct using 6 μl of FuGENE6 reagent (Roche, catalog number: 11-814-443-001) diluted in 94 ul of DMEM. This mixture was incubated for 15 minutes at room temperature. The complex mixture was added dropwise to the cells and stirred. The cells were placed in an incubator maintained at 37 ° C. with a CO 2 concentration of 5%.

  Forty-eight hours after transient transfection, cells were further processed for analysis by confocal microscopy. Coverslips were washed 3 times with phosphate buffered saline (PBS) and fixed with 3.7% or 1% paraformaldehyde (PFA) (Sigma, catalog number: P-6148) for 15 minutes. After washing twice with PBS, the fixed cover glass was adhered onto a slide glass using a mounting solution (Sigma, catalog number: G0918), and the cells were observed for the presence of the fluorescent product using a confocal microscope. . The results are shown in FIGS.

  FIG. 58A shows that AI216611_P0_EGFP (SEQ ID NO: 89) and AI216611_P1_EGFP (SEQ ID NO: 90) fusion proteins are localized to the cell membrane after expression in HEK293T cells. The images were obtained using a 40x objective lens of a confocal microscope.

  FIG. 58B shows that FXYD3_P0_EGFP (SEQ ID NO: 87) and FXYD3_P14_EGFP (SEQ ID NO: 88) fusion proteins are localized to the cell membrane after expression in HEK293T cells. The images were obtained using a 40x objective lens of a confocal microscope.

  FIG. 58C shows that the C1ORF32_P8_EGFP (SEQ ID NO: 91) fusion protein is localized to the cell membrane; the endoplasmic reticulum (ER) membrane, and the intercellular junction after expression in HEK293T cells. The images were obtained using a 40x objective lens of a confocal microscope.

  FIG. 58D shows that the LOC253012_P5_EGFP (SEQ ID NO: 92) fusion protein is localized to the cell membrane and the endoplasmic reticulum (ER) membrane after expression in HEK293T cells. The images were obtained using a 40x objective lens of a confocal microscope.

  FIG. 58E shows that the VSIG1_P5_EGFP (SEQ ID NO: 95) and VSIG1-_P4_EGFP (SEQ ID NO: 96) fusion proteins are localized to the nuclear and endoplasmic reticulum membranes after expression in HEK293T cells. The images were obtained using a 40x objective lens of a confocal microscope.

  FIG. 58F shows that ILDR1_P3_EGFP (SEQ ID NO: 93) and ILDR1_P5_EGFP (SEQ ID NO: 94) fusion proteins are localized to the plasma and endoplasmic reticulum membranes after expression in HEK293T cells. The images were obtained using a 40x objective lens of a confocal microscope.

Example 10: Cloning and expression of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, and FXYD3 extracellular region (ECD) fused to mouse FC The purpose of this analysis is to determine VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, and FXYD3. Cloning VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, and FXYD3 ECD fused to mouse Fc (mFc) via their corresponding C ′ ends for further use in the production of ECD antibodies and functional evaluation, The fusion ECD was expressed in HEK293T cells (ATCC-CRL-11268).

  The coordinates of the cloned ECD are shown in Table 137:

Table 137:

  Cloning of the fusion protein (ECD_mFc) was performed in two steps:

1. Cloning of ECD into pIRESpuro3 In-frame subcloning of mouse Fc IgG2a to the C ′ end of ECD previously cloned into pIRESpuro3 from step 1

  Cloning of ECD into pIRESpuro3 was performed as follows:

  The cloning of ECD for each of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, and FXYD3 uses the full-length sequence as a template, as shown in Table 137, by PCR with a partial amino acid sequence of the ECD as shown in Table 137. And with the primers listed in Table 138.

Table 138: ECD cloning details

  In Table 138, the bold letters above represent gene-specific sequences, while the restriction site ranges used for cloning are italicized and the Kozak sequences are underlined.

  The PCR product was purified and digested with the appropriate restriction enzymes shown in Table 138. To increase secretion, PCR products for FXYD3, AI216611, C1ORF32, and LOC253012 were ligated to pIRESpuro3, whereas PCR products for VSIG1 and ILDR1 were ligated to IL6sp_pIRESpuro3. The ligation mixture was transformed into DH5a competent cells. Positive transformants were screened and confirmed by DNA sequencing.

Cloning of ECD-mFc pIRESpuro3 Codon optimization of the mouse Fc (IgG2a) (accession number-CAA49868 aa 237-469) protein sequence followed by a TEV cleavage site sequence was performed to promote protein expression in mammalian systems. The optimized sequence was synthesized by GeneArt (Germany) with a BamHI restriction site adjacent to the N ′ end and a NotI restriction site at the C ′ end. The DNA fragment was digested with BamHI / NotI and ligated in frame to an ECD_pIRESpuro3 construct that had been digested with the same enzyme in advance to obtain ECD_mFc_pIRESpuro3. The ligation mixture was transformed into DH5a competent cells. Positive transformants were screened and confirmed by DNA sequencing.

  The nucleotide sequence of the obtained ECD_mFc ORF is shown in FIGS. 59A to F: the gene-specific sequence corresponding to the ECD sequence is shown in bold and underlined is the TEV cleavage site sequence, and the mFc sequence is not bold Italic, and the IL6sp sequence is bold italic. FIG. 59A shows the FXYD3_T25_P14_ECD-_mFc DNA sequence (924 bp) (SEQ ID NO: 97); FIG. 59B shows the AI216611_T0_P0_ECD_mFc DNA sequence (1170 bp) (SEQ ID NO: 98); FIG. 59D shows the LOC253012_T4_P5_ECD_mFc DNA sequence (1740 bp) (SEQ ID NO: 100); FIG. 59E shows the ILDR1_T0_P3_ECD_mFc DNA sequence (1167 bp) (SEQ ID NO: 101), and FIG. 59F shows the VSIG1_T6_P6_Pc (1641 bp) (SEQ ID NO: 102) is shown.

  The sequence of the obtained ECD_mFc fusion protein is shown in FIGS. 60A to 60F; the gene-specific sequence corresponding to the ECD sequence is shown in bold and underlined is the TEV cleavage site sequence, and the mFc sequence is not bold Italic, and the IL6sp sequence is bold italic. FIG. 60A shows the FXYD3_T25_P14_ECD_mFc amino acid sequence (307aa) (SEQ ID NO: 103); FIG. 60B shows the AI216611_T0_P0_ECD_mFc amino acid sequence (389aa) (SEQ ID NO: 104); FIG. FIG. 60D shows the LOC253012_T4_P5_ECD_mFc amino acid sequence (579aa) (SEQ ID NO: 106); FIG. 60E shows the ILDR1_T0_P3_ECD_mFc amino acid sequence (388aa) (SEQ ID NO: 107), and FIG. ) (SEQ ID NO: 108).

  To generate ECD-mFc expressing cells, HEK-293T cells were transfected with the above constructs corresponding to VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, and FXYD3 extracellular regions fused to mouse Fc. A stable pool was created as follows, 48 hours after transfection, cells were trypsinized and transferred to a T75 flask containing selective medium (DMEM 10% FCS and 5 μg / ml puromycin). Got. The medium was changed every 3 to 4 days until colonies formed.

  Genomic PCR was performed to confirm cell identity and showed that the correct sequence was integrated into the cell genome (data not shown).

  The media from which the cells were removed was collected and purified by Protein A Sepharose beads (Amersham, catalog number 17-5280-04) as follows: 1 ml of media from which the cells had been removed was brought to room temperature with 50 μl of Protein A Sepharose beads. And incubated for 45 minutes. At the end of the incubation period, the protein was eluted from the bead pellet with 50 μl of sample buffer containing 100 mM citrate phosphate pH 3.5 and 10 mM DTT. These samples were boiled for 3 minutes and 25 μl was loaded onto a 12% NuPAGE Bis Tris gel (Invitrogen, catalog number NPO342). The protein was transferred to a nitrocellulose membrane and blocked with 10% low fat milk in PBST (PBS supplemented with 0.05% tween-20). The membrane was then blotted with a goat anti-mouse IgG Fc fragment HRP (Jackson, catalog number 115-035-206) (1: 40,000 in blocking solution) for 1 hour at room temperature. After incubation with ECL solution (Amersham Biosciences, catalog number RPN2209), the membrane was exposed to film.

  FIG. 61 shows the expressed FXYD3_ECD_mFc (SEQ ID NO: 103), AI216611 ECD_mFc (SEQ ID NO: 104), C1ORF32_ECD_mFc (SEQ ID NO: 105), LOC253012_ECD_mFc (SEQ ID NO: 106), ILDR1_ECD_mFc (SEQ ID NO: 107), VSFIG1 108) shows the results of Western blotting for 108).

  Lanes are as follows: Lane 1 molecular weight markers (Amersham, full range ranbow, catalog number RPN800); Lane 2—LOC253012_ECD_mFc (SEQ ID NO: 106); Lane 3—FXYD3_ECD_mFc (SEQ ID NO: 103); Lane 4—AI216m1ECD Lane 5-C1ORF32_ECD_mFc (SEQ ID NO: 105); Lane 6-ILDR1_ECD_mFc (SEQ ID NO: 107); Lane 7-VSIG1_ECD_mFc (SEQ ID NO: 108).

Example 11: Production of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, and FXYD3 extracellular region (ECD) proteins fused to mouse FC Production of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, and FXYD3 ECD fused to mouse FC For this, a pool of HEK293T cells stably transfected with the corresponding construct described herein above was used. Transfected cells, normally maintained in 10% serum-supplemented medium, are transferred to serum-free medium (EX-CELL293, SAFC) supplemented with 4 mM glutamine and selected antibiotics (5 ug / ml puromycin). Transfer and grow in suspension culture in shake flask at 37 ° C. with agitation. The culture volume was increased by serial dilution until a 3-4 day production phase was performed in a 2 L spinner flask. The medium was collected from the spinner, the cells were removed by centrifugation, filtered through a 0.22 μm filter, and stored at −20 ° C.

  VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, and FXYD3 ECD fused to mouse Fc were purified using n-Protein A affinity chromatography as described below.

  The harvest was concentrated approximately 10-fold using a PALL ultrafiltration system on two 10 kD cassettes. Next, the pH was adjusted to 7.5 by adding 5 M NaOH to the concentrate and filtered through a 0.2 μm Stericup filter.

  The purification process was performed using an AKTA Explorer (GE Healthcare). 2 ml of nProtein A Sepharose ™ Fast Flow resin (Catalog No. 17-5280-02) on a Poly-prep chromatography column under reduced pressure, 10 column volumes (CV) of 70% ethanol, 10 CV WFI ( Washed with sterile water for irrigation (TEVA)) followed by 10 CV buffer A. 2 ml of resin was transferred to two 500 ml tubes (1 ml each) and concentrated collection was added. The tube was incubated overnight at 4 ° C. on a roller to allow protein binding. The bound resin was then transferred to a XK16 column (GE Healthcare, catalog number 18-8773-01) under a constant flow rate and packed. This column was washed with 20 CV of buffer A (100 Mm Tris pH 7.4), and one-step elution was performed using 100% buffer B (citric acid / phosphoric acid pH 3.0). Fractions were titrated with 12.5% (v / v) buffer C (2M Tris pH 8.5), pH adjusted to about 7.5 and pooled.

  Using 53 ml of HiPrep ™ (GE Healthcare, catalog number 17-5087-01) desalting column, the final buffer was DPBS (Dulbecco phosphate buffered saline pH 7.4, no Ca, No Mg) pH 7.4 No Ca, no Mg. The protein was filtered through a 0.22 μm filter, aliquoted under sterile conditions and stored at -800C.

  The final protein concentration was determined by BCA total protein assay and the protein was analyzed by Coomassie-stained reducing SDS / PAGE (data not shown). Endotoxin levels were measured by a colorimetric LAL assay (Homoptera blood cell extract, QCL-1000, Cambrex). Identification of specific proteins was confirmed by MS (Smoler Proteomics Center, Technology, Haifa, data not shown).

  The obtained protein analysis results are summarized in Table 139.

Table 139

Example 12: Binding of ECD Fc-fusion proteins of the invention to activated cells The VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, and FXYD3 Fc-fusion ECDs described above are putative counter receptors on T cells. These Fc-fusion ECDs were tested on resting or activated T cells to examine their ability to bind (receptor). Purified T cells were activated with ConA (Sigma Aldrich, catalog number C5275) and subsequently incubated with Fc-fusion ECD of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 and analyzed by flow cytometry.

  T cells were purified from whole blood by negative selection using RosetteSep ™ Human T Cell Enrichment Cocktail (StemCell Technologies, catalog number 15061). This resulted in a population of T (CD3 +) cells that was approximately 90% pure. Purified T cells (1X 105) were either unactivated or activated with ConA (Concovallin A, 10 ug / ml, Sigma Aldrich, catalog number C5275) in 100 ul containing 10% FBS. The cells were cultured in RPMI 1640 complete medium for 48 hours. Cultures were harvested and stained with ECD Fc-fusion protein for 1 hour at 4 ° C. (VSIG1, ILDR1, LOC253012, AI216611, FXYD3, or C1ORF32 ECD fused to mouse IgG2 Fc). Bound protein was detected with FITC-conjugated F (ab) 2 goat anti-mouse Fc for 30 minutes at 4 ° C. (Jackson ImmunoResearch Laboratories, catalog number 115-096-071). Sample analysis was performed using FACSCalibur (BD Immunocytometry Systems) and CellQuest software.

  Figures 62A-D show activity of ECD Fc-fusion proteins (VSIG1 (SEQ ID NO: 108), LOC253012 (SEQ ID NO: 106), AI216611 (SEQ ID NO: 104), or C1ORF32 (SEQ ID NO: 105)) on resting T cells or ConA. The binding to activated T cells is shown for various times. Primary human T cells from three different donors were cultured in the absence of stimulation (0 hours) or in the presence of ConA for a total of 48 hours, where ConA was the last 6, 18, 24, or 48 hours The culture was added to a final concentration of 10 μg / ml (T cells from donor 5 were cultured with ConA for 0, 6, 18, and 24 hours, while donors 6 and 7 were 0, Cultured for 6, 24, and 48 hours). Cells were then harvested and incubated with 10 μg / ml of the indicated ECD Fc-fusion protein. FIG. 62A shows the binding result of Fc-fusion VSIG1 ECD; FIG. 62B shows the binding result of Fc-fusion LOC253012; FIG. 62C shows the binding result of Fc-fusion C1ORF32 ECD; FIG. The binding result of fusion AI216611 ECD is shown, and FIG. 62E shows the binding result of Fc-fusion FXYD3 ECD. The percentage of positive cells was calculated as the difference between the positive cells with the indicated protein and the positive cells obtained with FITC-conjugated F (ab) 2 goat anti-mouse Fc. FIG. 63 shows the dose response for binding of B7-like protein to activated T cells. Purified T cells were cultured for 48 hours. ConA was added for the last 24 hours. Cells were then harvested and stained with Fc-fusion VSIG1, LOC253012, C1ORF32, AI216611, or ILDR1 ECD at increasing concentrations (3, 6, 12, 25, and 50 μg / ml). As a negative control, mouse IgG2a was used at the same concentration.

  The results shown in FIGS. 62A-D and 63 show that all tested ECD Fc-fusion proteins (VSIG1, ILDR1, LOC253012, AI216611, or C1ORF32 ECD fused to mouse IgG2 Fc, SEQ ID NOs: 108, 107, 106, 104 or 105) binding is shown to be higher than the negative control as an isotype control: mouse IgG2a (R & D Systems, catalog number MAB003). For Fc-fused VSIG1 ECD and LOC253012 ECD-Fc, significant binding to T cells stimulated with ConA was detected. As can be seen from FIGS. 62A-D and 63, the C1ORF32 and AI216611 Fc-fusion ECD showed weaker binding to these cells. Each protein was found to bind to a certain percentage of activated T cells. The order of binding levels was as follows: VSIG1> LOC253012> ILDR1 = AI216611> C1ORF32. Neither protein bound to resting T cells (i.e., ConA, 0 hours in Figures 62A-D).

Effect of the ECD Fc-fusion protein of the present invention on T cell activation The soluble protein of the present invention, VSIG1, ILDR1, LOC253012, AI216611, FXYD3, or C1ORF32 ECD fused to mouse IgG2 Fc, SEQ ID NOs: 108, 107, 106, respectively. , 104, or 105, human T cells were tested for anti-CD3 (clone OKT3, eBioscience, Catalog No. 16-) to test the potential for costimulatory or / and co-inhibitory activity on T cell proliferation and IL-2 secretion. 0037-85) and the aforementioned B7-like protein of the present invention. Recombinant human B7-1 protein (R & D Systems, catalog number 140-B1) was used as a positive control for costimulatory activity. Recombinant mouse B7-H4 protein (R & D Systems, catalog number 4206-B7) was used as a positive control for co-inhibitory activity.

  First, flat bottom 96-well plates were coated overnight with 3 μg / ml anti-CD3 mAb (clone OKT3) at 4 ° C., followed by the indicated concentrations of human B7-1 (R & D, 3 μg / ml), mouse Coated with B7-H4 (R & D, 10 μg / ml), or VSIG1, ILDR1, LOC253012, AI216611, FXYD3, or C1ORF32 ECD fused to ECD Fc-fusion protein, mouse IgG2 Fc of the present invention for 4 hours at 37 ° did. Human T cells were purified from whole blood as described above and cultured in pre-coated 96-well plates (1 × 10 5 cells / well) in 250 μl of RPMI 1640 complete medium containing 10% FBS for 48 hours. . The coated plate was washed 3 times with PBS and then seeded with cells. T cell proliferation was measured from BrdU incorporation by cell proliferation ELISA, BrdU (colorimetric) (Roche). Cells were labeled with a final concentration of 100 μM BrdU labeling reagent for the last 18 hours. The plates are then centrifuged (300 g, 10 min), the supernatant is aspirated, followed by -20 for IL-2 measurements using a human IL-2 ELISA (Diacron, catalog number 850.010 096). Stored at ° C. BrdU incorporation was measured according to the manufacturer's instructions for the cell proliferation ELISA, BrdU (colorimetric) (Roche, catalog number 11-647-229).

  FIGS. 64A-B show the effect of the ECD Fc-fusion protein of the invention on T cell proliferation or IL-2 secretion when activated with anti-CD3 Ab. FIG. 64A shows the level of BrdU incorporation. FIG. 64B shows the level of IL-2 secretion.

  The results shown in FIGS. 64A-B indicate that none of the ECD-Fc fusion protein, VSIG1, ILDR1, LOC253012, AI216611, FXYD3, or C1ORF32 ECD fused to mouse IgG2Fc showed costimulatory activity. Positive control B7-1 showed strong costimulatory activity as expected. Fc-fused ILDR1 ECD and Fc-fused AI216611 ECD have co-inhibitory activity because they inhibited cell proliferation as did B7-H4 compared to those obtained in the presence of negative control: mouse IgG2a It seems (Figure 64A). However, no significant effect on IL-2 secretion was observed with any of the ECD-Fc fusion proteins, VSIG1, ILDR1, LOC253012, AI216611, FXYD3, or C1ORF32 Fc-fusion ECD (FIG. 64B).

Example 13: Binding of ECD Fc-fusion proteins of the invention to lymphocytes and CD4 positive cells VSIG1, ILDR1, LOC253012, AI216611, FXYD3, and C1ORF32 Fc-fusion ECD bind to putative counter-receptors on T cells To further investigate the ability, these Fc-fusion ECDs were first tested on lymphocytes. PBMC were prepared from human peripheral blood at 1 × 10e7 / ml in FACS buffer. 30 ug / ml Fc blocker (hIgG (16D10), lot number 080706, 1.3 mg / ml) was added and cells were incubated with blocker on ice for 30 minutes. The fusion protein was added at 1 ug / 10e6 per stain for 30 minutes on ice. The second Ab was added at 1 ug / 100 ul / stained portion over 25-30 minutes (G @ mIgG-Fc-FITC: Jackson Immunol Lab, 1 mg / ml, code number 115-096-071, lot number 71453, Used at 1.0 mg / ml, 1 ug / stained part). Cells were washed with buffer at each step outlined above. Binding was analyzed by flow cytometry.

  FIG. 65 shows binding of VSIG1, ILDR1, LOC253012, AI216611, FXYD3, or C1ORF32 ECD Fc-fusion to lymphocytes. As can be seen from FIG. 65, C1ORF32, AI216611, and ILDR1 bind to their counterparts expressed on lymphocytes.

  Next, binding of VSIG1, ILDR1, LOC253012, AI216611, FXYD3, and C1ORF32 Fc-fusion ECD to CD4 + cells. Fc blocker (hIgG (16D10), lot number 080706, 1.3 mg / ml) was added at 30 ug / ml and cells were incubated with blocker on ice for 30 minutes. The fusion protein was added at 1 ug / 10e6 per stained area for 30 minutes on ice. The second Ab was added at 1 ug / 100 ul / stained portion over 25-30 minutes (G @ mIgG-Fc-FITC: Jackson Immunol Lab, 1 mg / ml, code number 115-096-071, lot number 71453, Used at 1.0 mg / ml, 1 ug / stained part). @ CD4 (m @ hCD4-APC: BD, catalog number 3555349, lot number 44331) was added at 20 ug per stained area for 30 minutes on ice.

  Cells were washed with buffer at each step outlined above. Binding was analyzed by flow cytometry.

  FIG. 66 shows binding of ILDR1, C1ORF32, and AI216611 ECD Fc-fusion to CD4 + cells.

Example 14: Effect of the ECD Fc-fusion protein of the invention on T cell activation To test the possible costimulatory or / and co-inhibitory activity of the B7-like protein of the invention, VSIG1, fused to mouse IgG2 Fc, The effect of ILDR1, LOC253012, AI216611, or C1ORF32 ECD on T cell proliferation was tested. T cells were purified from whole blood by positive sorting using CD3 microbeads (MACS Whole Blood CD3 Microbeads # 130-090-874) coupled to monoclonal anti-human CD3 antibody (isotype: mouse IgG2a). Coated with CD3 +/− B7 with M-450 Epoxy Dynabeads (Invitrogen Cat # 140.11) For activation of CD3 T cells, purified CD3 T cells can be treated with CD3 + CD28 coated beads as needed. Stimulate at a ratio of 1: 1 or 1:05 at time points.Cells are weighed in the presence or absence of CD3 + CD28 (2 ug / ml each) coated beads. Cell proliferation was measured by tritium-thymidine incorporation 72 hours later, and the results are shown in Fig. 67. "CD3" in Fig. 67 shows no costimulatory or co-inhibitory molecule, “CD3 + B7.2” means CD3 + known B7 stimulation control, B7.2; “CD3 + B7H4” means CD3 and B7H4, known B7 inhibition control; “CD3 + B7H3” means CD3 and B7H3, known B7 stimulating proteins; “CD3 + 702” means CD3 + LOC253012-ECD-Fc fusion (SEQ ID NO: 106); “CD3 + 721” means CD3 + AI216611-ECD-Fc fusion ( SEQ ID NO: 104); “CD3 + 754” means CD3 + 1ORF32-ECD-Fc fusion (SEQ ID NO: 105); "CD3 + 768" means CD3 + VSIG1-ECD-Fc fusion (SEQ ID NO: 108); "CD3 + 770" means CD3 + ILDR1-ECD-Fc fusion (SEQ ID NO: 107) “CD3 + 789” means a CD3 + FXYD3-ECD-Fc fusion (SEQ ID NO: 103).

  As can be seen from FIG. 67, LOC253012-ECD-Fc, AI216611-ECD-Fc, VSIG1-ECD-Fc, and FXYD3-ECD-Fc were compared to CD3 alone in three different experiments compared to CD3 alone. It had an inhibitory effect (FIGS. 67A, B, and C).

Example 15: Interaction of ECD-Fc fusion proteins of the invention with resting B cells, activated B cells, and B cell-derived lymphoma cell lines Following the demonstrated binding of the protein of the invention to lymphocytes (Examples 12 and 13 herein), the ability of the soluble proteins of the invention to bind to B cells was examined.

  PBMC were prepared from human peripheral blood at 1 × 10e7 / ml in FACS buffer. 30 μg / ml Fc blocker (hIgG (16D10), lot number 080706, 1.3 mg / ml) was added and cells were incubated with blocker on ice for 30 minutes. A fusion protein of the invention, ILDR1-ECD-Fc (SEQ ID NO: 107), C1ORF32-ECD-Fc (SEQ ID NO: 105), AI216611-ECD-Fc (SEQ ID NO: 104), LOC253012-ECD-Fc (SEQ ID NO: 106), FXYD3-ECD-Fc (SEQ ID NO: 103) and VSIG1-ECD-Fc (SEQ ID NO: 108) were added at 1 μg / 10e6 per stained area for 30 minutes on ice. The second Ab was added at 1 μg / 100 ul / stained portion over 25-30 minutes (G @ mIgG-Fc-FITC: Jackson Immunol Lab, 1 mg / ml, code number 115-096-071, lot number 71453, Used at 1.0 mg / ml, 1 ug / stained part). Cells were washed with buffer at each step outlined above. Binding was analyzed by flow cytometry. The cells were then stained with @human IgM-PE (BD Bioscience, California, USA, catalog number 555783) that is specific for B cells. Stained cells were analyzed by flow cytometry. @Human IgM positive cells were gated and analyzed for binding of the fusion protein of the invention to B cells.

  As shown in FIG. 68A, ILDR1-ECD-Fc and C1ORF32-ECD-Fc bound to B cells from all three donors tested. AI216611-ECD-Fc showed binding only to B cells of one donor.

  To confirm the presence of the counterpart on activated B cells, PBMC were activated with LPS for 72 hours. Thereafter, the ECD Fc-fusion protein of the present invention, ILDR1-ECD-Fc (SEQ ID NO: 107), C1ORF32-ECD-Fc (SEQ ID NO: 105), AI216611-ECD-Fc (SEQ ID NO: 104), LOC253012-ECD-Fc ( SEQ ID NO: 106), FXYD3-ECD-Fc (SEQ ID NO: 103), and VSIG1-ECD-Fc (SEQ ID NO: 108) were conjugated as described above and the cells were treated with mouse @ human CD86-Cy5PE (BD Bioscience, California, USA, S, Catalog No. 555659) and mouse @ human CD19-PE (BD Bioscience, CA, USA) antibody. Activated cells were defined as a double positive CD19 + / CD86 + cell population.

  As shown in FIG. 68B, ILDR1-ECD-Fc (SEQ ID NO: 107), C1ORF32-ECD-Fc (SEQ ID NO: 105), and AI216611-ECD-Fc (SEQ ID NO: 104) bind to activated B cells. Indicated.

  To confirm the presence of counterparts in B cell malignancies, the ECD Fc-fusion protein of the invention, ILDR1-ECD-Fc (SEQ ID NO: 107), C1ORF32-ECD-Fc (in a B cell lymphoma cell line) SEQ ID NO: 105), AI216611-ECD-Fc (SEQ ID NO: 104), LOC253012-ECD-Fc (SEQ ID NO: 106), FXYD3-ECD-Fc (SEQ ID NO: 103), and VSIG1-ECD-Fc (SEQ ID NO: 108) Binding was analyzed. Raji (ATCC number CCL-86) and Daudi (ATCC number CCL-213) cells were purchased from ATCC and maintained in RPMI + 10% FBS. Cells were stained with B7 protein or control at 10 μg / ml, followed by FITC-conjugated goat anti-mouse IgG Fc (Jackson Immunol Lab, NJ, USA, catalog number 115-096-071, lot number 71453).

  FIG. 68C shows Fc-fusion ECD of the B7-like protein of the present invention (ILDR1-ECD-Fc (SEQ ID NO: 107), C1ORF32-ECD-Fc (SEQ ID NO: 105), AI216611-ECD-Fc (SEQ ID NO: 104), LOC253012 -ECD-Fc (SEQ ID NO: 106), FXYD3-ECD-Fc (SEQ ID NO: 103), and VSIG1-ECD-Fc (SEQ ID NO: 108)) binding to B cell lymphoma cell lines. ILDR1-ECD-Fc (SEQ ID NO: 107) showed clear binding to both B cell lymphoma cell lines.

Example 16: Interaction of ECD-Fc fusion protein of the present invention with known B7 The interaction of the AI216611 protein of the present invention with various known ligands of the B7 family was analyzed. Since AI216611 was predicted to be a putative CD28 receptor, it was hypothesized to bind to the known B7 ligand, B7H4, which is considered to be an orphan (its corresponding receptor has not yet been identified).

  Analysis of the interaction between B7H4-Ig (R & D Systems, Inc., catalog number 4206-B7) and Fc fusion AI216611 ECD (SEQ ID NO: 104) directly measures intermolecular interactions using surface plasmon resonance BIAcore 3000 system (Uppsala, Sweden) (Pharmacia Biosensor, Uppsala, Sweden). Fc fusion AI216611 ECD (SEQ ID NO: 104) (400-500 resonance unit (RU)) was directly immobilized on the sensor CM5 chip. Solutions containing 5 different concentrations of B7H4-Ig (5 and 10 micromolar) were injected. As a control, this solution was also injected into an empty flow cell in which no ligand was immobilized.

  Data analysis was performed using BIAevaluation software (GraphPad Software Inc., San Diego, Calif.). Zero baseline levels were obtained by subtracting the background response from the analyte injection through the control flow cell without ligand fixation.

  As can be seen from FIG. 69, a slight interaction between Fc-fusion AI216611 ECD (SEQ ID NO: 104) and B7H4 was seen with 5 and 10 μM AI216611.

Example 17: Development of mouse monoclonal anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, and anti-FXYD3 antibodies To test the expression of B7-like proteins in various cancer tissues by immunohistochemistry, the present invention. Monoclonal mouse antibodies that are specific for the Fc-fusion ECD of these proteins have been developed.

Development of mouse monoclonal antibodies:
Four groups of Balb / c mice (three mice per group) were prepared from the four Fc-fusion ECD proteins of the invention: VSIG1 (SEQ ID NO: 108), LOC253012 (SEQ ID NO: 106), C1ORF32 (SEQ ID NO: 105), and Immunization was performed with FXYD3 (SEQ ID NO: 103). Immunization was performed 8 times at one week intervals in multiple sites, subcutaneously and intraperitoneally. Mice were bled 10 days after the fourth and eighth immunizations. Sera were screened for antibody titers using the direct ELISA protocol described below.

  ELISA plate was diluted into 50 μl / well of 2.5 μg / mL Fc-fusion protein (VSIG1, LOC253012, C1ORF32, FXYD3 ECD fused to mouse IgG2 Fc, SEQ ID NOs: 108, 106, 105, 103, respectively) diluted in DPBS. For 1 hour at room temperature (RT). Human IgG fused to the mouse Fc region was used as a negative control. The plates were then blocked with 300 μl / well of 1% BSA / DPBS for 15 minutes at RT. Following the blocking step, serially diluted serum from immunized mice and unrelated mouse IgG were transferred to the blocked ELISA plate and incubated for 1 hour at RT. The plate was then washed 3 times with 300 μl / well wash buffer (DPBS with 0.05% Tween 20, pH 7.2-7.4). For detection, plates were incubated with 50 μl / well goat anti-mouse kappa light chain antibody diluted 1: 1000 for 1 hour at RT, followed by extensive washing (6 with 300 μl / well wash buffer). Times) and incubated with the substrate. Substrate 2,2′-Azino-bis- (3-ethylbenzthiazoline-6-sulfonic acid (ABTS) was added at 100 μL / well, incubated for about 5 minutes at RT, and then Molecular Devices SPECTRAmax 340 PC plate reader. And plate readings at 414 nm using SOFTmax PRO software.

  Serum antibody titer was defined as the dilution of serum that produced a signal that was twice the background.

  The results of the ELISA test of immunized serum after 4 immunizations are summarized in Table 140. The data show that after 4 immunizations, two groups of mice (immunized with LOC253012 and VSIG1 Fc fusion protein ECD) generated antibody titers sufficient for hybridoma production.

  Mice that showed the highest antibody serum titers were selected for hybridoma production. Splenocytes were fused with the mouse myeloma cell line Ag8.653. Hybridoma clone supernatants were tested by direct ELISA (as described above) using plates coated with related and unrelated coating agents. The results are summarized in Table 141A and Table 141B.

  The results show that the production of hybridoma cell lines yielded 14 clones that specifically recognize LOC253012 (Table 141A, bold) and 14 clones that specifically recognize VSIG1 (Table 141B, bold). Show.

  The remaining proteins were further immunized four times to promote the generation of serum antibody titers against the remaining proteins. Serum titers after the 8th immunization were tested by direct ELISA. The results are summarized in Table 142. The results show that after 8 immunizations, mice immunized with FXYD-Fc fusion ECD (SEQ ID NO: 103) and C1ORF32-Fc fusion ECD (SEQ ID NO: 103) generate antibody titers that are sufficient for hybridoma production. It was. In the next step, the best responders are selected for hybridoma production and monoclonal antibody production.

  Mouse monoclonal anti-ILDR1 and anti-AI216611 antibodies are generated in a similar manner.

  Using monoclonal antibodies against each antigen of the present invention (VSIG1, LOC253012, C1ORF32, FXYD3, AI216611, and ILDR1, SEQ ID NOs: 108, 106, 105, 103, 104, and 107, respectively), these in cancer and normal tissues To confirm the expression profile of each putative protein, an immunohistochemical analysis is performed.

Table 140: Antibody serum titers of immunized mice after 4 immunizations

Table 141A: post fusion clones obtained from mouse number 167223 immunized with LOC253012

Table 141B: Post-fusion clones obtained from mouse number 167228 immunized with VSIG1

Table 142: Antibody serum titers of immunized mice after 8 immunizations

Immunohistochemistry analysis Immunohistochemistry allows visualization of tissue distribution of specific antigens (or epitopes) using optical or confocal microscopy. In this process, the location of the protein target of interest is determined by applying specific monoclonal or polyclonal antibodies to the tissue surface in a process called antibody incubation.

  This method involves the in situ detection of the substrate in immobilized cells by a substrate specific antibody. The substrate specific antibody may be conjugated to an enzyme or conjugated to a fluorophore. Detection is performed by subjective evaluation by microscopic observation. When enzyme-linked antibodies are used, a colorimetric reaction may be necessary.

  The immunohistochemical analysis performed on the antigens of the invention (VSIG1, LOC253012, C1ORF32, FXYD3, AI216611, and ILDR1, SEQ ID NOs: 108, 106, 105, 103, 104, and 107, respectively) Consists of:

  Phase I: Antibody Calibration: Experiments are performed on serial dilutions of each antibody raised against a specific protein antigen using selected formalin fixed paraffin embedded (FFPE) control tissues and cell lines. Select the antibody with the best results and proceed to Phase II.

  Phase II: Protein distribution and localization analysis: Using the optimal antibody concentrations selected in Phase I, the distribution and localization analysis of VSIG1, LOC253012, C1ORF32, FXYD3, AI216611, and ILDR1 proteins were performed in cancer and normal tissues. Determine the differential expression in some of the cancer samples compared to normal samples.

Example 17: Development of fully human anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, and anti-FXYD3 antibodies Production of human monoclonal antibodies against VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, and FXYD3 antigens

  A fusion protein consisting of the extracellular regions of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, and FXYD3 conjugated to mouse IgG2 Fc polypeptide is generated by standard recombinant methods and used as an antigen for immunization.

Transgenic HuMab mice Fully human monoclonal antibodies against VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, and FXYD3 are generated using mice from the HCo7 line of transgenic HuMab mice® that express human antibody genes. In this mouse strain, Chen et al. (1993) EMBO J. et al. 12: 811-820, the endogenous mouse kappa light chain gene has been homozygously disrupted, and the endogenous mouse heavy chain gene is as described in Example 1 of PCT Publication WO01 / 09187. It has been homozygously destroyed. In addition, this mouse system is described in Fishwild et al. (1996) Nature Biotechnology 14: 845-851, having a human kappa light chain transgene, KCo5, and US Pat. Nos. 5,545,806; 5,625,825; As described in US Pat. No. 545,807, it has a human heavy chain transgene, HCo7.

HuMab immunization:
To generate fully human monoclonal antibodies against VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, and FXYD3, this strain is a mammal transfected with an expression vector containing a gene encoding the fusion protein. Can be immunized with purified recombinant VSIG1 fusion protein from cells). A general immunization scheme for HuMab mice® is described in Lonberg, N .; et al (1994) Nature 368 (6474): 856-859; Fishwild, D. et al. et al. (1996) Nature Biotechnology 14: 845-851, and PCT Publication 98/24884. The age of the mouse at the first injection of the antigen is 6 to 16 weeks. HuMab mice are immunized intraperitoneally with purified recombinant VSIG1 antigen preparation (5-50 mug, purified from transfected mammalian cells expressing VSIG1 fusion protein).

  Transgenic mice are immunized intraperitoneally twice with antigen in complete Freund's or Ribi adjuvant, followed by 3-21 days of intraperitoneal immunization with antigen in incomplete Freund's or Ribi adjuvant (total) Up to 11 immunizations). The immune response is monitored by retroorbital bleeds. Plasma is screened by ELISA (as described below) and mice with sufficient titers of anti-VSIG1 human immunoglobulin are used for fusion. Mice are boosted intravenously with antigen and sacrificed and spleen removed 3 days later.

Selection of HuMab mice ™ that produce anti-VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, and FXYD3 antibodies:
To screen for HuMab mice ™ that produce antibodies that bind to VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3, Fishwild, D. et al. et al. Serum from immunized mice is tested in a modified ELISA as first reported by (1996). Briefly, microtiter plates were coated at 50 mul / well with 1-2 mug / ml of purified recombinant VSIG1 fusion protein in PBS, incubated overnight at 4 ° C., followed by Block with 200 mul / well of 5% BSA. Dilutions of plasma from mice immunized with VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 are added to each well and incubated for 1-2 hours at ambient temperature. Plates are washed with PBS / Tween and then incubated with goat anti-human kappa light chain polyclonal antibody conjugated with alkaline phosphatase for 1 hour at room temperature. After washing, the plates are developed with pNPP substrate and analyzed with a spectrophotometer at OD 415-650. Mice with the highest anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, or anti-FXYD3 antibody titers are used for fusion. Fusions are performed as described below and hybridoma supernatants are tested by ELISA for anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, or anti-FXYD3 activity.

Generation of hybridomas producing human monoclonal antibodies against VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 Mouse splenocytes isolated from HuMab mice are fused to a mouse myeloma cell line with PEG according to standard protocols Let The resulting hybridomas are then screened for production of antigen specific antibodies. Single cell suspensions of splenic lymphocytes from immunized mice were combined with 50% PEG (Sigma) for P3X63 Ag8.6.53 (ATCC CRL 1580), a quarter number of nonsecreting mouse myeloma cells. To fuse. Flat bottom microtiter plates were seeded with cells at approximately 1 × 10 −5 / well, followed by 10% fetal calf serum, origen (IGEN), L-glutamine, sodium pyruvate, HEPES in RPMI, Incubate for about 2 weeks in selective medium supplemented with penicillin, streptomycin, gentamicin, 1 × HAT, and beta-mercaptoethanol. After 1-2 weeks, the cells are cultured in a medium in which HAT is replaced with HT. Individual wells are then screened by ELISA for human anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, or anti-FXYD3 monoclonal IgG antibodies (described above). The medium is monitored usually after 10-14 days after the hybridoma has been greatly propagated. If the antibody-secreting hybridoma is replated and screened again and still positive for human IgG, anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, or anti-FXYD3 monoclonal antibody is at least twice by limiting dilution Subclone. The stable subclone is then cultured in vitro and a small amount of antibody is produced in the tissue culture medium for further characterization.

  Hybridoma clones are selected for further analysis.

Structural characterization of desired anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, or anti-FXYD3 human monoclonal antibody The resulting anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, or anti-FXYD3 monoclonal CDNA sequences encoding the heavy and light chain variable regions of the antibody are each obtained from the resulting hybridoma using standard PCR techniques and sequenced using standard DNA sequencing techniques.

  The nucleotide and amino acid sequences of the heavy and light chain variable regions are identified. These sequences can be compared to known human germline immunoglobulin light and heavy chain sequences and of the resulting anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, or anti-FXYD3 sequences The CDRs for each heavy and light chain are identified.

Characterization of binding specificity and binding kinetics of anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, or anti-FXYD3 human monoclonal antibody Anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, or anti-FXYD3 antibody The binding affinity, binding kinetics, binding specificity, and cross-competition of are examined by Biacore analysis. In addition, binding specificity is examined by flow cytometry.

Binding Affinity and Kinetics Anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, or anti-FXYD3 antibodies produced by the present invention were analyzed for affinity and binding kinetics by Biacore analysis (Biacore AB, Uppsala, Sweden). Determine characteristics. Purified recombinant human VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 fusion protein is converted to a CM5 chip (carboxymethyldextran coated chip via primary amine using standard amine coupling chemistry and a kit from Biacore. ). Binding is measured by flowing the antibody in HBS EP buffer (BIAcore AB) at a concentration of 267 nM and a flow rate of 50 mul / min. The binding kinetics of the antigen-binding antibody are followed for 3 minutes and the dissociation rate is followed for 7 minutes. The binding and dissociation curves are fitted to a 1: 1 Langmuir binding model using BlAevaluation software (Biacore AB). In order to minimize the influence of binding activity in the calculation of the binding constant, only the first data segment corresponding to the binding and dissociation phase is used for fitting.

Epitope mapping of the resulting anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, or anti-FXYD3 antibody using Biacore, anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, or anti-FXYD3 HuMAb Epitope grouping is determined. The resulting anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, or anti-FXYD3 antibody is used to map those epitopes on the VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 antigen, respectively. These different antibodies are coated on three different surfaces of the same chip, each up to 8000 RU. Starting with 10 mug / mL, each dilution of mAb is made and incubated with Fc fusion VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 (50 nM) for 1 hour. Incubated complexes are injected simultaneously on all three surfaces (and the blank surface) for 1.5 minutes at a flow rate of 20 muL / min. The signal from each surface at the end of 1.5 minutes was plotted against the mAb concentration in the complex after drawing an appropriate blank. Based on the analysis of the data, anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, or anti-FXYD3 antibodies are classified into different epitope groups depending on the result of epitope mapping. Its functional characteristics are also compared.

  A Chinese hamster ovary (CHO) cell line expressing the VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 protein is generated on the cell surface and used to produce VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3HMAb Specificity is measured by flow cytometry. CHO cells are transfected with an expression plasmid containing a full-length cDNA encoding the transmembrane form of VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 antigen or variants thereof. Transfected proteins containing an epitope tag at the N-terminus are used for detection with antibodies that are specific for the epitope. Assessment of binding of anti-VSIG1, anti-ILDR1, anti-LOC253012, anti-AI216611, anti-C1ORF32, or anti-FXYD3 MAb was performed using transfected cells at a concentration of 10 mug / ml VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3Ab By incubating with each of the above. Cells are washed and binding is detected with FITC-labeled anti-human IgG Ab. As a positive control, mouse anti-epitope tag Ab followed by labeled anti-mouse IgG is used. Non-specific human and mouse Ab are used as negative controls. The resulting data will be used to assess the specificity of HuMAb for VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 antigen targets.

  These and other antibodies specific for VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 are VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 antigen, such as lung cancer, colon cancer, and ovarian cancer. Can be used for treatments related to the aforementioned anti-VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3, such as treatment of differentially expressed cancers, and / or such antibodies are desired, for example VSIG1 such as the treatment of cancer and autoimmune diseases that prevent negative stimulation of T cell activity on target cancer cells of the subject or prevent positive stimulation of T cell activity, thereby inducing a desired anti-autoimmune effect , I DR1, LOC253012, AI216611, or C1ORF32 antigen can be used for modulating (enhancing or inhibiting) participating B7 immune costimulatory.

  Production of anti-VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 antibodies desired to be used as therapeutic and diagnostic methods where the disease or condition is associated with VSIG1, ILDR1, LOC253012, AI216611, C1ORF32, or FXYD3 antigen and In connection with screening, the present invention has been described and a predictive embodiment has been provided. The invention is further described by the following claims.

Claims (3)

A monoclonal or polyclonal antibody comprising an antigen binding site that specifically binds to an isolated polypeptide selected from the group consisting of: (i) to (iii) , F (ab ′), F (ab), Fv or scFv fragment selected from the group consisting of fragments.
(I) a C1ORF32 extracellular region polypeptide consisting of the amino acid sequence of SEQ ID NO: 299, or a fragment thereof;
(Ii) a C1ORF32 extracellular region polypeptide consisting of the amino acid sequence of SEQ ID NO: 147, or a fragment thereof;
(Iii) C1ORF32 extracellular region polypeptide consisting of the amino acid sequence of SEQ ID NO: 148 or a fragment thereof   Said antibody is a fully human antibody, chimeric antibody, humanized or primatized antibody; Fab, Fab ′, F (ab ′) 2, F (ab ′), F (ab), Fv or scFv fragments thereof The antibody or fragment according to claim 1, which is an antigen-binding fragment selected from the group consisting of:   The antibody is linked to a moiety selected from a drug, radionuclide, fluorophore, enzyme, toxin, therapeutic drug, chemotherapeutic agent, or detectable marker, and the detectable marker is a radioisotope, metal chelator The antibody or fragment according to claim 1 or 2, which is an enzyme, a fluorescent compound, a bioluminescent compound, or a chemiluminescent compound.